GAINING INSIGHTS INTO THE MITOTIC TENSION SENSING MECHANISM IN SACCHAROMYCES CEREVISIAE: REGULATION OF CHROMATIN RECRUITMENT OF SHUGOSHIN PROTEIN SGO1P By Xiexiong Deng A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology – Doctor of Philosophy 2018 ABSTRACT GAINING INSIGHTS INTO THE MITOTIC TENSION SENSING MECHANISM IN SACCHAROMYCES CEREVISIAE: REGULATION OF CHROMATIN RECRUITMENT OF SHUGOSHIN PROTEIN SGO1P By Xiexiong Deng Faithful segregation of chromosomes to progeny cells is essential for species perpetuation. Eukaryotic cells use the spindle assembly checkpoint (SAC) to correct mistakes and prevent chromosome missegregation. SAC ensures that sister chromosomes are bioriented before segregation starts. Chromosome biorientation is manifested by tension between the two sister chromatids that are held together by the cohesin complex. The centromeric protein Shugoshin (Sgo1p in Saccharomyces cerevisiae) is an integral part of the tension sensing function of SAC. The underlying mechanism of how Shugoshin responds to tension, and relays this signal to the appropriate players in SAC remains largely unresolved. Here I use S. cerevisiae as a model to understand this tension-sensing mechanism by studying the recruitment of Sgo1p. Our lab has discovered that residues Lys42, Pro43, Gly44, and Thr45 of histone H3 form the core of a tension sensing motif, TSM, in S. cerevisiae. TSM functions by physically recruiting and retaining Sgo1p at pericentromere. I have identified a pair of antagonistic chromatin modifiers, Gcn5p and Rpd3p/Hos2p, that are critical for regulation of the H3-Sgo1p interaction in vivo. Deletion of GCN5 or overexpressing catalytically-dead mutants of Gcn5p suppresses the mitotic defects of tsm- mutants. In contrast, deleting RPD3 causes prominent chromosome instability phenotype in tsm- mutant G44S. In addition, both Gcn5p and Rpd3p are enriched at centromere and pericentromere. These results suggest the opposing enzymes Gcn5p and Rpd3p are parts of the mitotic checkpoint through regulating TSM/Sgo1p function. Genome-wide ChIP-seq assay reveals a unique TSM-dependent, tripartite domain of Sgo1p in each mitotic chromosome. This domain consists of one centromeric and two flanking peaks 3 – 4 kb away. Strikingly, this trident motif coincides with cohesin localization at centromeres and pericentromeres. The trident localization of Sgo1p requires both TSM and cohesin. Chromosome conformation capture assays reveal apparent looping at the centromeric and pericentric regions. The TSM-Sgo1p- cohesin triad is therefore at the center stage of higher-ordered chromatin architecture for error-free segregation. ACKNOWLEDGMENTS I’m deeply grateful to my advisor, Dr. Min-Hao Kuo, for his continuous support and guidance during my PhD training. He inspired me with his passion and rigorous attitude toward pursuing challenging scientific problems. He is also a role model of critical thinking for me and taught me how to do good science. After seven years of training, I’m confident to say I have improved tremendously as an independent scientist, thanks to Min-Hao’s mentoring. I really appreciate your help and the email of recruitment seven years ago, which I believe has completely changed my life. Additionally, I need to express my gratitude to my committee members, Dr. Zachary Burton, Dr. Susan Conrad, Dr. Monique Floer and Dr. Lee Kroos. It has been fortunate to have them on my committee. They have always provided me with professional advice and insightful suggestions for my projects. Moreover, I also need to thank my colleagues from the Kuo laboratory, Dr. Xiaobo Li, Dr. Witawas Handee, Dr. Christopher Buehl, Dexin Sui, Dr. Stacy Hovde and Mengyu Liu, and friends within BMB department, Dr. Rance Nault and Dr. Senem Aykul. They have been very supportive and provided with considerable help on my research. In the end, I must say thank you to my loving family. The love from my parents and parents-in-law is priceless and accompanies me wherever I go. Most importantly, I must thank my beloved wife, Dr. Xiaoting Wu, who has been incredible by giving me endless support in the last 7 years. I also need to thank my three lovely children, Ethan, Franklin and Grace, who have inspired me a lot in various aspects. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... vi LIST OF FIGURES......................................................................................................... vii CHAPTER I LITERATURE REVIEW ............................................................................... 1 Part I: Chromatin structure, function and dynamics .............................................. 2 Part II: Spindle Assembly Checkpoint (SAC), Shugoshin and tension sensing . 15 Part III: Research interests and significance....................................................... 24 REFERENCES ................................................................................................... 26 CHAPTER II IDENTIFICATION OF TENSION SENSING MOTIF OF HISTONE H3 IN SACCHAROMYCES CEREVISIAE AND ITS REGULATION BY HISTONE MODIFYING ENZYMES .......................................................................... 42 ABSTRACT ........................................................................................................ 44 INTRODUCTION ................................................................................................ 45 MATERIALS AND METHODS ............................................................................ 48 RESULTS ........................................................................................................... 57 DISCUSSION ..................................................................................................... 83 REFERENCES ................................................................................................... 87 CHAPTER III TRIPARTITE CHROMATIN LOCALIZATION OF BUDDING YEAST SHUGOSHIN INVOVLES HIGHER-ORDERED ARCHITECTURE OF MITOTIC CHROMOSOMES................................................................... 94 ABSTRACT ........................................................................................................ 96 INTRODUCTION ................................................................................................ 97 RESULTS ......................................................................................................... 100 DISCUSSION ................................................................................................... 123 MATERIALS AND METHODS .......................................................................... 129 ACKNOWLEGEMENTS ................................................................................... 138 REFERENCES ................................................................................................. 139 v LIST OF TABLES Table 2-1. Yeast strains used in this study ................................................................. 49 Table 2-2. Plasmid constructs used in this study ....................................................... 52 Table 2-3. Oligos used in this study ........................................................................... 53 Table 3-1. Yeast strains used in this study ............................................................... 131 Table 3-2. Plasmid constructs used in this study ..................................................... 133 Table 3-3. Oligos used in this study ......................................................................... 134 vi LIST OF FIGURES Figure 1-1. Centromere features in budding yeast and human cells ............................... 6 Figure 1-2. Composition of cohesin and condensin complexes .................................... 11 Figure 1-3. Spindle assembly checkpoint and shugoshin protein .................................. 16 Figure 1-4. Tension sensing motif (TSM) of histone H3 in nucleosome ........................ 23 Figure 2-1. Identification of the tension sensing motif of histone H3 ............................. 58 Figure 2-2. Mutations introduced to the tension sensing motif cause chromosome instability ...................................................................................................... 62 Figure 2-3. K42A and T45A mutations diminish the ability to activate spindle assembly checkpoint when tension is absent .............................................................. 66 Figure 2-4. Defective tension sensing motif fails to retain Sgo1p in pericentromeres ... 68 Figure 2-5. Gcn5p histone acetyltransferase is a negative regulator of the histone H3 tension sensing motif ................................................................................... 71 Figure 2-6. Gcn5p and Rpd3p affect chromosome stability via genetic interaction with the tension sensing motif ............................................................................. 73 Figure 2-7. Histone deacetylases Rpd3p and Hos2p functionally interact with the H3 tension sensing motif ................................................................................... 76 Figure 2-8. Both Gcn5p and Rpd3p are present in centromeres and pericentromeres . 78 Figure 2-9. Overexpression of HAT-null gcn5p-E173H doesn’t restore pericentric localization of Sgo1 in benomyl-arrested G44S mutant .............................. 82 Figure 3-1. Sgo1p is localized only to the centromeric area in each chromosome ...... 101 vii Figure 3-2. Sgo1p is recruited to centromeres and pericentromere to form a tripartite localization domain on each mitotic chromosome ...................................... 102 Figure 3-3. Sgo1p enrichment overlaps with cohesin domains at the centromeres and pericentromere ........................................................................................... 104 Figure 3-4. The histone H3 tension sensing motif is essential for pericentric Sgo1p localization but not Mcd1p ......................................................................... 107 Figure 3-5. ChIP-qPCR to verify the ChIP-Seq findings .............................................. 109 Figure 3-6. Gcn5p is enriched in centromeres but shows no overlap with cohesin elsewhere .................................................................................................. 111 Figure 3-7. Sgo1p recruitment requires cohesin ......................................................... 113 Figure 3-8. Pericentric localization of Sgo1p and Mcd1p is susceptible to ectopic transcription through the region ................................................................. 114 Figure 3-9. Dynamic recruitment of Sgo1p and cohesin at centromere and pericentromere through cell cycle .............................................................. 117 Figure 3-10. Dynamics of Sgo1p (A) and Mcd1p (B) localization at CEN1 region ....... 118 Figure 3-11. Sgo1p tripartite localization domain is associated with high-ordered chromatin architecture in mitosis .............................................................. 121 Figure 3-12. Model for the formation and dynamics of Sgo1p chromatin domain ....... 128 viii CHAPTER I LITERATURE REVIEW 1 Part I: Chromatin structure, function and dynamics Chromatin composition and structure DNA is the primary material passing genetic information through generations of most organisms. Unlike prokaryotes with less organized DNAs, eukaryotes incorporate various proteins with their DNAs to form chromatin and pack them into the nucleus. Chromatin is a highly-ordered nucleoprotein complex containing DNA, protein and RNA. The prominent proteins involved in this assembly are histones. Histones are highly conserved basic proteins in eukaryotes (1). Two molecules of each of the four core histones, H3, H4, H2A and H2B form an octamer that is wrapped by 147 base pair (bp) of DNA in 1.65 superhelical turns (2), forming a nucleosomal core particle that serves as the fundamental building block of chromatin. Neighboring nucleosomes are connected by 20 to 75 bp of linker DNA (3), resembling a structure of “beads on a string’. This 11 nm “beads-on-a-string” polymer of nucleosomes can be coiled and organized to a 30 nm fiber structure with the presence of histone H1 that binds to linker DNA. There are two different models of 30 nm fiber formation, solenoid and zigzag fiber models. The solenoid model suggests the linker DNA continues the curvature established in the nucleosome and nucleosomes coil to form the fiber with 33 nm diameter, positioning six nucleosomes along the fiber axis in every 11 nm (4). In contrast, the zigzag fiber model proposes the linker DNA remains straight to some extent and nucleosomes appear as a double row with the linkers crisscrossing between the rows. The zigzag fiber has a diameter between 27.2 to 29.9 nm, with five to six nucleosomes deposited in every 11 nm (5, 6). The 30 nm chromatin fiber is thought to form loops that are assembled into higher-order structures including 120-nm chromonema, 300- and 700-nm chromatids, 2 and metaphase chromosomes (7). In this way, DNA is compacted 10,000- to 20,000- fold and ready for passing on to daughter cells. Heterochromatin and euchromatin For most cells, the metaphase chromosome is the highest compressed state of the chromatin, only existing for a short period of time during segregation. During interphase, chromatins are organized in relatively loosened forms, but the levels of compaction vary across the genome (8). The Drosophila polytene chromosome is a great example of heterogeneous compaction of DNA. There are characteristic transverse bands of darker staining along the polytene chromosome, representing the tightly compacted regions called heterochromatin, and interbands of lighter staining, representing the loosely packed regions referred to as euchromatin (9, 10). Even chromosome puffs can be observed at those active transcription regions due to local chromatin expansion by transcriptional machinery accumulation. In mammalian cells, different degrees of compaction have been inferred from Fluorescence in situ Hybridization (FISH) experiments, where the signal from a gene-rich region occupies a larger area of the nucleus than does the signal from a gene-poor region of equivalent size (11). Direct in vivo evidence has also been acquired by advanced imaging technique, ChromEMT, for visualizing the three-dimentional (3D) chromatin structure and compaction (12). By combining electron microscopy tomography (EMT) with a labeling method (ChromEM), the authors show chromatin in human cells is packed together at different concentration densities in both mitotic chromosomes and interphase nuclei. 3 Heterochromatin is highly condensed, exhibits low-accessibility and is composed of rigidly spaced nucleosomal arrays, whereas euchromatin is less condensed, more accessible and less ordered in nucleosomal spacing. These structural distinctions cause functional differences of heterochromatin and euchromatin (13). While euchromatin supports efficient transcription, heterochromatin is generally transcriptionally repressed, or even silenced. In mammalian X-chromosome inactivation, heterochromatin is formed at the nucleation site and spreads out, leading to silencing of one entire X-chromosome in female animals to compensate for the extra gene dosage (14). Heterochromatin is thought to serve as genome defender as well, by preventing illegitimate recombination between repetitive DNA elements, such as centromeres, telomeres and ribosomal DNA loci (15, 16). The genome defense provided by heterochromatin also comes from silencing ‘parasitic’ transposable elements (15, 17). Besides protecting genome stability, heterochromatin has been shown to be involved in nuclear organization (18, 19) and in promoting long-range chromatin interactions for mating-type switching (20). Heterochromatins that remain condensed throughout the cell cycle and development are called constitutive heterochromatin, such as those at centromeres and telomeres. However, heterochromatins can also be found at loci of cell-type-specific genes. These heterochromatins can be unpacked in response to developmental signaling and are referred as to facultative heterochromatin (16). In fission yeast, Schizosaccharomyces pombe, and higher eukaryotes, heterochromatin is highly associated with DNA methylation and histone posttranslational modifications (PTMs) related to transcription silencing (21). The H3K9me/HP1 and Polycomb pathways are the two major pathways involved in 4 heterochromatin assembly (22). H3K9me is the methylated lysine 9 of histone H3, which can serve as a molecular anchor to attract heterochromatin protein 1 (HP1) binding through its amino-terminal chromodomain (23). The H3K9me bound HP1 can then recruit multiple downstream effectors via its carboxy-terminal chromoshadow domain. These effectors include histone methyltransferase and histone deacetylase (HDAC) that help maintain and propagate heterochromatin formation (24-26). The Polycomb repressive pathway includes two complexes, Polycomb repressive complex 1 and 2 (PRC1 and PRC2), catalyzing two distinct histone PTMs, H2AK119ub1 and H3K27me3, respectively (27-29). PRC1 and PRC2 work together on modifying the nucleosomes and ultimately lead to chromatin compaction and heterochromatin formation (30). The budding yeast, Saccharomyces cerevisiae does not possess a HP1 homolog, which is essential for heterochromatin assembly in metazoans. Instead, the silencing and heterochromatin formation at its telomeres, mating-type loci and rDNA loci depend on the Silent Information Regulator (SIR) complex (31). The SIR complex has three subunits, Sir2p, Sir3p and Sir4p. The Sir2p is a nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that deacetylates lysines in histone tails (32, 33). It is proposed that the recruitment of SIR complex at silencer triggers a sequential deacetylation and SIR complex recruitment on the neighboring nucleosomes (34). The resultant hypoacetylated chromatin and SIR complex are compacted together, forming the heterochromatin. 5 Figure 1-1. Centromere features in budding yeast and human cells. The point centromere of budding yeast is around 120 bp in length and has only one centromeric nucleosome. In contrast, the regional centromere of human cells is up to 5 Mb long and has both canonical and centromeric nucleosomes. 6 Centromere The centromere is a specialized chromatin domain that provides the foundation for kinetochore assembly (35). The size of centromeres varies from 125 bp in S. cerevisiae, to 40-100kb in S. pombe to several megabases in human cells [(36) and Figure 1-1]. Unlike most eukaryotic centromeres which are maintained epigenetically (37, 38), the centromere of S. cerevisiae is genetically specified by DNA sequence (39). The 125 bp ‘point’ centromere of S. cerevisiae is composed of three conserved centromere-determining elements (CDE), CDEI, CDEII and CDEIII. CDEI is a palindrome sequence of 8 bp that binds to Cbf1p, whereas CDEII is a stretch of AT-rich sequence, 78 to 86 bp long, bound by Cbf2p. CDEIII is a conserved non-perfect palindromic sequence of 26 bp and binds to the Centromere Binding Factor 3 (CBF3) complex (40). The CBF3 complex includes Ndc10p, Cep3p, Ctf13p and Skp1p, and coordinates the deposition of a centromeric nucleosome (41). CDEII and CDEIII are essential for centromere function (42, 43). Although its full sequence remains to be fully delineated, the human centromere is composed of repetitive alpha satellite DNA arrays, which occupy approximately 5% of the genome (44). The basic alpha satellite is a 171 bp element that shares only 50%-70% identity (45). They are arranged in head-to-tail fashion to form long tandem, called ‘regional centromere’, spanning from 250 to 5,000 kb. Another conserved element can be found in human centromeres is the B-box, a 17 bp motif for centromere associated protein B (CENP-B) binding (46). It is sporadically distributed in the alpha satellite array and thought to be important for nucleosome assembly at centromere and centromere function (47). 7 Besides sequences characteristic of centromeres, the nucleosomes bound to centromeres are distinct from the canonical nucleosome (48). Although the exact composition and structure of centromeric nucleosomes is a subject of debate (49-54), it is well-accepted that the canonical histone H3 is replaced by a centromere-specific variant, centromere-associated protein A (CENP-A, Cse4p in S. cerevisiae) (55). The deposition of CENP-A containing nucleosome at centromeres is regarded as the nucleating event leading to the assembly of kinetochore, a highly conserved protein- DNA complex at centromeres (48). Like other histones, CENP-A requires a chaperone protein for proper chromatin loading. The CENP-A-specific chaperone is Scm3p in yeast (56), and HJURP in humans (49), which recognizes the centromere-targeting domain (CATD) of CENP-A and mediates its incorporation into centromere. In S. cerevisiae, Scm3p also interacts with the Ndc10p subunit of CDEIII-bound CBF3 complex, rendering the centromeric specificity of Cse4p (56). By using various techniques, including chromatin immunoprecipitation (ChIP), it was shown that a single Cse4p nucleosome was well positioned at the CDEII element of budding yeast centromere (57- 60). The chromatin remodeling factor SWI/SNF complex was also shown to be capable of constraining the distribution of Cse4p to the centromere (61). In higher eukaryotes with regional centromeres, like humans, it’s more complicated and largely remains unclear how the deposition of centromeric nucleosome forms. The CENP-A containing nucleosome is only incorporated into a portion of the centromeric chromatin, and the rest is deposited with canonical H3 containing nucleosomes (62, 63). It’s still a conundrum, mainly due to missing a contiguous full sequence of a centromere, as to 8 how the higher eukaryotic cells distinguish the highly repetitive alpha satellite sequences and incorporate CENP-A nucleosomes specifically at certain locations (44). Structure Maintenance Chromosome (SMC) complex, cohesin and condensin To assemble the higher-ordered chromatin structure, eukaryotic cells share a group of chromatin binding proteins, called Structure Maintainence Chromosome (SMC) (64). There are two major SMC complexes, the cohesin complex and the condensin complex (65, 66). The core cohesin complex contains two SMC proteins, Smc1 and Smc3, and two non-SMC proteins, Scc1 (also known as Mcd1 or Rad21) and Scc3 (called SA in mammalian cells) [(65) and Figure 1-2]. Smc1 and Smc3 are rod-shaped proteins with a globular hinge domain at one end and a nucleotide-binding domain (NBD) at the other end. Smc1 and Smc3 form a V/U-shaped heterodimer through interaction of their hinge domains, functioning as the backbone of the cohesin ring structure (67, 68). The evolutionarily-conserved lysine residues 112 and 113 within the NBD of Smc3 can be acetylated by Eco1, an acetyltransferase that critically promotes the loading of cohesin on chromatin duing S phase (69). A histone deacetylase Hos1 (HDAC8 in human) has been shown to antagonize this loading effect of Eco1 by deacetylating the same pair of lysines of Smc3 (70-72). Scc1/Mcd1 is an alpha-kleisin protein, which has an amino- terminal domain and a carboxyl-terminal domain that bind tightly to the NBD domains of Smc3 and Smc1 respectively (67, 73). The binding of Scc1/Mcd1 to the Smc1/Smc3 heterodimer creates a large tripartite ring structure that encircles the sister chromatids and generates cohesion. The Scc1/Mcd1 also recruits the regulatory subunit Scc3, a hook-shaped protein with a tandem of alpha helices (HEAT repeats), which maintains 9 the association of cohesin with chromatin during G2/M phase and promotes cohesin’s dissociation upon its cleavage (74). In addition to establishing sister chromatid cohesion, cohesin complexes also play important roles in DNA double-strand break repair (75, 76), DNA replication (77) and transcription control (78-81). Most of these functions are based on cohesin-mediated chromatin looping to generate long-range DNA interaction (82). Condensin complexes share many similarities with cohesin complexes in subunit organization (83). The core condensin complex also contains two SMC subunits and one kleisin subunit, but has two HEAT subunits (Figure 1-2). Most eukaryotes possess two different condensin complexes, condensin I and condensin II, whereas fungi (both S. cerevisiae and S. pombe) only have condensin I (66).The two SMC subunits, Smc2 and Smc4, also form a V-shaped heterodimer that is bound by the alpha-kleisin subunit, CAP-H in condensin I and CAP-H2 in condensin II. Subsequently the kleisin subunit recruits two HEAT proteins, CAP-D2 and CAP-G in condensin I and CAP-D3 and CAP- G2 in condensin II. However, the hinge structure of the condensin’s SMC heterodimer is much narrower than that of conhesin, leading to a rod-shaped complex (84). This structural difference is believed to cause a different mode of DNA interaction for condensin (83). The primary function of the codensin complex is in chromatin condensation and organization, which can be important for chromosome segregation (85) and gene expression (86, 87). 10 Figure 1-2. Composition of cohesin and condensin complexes. Generally, cohesin and condensin complexes are composed of two SMC proteins, one kleisin protein and one or two HEAT protein(s). 11 Chromatin modifiers Eukaryotic chromatin is highly dynamic to achieve adaptive regulation of gene expression in response to cell signaling and the changing environment. The fundamental contribution to this dynamic is from chromatin modifiers that covalently modify DNA or histones, facilitating or restricting access of specific genes. One of the best-studied DNA modifications is 5-methylcytosine (5mC), catalyzed by DNA methyltransferases (DNMT). DNMTs can both introduce methylation on cytosine (called de novo methylation) and maintain the methylation pattern after semiconservative DNA replication (called maintenance methylation) (88). The DNMT- mediated 5mC is wildly spread in mammalian genomes and enriched in the CpG dinucleotide context (89). 5mC can promote the compaction of chromatin and lead to gene silencing, a mechanism associated with genomic imprinting, X-chromosome inactivation and suppression of transposons (90). However, DNA methylation is rarely detected in Drosophila, S. cerevisiae and S. pombe (91), though DNMT-like genes are found in their genomes. The enzymes responsible for 5mC DNA demethylation were found to be the ten-eleven-translocation (TET) protein family (92, 93) and thymine DNA glycosylase (TDG) (94, 95). The demethylation of 5mC is a TET-mediated oxidation process followed by TDG-mediated base excision repair, which ultimately regenerates an unmodified cytosine (88). These enzymes have been shown to be important for embryonic stem cell development (96), reprogramming (97) and tumorigenesis (98, 99). Histones are also ideal substrates for the chromatin modifying enzymes. Histones possess a large number and various types of residues that can be modified posttranslationally, particularly in their accessible and unstructured N-terminal tails 12 (100). There are at least eight different types of histone modifications identified with significant biological functions, including acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, deamination and proline isomerization (100). These modifications involve histone acetyltransferases (HATs), deacetylases (HDACs), methyltransferases (HMTs), demethylases (HDMs), kinases, ubiquitylases and proline isomerases (100). Most of these modifications happen on charged residues such as lysine, or on polar residues such as serine and threonine, thereby causing a change in the net charge that results in conformational change of the nucleosome particle. The charge change by acetylation of histone H4 lysine 16 (H4K16ac), for example, has been shown to affect inter-nucleosomal interactions and lead to decompaction of chromatin (101, 102). Moreover, the modified residues can also influence chromatin packaging via recruitment of other chromatin binding proteins, like HP1 binding to H3K9me and PRC2 binding to H3K27me in heterochromatin formation (24) (30). Gcn5p and Rpd3p Gcn5p and Rpd3p (called HDAC1 in mammals) in S. cerevisiae are the very first HAT and HDAC, respectively, to be identified that function in chromatin-mediated gene regulation (103, 104). Gcn5p preferentially targets lysine 14 of histone H3 in free histones in vitro (105), but is unable to modify nucleosomes by itself (106). To target H3 in a nucleosome particle, Gcn5p needs to incorporate with other proteins to form multisubunit HAT complexes, such as the ADA complex and Spt-Ada-Gcn5- Acetyltransferase (SAGA) complex (107). The Gcn5p-containing complexes are associated with transcription activation (108). In contrast, the HDAC Rpd3p seems to 13 lack strict substrate specificity on histones in vivo. The deletion of RPD3 causes an increase of acetylation on most sites of histones, except lysine 16 of histone H4 (109). Rpd3p can be found in two multisubunit complexes, Rpd3L and Rpd3S, both sharing a heterotrimer Rpd3p-Sin3p-Ume1p (110). Rpd3L is believed to target histones at promoter regions for gene repression (111), while Rpd3S deacetylates transcribed regions to suppress spurious intragenic transcription (112). Beyond transcription, Gcn5p was also found to be involved in chromatin structure assembly in centromeric regions, which is important for timely mitotic progression (113). At the same time, Rpd3p can antagonize Sir2p-dependent silencing at telomeres (114), as well as regulating replication initiation timing or efficiency (115). 14 Part II: Spindle Assembly Checkpoint (SAC), Shugoshin and tension sensing Spindle Assembly Checkpoint (SAC) In mitosis, the sister chromatids synthesized during S phase must be faithfully partitioned into the daughter cells to preserve genome stability. Mistakes in this process can lead to uneven numbers of chromosomes in the offspring, referred to as aneuploidy. Aneuploidy is causally linked to developmental diseases, like Down’s syndrome, and tumorigenesis (116). To avoid aneuploidy, eukaryotic cells utilize a highly accurate and well-conserved mechanism called the spindle assembly checkpoint (SAC) (117). The SAC primarily monitors kinetochore-microtubule attachment and the tension between the sister kinetochores, which is generated by bipolar pulling of spindles on cohesin-linked sister chromatids (Figure 1-3A). Defects in either attachment or tension activates the SAC that halts cell cycle progression and keeps the cell in metaphase. SAC does so by forming a mitotic checkpoint complex (MCC), which traps and inhibits Cdc20p, the E3 ligase subunit of the anaphase promoting complex (APC) (118). The erroneous attachment in the halted cell is then subjected to a ‘detach and re- attach’ cycle, a process known as ‘error and correction’ mechanism (119), which involves a key kinase activity of Aurora B (Ipl1p in S. cerevisiae) (120) and centromeric adaptor protein Shugoshin (Sgo1p in S. cerevisiae) (121). Only until all sister chromatids are under tension and properly aligned, so-called bioriented, is the SAC silenced, which gives the cell permission to undergo the metaphase-anaphase transition. Detailed mechanisms of attachment, checkpoint activation and silencing will be discussed in the following sections based on the current understanding. 15 Figure 1-3. Spindle assembly checkpoint and shugoshin protein. (A) Monotelic, syntelic and amphitelic attachment are shown in metaphase. (B) Features of Shugoshin protein and domain interaction. Shugoshin protein has a conserved N-terminal coiled coil domain and a C-terminal basic domain. Protein phosphatase 2A (PP2A) and the chromosome passenger complex (CPC) can be recruited by the coiled coil domain of shugoshin. 16 Kinetochore-microtubule attachment and SAC activation The kinetochore is a hierarchical protein structure of around 100 subunits built on the centromere, mediating the attachment of spindle microtubule to chromosome (122). It couples microtubule dynamics to chromosome movement during cell division. The core complex of the kinetochore is evolutionally conserved across eukaryotes and is conventionally divided into two layers, inner kinetochore and outer kinetochore (118). The inner kinetochore is composed of proteins that closely associate with the centromeric chromatin, also known as the constitutive centromere associated network (CCAN). The outer kinetochore possesses the microtubule binding activity and serves as a platform for SAC regulation. The essential components of outer kinetochore are Knl1 (Spc105 in S. cerevisiae), the Mis12 complex and the Ndc80 complex, collectively termed as KMN network (123). Knl1/Spc105 and the Ndc80 complex directly bind to microtubule through electrostatic interactions (124, 125), while the Mis12 complex bridges the inner and outer kinetochore (126). Furthermore, the Knl1/Spc105 complex can function as a scaffold to recruit the SAC checkpoint proteins, Bub1p and Bub3p (127, 128). In budding yeast, there is an additional complex called Dam1, which can strengthen the attachment by forming a ring structure that caps at the joint area of the kinetochore and microtubule (129, 130). A microtubule is a dynamic polymer that can grow or shrink by adding or removing the a tubulin dimer at its tip, known as plus end. Its minus end is imbedded in the microtubule-organizing center (MTOC), also known as centrosomes in mammalian and spindle pole bodies (SPB) in S. cerevisiae (131). When the plus end of microtubule reaches the outer kinetochore, the acidic E-hook domain (negative charge) 17 b of tubulin can provide electrostatic affinity for binding to the basic N-terminus (positive charge) of the Ndc80 complex (132, 133). However, this attachment of microtubule to the kinetochore is not always stable and subjected to selective regulation in a tension- dependent manner. It has been shown that attachments lacking tension are highly unstable and eliminated in vivo, whereas attachments under tension are stably maintained (134). The kinetochore-microtubule attachment for biorientation is prone to errors. In budding yeast, one microtubule spindle fiber attaches to each kinetochore (135), whereas 20-25 fibers attach to each kinetochore in humans (136). Due to the highly simplified microtubule-kinetochore stoichiometry in yeast, three types of attachment can occur in budding yeast (Figure 3A), monotelic, syntelic and amphitelic (bipolar). In monotelic attachment, only one of the two sister kinetochores is attached to the microtubule. The other unoccupied kinetochore can recruit Mps1p kinase that phosphorylates Knl-1/Spc105 (137, 138). Phosphorylated Knl-1/Spc105 consequently recruits SAC checkpoint proteins Bub1p kinase, Bub3p, BubR1/Mad3p, Mad1p and Mad2p (128, 139). The docking of Mad1p and Mad2p on the kinetochore triggers the conformational change in Mad2p from an ‘open’ to a ‘closed’ state (140). The ‘closed’ Mad2p then binds to Cdc20p of the APC complex, together with Bub3p and BubR1/Mad3p, forming the inhibitory complex MCC (141). Without an active Cdc20p, the APC complex is unable to destroy securin (Pds1p in S. cerevisiae), which prevents its binding partner separase from cutting on Mcd1p of the cohesin complex (142). The blocking of APC activity by MCC ultimately arrests the cell in metaphase until the attachment defect is resolved. 18 In the syntelic condition, both sisters are occupied by microtubules originated from the same spindle pole body. Even though the occupation requirement is met, there is no tension generated between the sister chromatids as they are pulled in the same direction. Cells need Shugoshin 1 (Sgo1p) to respond to this erroneous attachment (121). It is thought that Sgo1 localizes at these tensionless chromatids, serving as a versatile adaptor [(143) and Figure 1-3B]. It can recruit Aurora B/Ipl1p kinase to the centromeres to destabilize the erroneous attachment by phosphorylating Ndc80p and Dam1p (144, 145). The destabilized and detached kinetochore can further activate SAC by forming MCC. Meanwhile, Sgo1p can also recruit protein phosphatase 2A (PP2A) to protect cohesin from being cleaved, by keeping Mcd1p unphosphorylated (146, 147). The protected cohesin holds sister chromatids together and avoids premature segregation. Only when amphitelic attachment is established, then is tension generated between sister chromatids. This tension, via a yet-to-be-identified mechanism or molecule, silences the SAC. Shugoshin and tension sensing Shugoshin proteins, meaning “spirit of guardian” in Japanese, were first identified as a protector of chromosome cohesion in yeast meiosis (143). More shugoshin family proteins have been discovered in various eukaryotes with relatively low sequence identities (148). There are different numbers of shugoshin proteins in different species. S. cerevisiae and Drosophila have only one shugoshin, Sgo1p and Mei-S322 respectively. S. pombe and mammals possess two, Sgo1/Sgo1L and Sgo2/Sgo2L (148). Shugoshin proteins share two short, common domains (Figure 3B), a N-terminal coiled-coil domain and C-terminal basic domain (also known as the SGO domain) (143). 19 The coiled-coil domain is responsible for interaction with downstream effectors, including PP2A (149) and Chromosome Passenger Complex (CPC) (150). Subsequent studies revealed that it protects the cohesin complex from separase-mediated degradation via recruiting PP2A. The interaction with Aurora B-containing CPC complex links shugoshin to chromosome biorientation and SAC signaling in mitosis (151). Moreover, Sgo1p in S. cerevisiae was suggested to bias chromosome biorientation by recruiting condensin to shape the back-to-back geometry at the centromeres and pericentromeres (152). On the other end, the basic/SGO domain is required for centromeric targeting of shugoshin. It was shown the basic/SGO domain interacts with the Bub1p-phosphorylated histone H2A (Ser121 in yeast, Thr120 in human) in vitro and in vivo (153). Mutations of those conserved residues within the basic/SGO domain can abolish the centromeric localization of shugoshin. Besides being protected by shugoshin, the cohesin complex is a crucial recruitment factor for shugoshin as well (143). In budding yeast meiosis, Sgo1p colocalizes with the cohesin complex at a 50-kb domain flanking centromeres (154). Genetic disruption of pericentric cohesin significantly decreases the Sgo1p localization in that region. In human cells, Sgo1 is phosphorylated on Thr346 by Cdk1, a modification required for cohesin binding. The T346A mutant of Sgo1 loses the ability to bind to pericentromeres through coehsin (155). However, this residue is not conserved in yeast, and whether there is a similar regulatory mechanism for the yeast Sgo1p is unclear. To position shugoshin at the appropriate chromosomal domains, certain features of nucleosomes are required (156). The first critical nucleosomal recruiter for Sgo1 is phosphorylated S121 of H2A by Bub1p kinase at centromeres. The Bub1p 20 kinase is recruited by the kinetochore Knl1/Spc105 complex and phosphorylates the centromeric H2A (127). The phospho-H2A is thought to nucleate shugoshin localization at centromere (153). This idea is supported by the observation that S121A mutation of H2A totally diminishes the chromosome localization of shugoshin. In addition, our lab has previously identified a tension sensing motif (TSM, 42Lys-Pro-Gly-Thr) of histone H3 that is pivotal for Sgo1p localization to the pericentromeres in S. cerevisiae (157, 158). The TSM resides at the location that bridges the N-terminal tail and the a N helix of histone H3 (Figure 1-4). Mutations K42A, G44A and T45A of H3 display hypersensitivity to benomyl, a spindle poison that inhibits microtubule polymerization. Subsequent experiments revealed that the mitotic defect observed in the presence of benomyl is due to attenuated H3-Sgo1p interaction and the loss of pericentric localization of Sgo1p (157, 158). Later, two comprehensive screening studies of histones identified a number of residues on histone H3, H4, H2A and H2B, overlapping with our TSM, important for shugoshin related mitotic function (156, 159). It was proposed that these residues comprise a binding path for shugoshin association with chromatin (156). Increasing evidence suggests that shugoshin undergoes tension-dependent relocalization on chromosomes upon biorientation in budding yeast, mouse and human cells (155, 160, 161). Sgo1p is evicted from chromatin in a tension-dependent manner in budding yeast (160). Given the fact that Sgo1p physically interacts with the nucleosome (153, 157), Sgo1p association with the chromatin can potentially serve as a tension sensor. Two lines of evidence support this hypothesis. First, histone tails of neighboring nucleosomes can make physical contact in chromatin (2, 6). When tension is built up, the stretching of chromatin may cause conformational change of nucleosome 21 that disrupts Sgo1p-nuclesome interaction. Second, core histones at pericentromeres, where spindle exerts physical force on (162), have a higher turnover rate than histones in chromosome arm regions (163). This tension-dependent remodeling of histones at pericentromeric regions can lead to the eviction of Sgo1p that binds to the ‘old’ histones. This may ultimately result in SAC silencing by loss of effector activities of Sgo1p. The so-called “tension response” hypothesis of Sgo1p function will require future studies to demonstrate and clarify the underlying mechanism. 22 Figure 1-4. Tension sensing motif (TSM) of histone H3 in nucleosome. The budding yeast nucleosomal structure is shown (PDB 1ID3 (2)). TSM is highlighted in red. 23 Part III: Research interests and significance The tension response hypothesis of Sgo1p function motivates us to study its recruitment and retention. Sgo1p is thought to coat the 50 kb regions of centromeres and pericentromeres in budding yeast during G2/M phase (154). This recruitment requires phosphorylated Serine121 of H2A by Bub1 kinase in centromeres (153). Cohesin and the TSM of histone H3 are two other factors involved in pericentromeric recruitment of Sgo1p (155, 157). While it is highly likely that Sgo1p is first recruited to the centromeres and then spreads to pericentromeres (157, 164, 165), it is enigmatic as to how Sgo1p is confined at such loci when H3 is nearly ubiquitous in chromosomes. One possibility is that the H3-Sgo1p interaction is subject to direct or indirect regulation at or surrounding centromeres such that only the pericentric H3 is amenable to Sgo1p association. Because bacterially expressed Sgo1p and H3 interact well (157), it is possible that the H3-Sgo1p interaction might be negatively regulated in vivo by a reversible modification. Indeed, Gcn5p, a prototypical histone acetyltransferase well- known for its role in transcriptional regulation, is found as a negative regulator of H3- Sgo1p interaction (158). To further support this hypothesis, we predicted the opposing enzymes, histone deacetylases (HDACs), may be involved and identified Rpd3p and Hos2p. Our genome-wide mapping of Sgo1p localization shows that there is a trident domain of Sgo1p with discrete peaks flanking the centromeric regions. This trident domain of Sgo1p prompts us to investigate the underlying mechanism of recruitment and retention. 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PLoS Genet 3:e213. 41 CHAPTER II IDENTIFICATION OF TENSION SENSING MOTIF OF HISTONE H3 IN SACCHAROMYCES CEREVISIAE AND ITS REGULATION BY HISTONE MODIFYING ENZYMES 42 Jianjun Luo1, Xiexiong Deng1, Christopher Buehl2, Xinjing Xu1, and Min-Hao Kuo1* 1Department of Biochemistry and Molecular Biology, and 2Program in Cell and Molecular Biology, Michigan State University, East Lansing, MI 48824 Running title: chromatin role in mitosis Key words: histone H3, chromatin, mitosis, Shugoshin, Saccharomyces cerevisiae Figure 2-1 to 2-5, 2-6A and 2-6B are done by Dr. Jianjun Luo. *Corresponding author. 603 Wilson Road, 401 BCH Building, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824. E-mail: kuom@msu.edu; Phone: 517-355-0163; FAX: 517-353-9334 43 ABSTRACT To ensure genome stability during cell division, all chromosomes must attach to spindles emanating from the opposite spindle pole bodies before segregation. The tension between sister chromatids generated by the poleward pulling force is an integral part of chromosome bi-orientation. In budding yeast, the residue Gly44 of histone H3 is critical for retaining the conserved Shugoshin protein Sgo1p at the pericentromeres for monitoring the tension status during mitosis. Studies carried out in this work showed that Lys42, Gly44, and Thr45 of H3 form the core of a tension sensing motif, TSM. Similar to the previously reported G44S mutant, K42A, G44A, and T45A alleles all rendered cells unable to respond to erroneous spindle attachment, a phenotype suppressed by Sgo1p overexpression. TSM functions by physically recruiting or retaining Sgo1p at pericentromeres as evidenced by chromatin immunoprecipitation and by in vitro pulldown experiments. Intriguingly, the function of TSM is likely regulated by multiple histone modifying enzymes, including the histone acetyltransferase Gcn5p, and deacetylases Rpd3p and Hos2p. Defects caused by TSM mutations can be suppressed by the expression of a catalytically inactive mutant of Gcn5p. Conversely, G44S mutant cells exhibit prominent chromatin instability phenotype in the absence of RPD3. These results demonstrate that the tension sensing motif of histone H3 is a key component of a mechanism that ensures faithful segregation, and that interaction with chromatin modifying enzymes maybe an important part of the mitotic quality control process. 44 INTRODUCTION Faithful partitioning of the genome duplicates in mitosis requires that the sister chromatids be engaged in bipolar attachment to mitotic spindle. Once all chromosomes are appropriately captured by the spindles, anaphase starts with the action of separase that cleaves the cohesin complex, thus separating the two sister chromosomes (1). Premature anaphase onset causes aneuploidy, a common trait associated with spontaneous abortion, birth defects, and cancer. Chromosome biorientation is a result of sister chromatid cohesion by the cohesin complex, stable attachment of spindles to kinetochores, and that the two sister kinetochores each attach to spindles emanating from different spindle pole bodies (2). Erroneous attachment of spindles to kinetochore activates the spindle assembly checkpoint (SAC), which prevents metaphase-to- anaphase transition so that errors can be corrected (3). The two essential elements of biorientation are the spindle-kinetochore interaction and the tension between sister chromatids (4-6). The latter results from the physical cohesion of the sister chromatids that resists the poleward pulling force from opposing mitotic spindles, a scenario also called amphitelic attachment. If one of the two sister kinetochores is not attached (monotelic), or if both kinetochores are attached to spindles from the same spindle pole body (syntelic), tension will not be produced, and both copies of sisters will co- segregate, leading to aneuploidy (6). The physical form of tension detectable by cells remains a subject of investigation (7). Tension-dependent conformational changes of chromatin and cohesin near kinetochores are likely candidates (8-10). Besides the biorientation-induced separation of sister kinetochores within the confinement of cohesion (11), intrachromosomal extension of the distance between adjacent 45 nucleosomes in the pericentric regions has also been suggested to be an outcome of bipolar attachment (12). On the other hand, how cells interpret such structural changes induced by tension is unclear. One key player in tension sensing is the Shugoshin protein (13-16). Homologues of Shugoshin are found in eukaryotes ranging from yeast to humans and are important for both meiotic and mitotic chromosome segregation (17, 18). Deleting SGO1, which encodes the sole copy of Shugoshin in the budding yeast, renders cells unable to detect or to respond to tensionless crises (13, 19). During mitosis, Shugoshin is enriched at the centromeres and pericentromeres (20-22), from which tension originates (23). Centromeric recruitment of Shugoshin depends critically on the phosphorylation of Ser121 of H2A by the Bub1p kinase, as well as several heterochromatic marks at the pericentromeres (14, 19, 20, 24, 25). Genetic and biochemical experiments revealed that Shugoshin interacts with Ipl1p, the kinase subunit of the chromosomal passenger complex (26, 27), protein phosphatase 2A (PP2A) (28-33), and cohesin (30). It is possible that Shugoshin proteins participate in the detection and/or correction of attachment error. Consistently, evidence has been presented for the biorientation-dependent removal of Shugoshin from pericentromeres (28, 34), suggesting that retaining this protein at centromeres and pericentromeres may be a crucial element that keeps the spindle assembly checkpoint at an “on” state before the establishment of biorientation. However, how Shugoshin interacts with SAC remains an open question. Previously we reported that histone H3 plays a critical role in mitotic tension surveillance in budding yeast (35). Yeast cells harboring the Gly44-to-Ser (G44S) mutant allele of H3 exhibit phenotypes typical of those resulting from tension sensing 46 defects, including chromosome instability, missegregation, and inability to activate the SAC when tension buildup is perturbed (13). This mutation apparently impairs the recruitment and retention of Sgo1p at pericentromeres, whereas the centromeric Sgo1p localization remains in large normal (35). Moreover, scanning mutagenesis of H3 helped uncover multiple residues, including Gly44, required for faithful segregation of chromosomes (26, 36). Together, these reports attest to the indispensable, yet frequently overlooked function of nucleosomes in the regulation of mitosis. Nucleosome are the basal components specifying both the structures and functions of chromatin. Dynamic changes in nucleosomes, including their posttranslational modifications, critically affect nuclear activities including transcription, replication, and recombination. Comparatively, how mitosis might be regulated by chromatin is only beginning to be understood. Here we present evidence that Gly44 of histone H3 is part of the tension sensing motif 42KPGT that bridges the N’ tail domain and the central histone-fold domain of H3. Genetic assays also revealed that the function of H3 tension sensing motif is regulated by two opposing chromatin modifying activities, the histone acetyltransferase Gcn5p and deacetylases Rpd3p and Hos2p. Together, these results further demonstrate that chromatin, besides itself being the cargo of genome partitioning, plays an active role in ensuring faithful segregation. 47 MATERIALS AND METHODS Yeast strains and plasmid constructs. The yeast strains, plasmids, and primers used in this work are listed in Tables 2-1 to 2-3. All materials are available upon request. To assess the effects of histone H3 mutations from lysine 36 to lysine 56 on the benomyl sensitivity, pJL74 (a LEU2 plasmid bearing histones H2A and H2B) was co- transformed with H3 mutant collection from the Boeke group (TRP1 plasmids harboring H3 and H4 genes) that contains specific H3 mutations (37). 5-FOA selection was conducted to select for yeast cells that had lost the plasmid pMK440 (a URA3 plasmid bearing all four core histone genes). All other studies were carried out with histone mutations generated in pMK439 by two-step PCR site-directed mutagenesis (35). To study the functions of GCN5 on the H3-Sgo1p tension sensing mechanism, H3 mutations (on pMK439 LEU2+) were introduced into yJL486 (gcn5∆, pMK440 URA3+) via plasmid shuffling and 5-FOA selection, resulting in strains yJL506 to yJL510 (see Table 2-1). yJL486 was constructed by transforming a 4.6kb gcn5::URA3 fragment, which is released from pMK147 by Xho I and Xba I, into yMK1141 (pMK440 URA3+). Dominant negative HAT inactive mutants of gcn5, gcn5E173H, or gcn5F221A were carried on pMK144 and were directly transformed into cells bearing H3 mutations. To delete RPD3, primers oXD37 and oXD38 were used to amplify the Kluyveromyces lactis TRP1 selective marker from plasmid pBS1479. The PCR product was transformed into yMK1141 for tryptophan prototroph selection. The same strategy was used for knockout of SIN3 (oXD33 and oXD34), HDA1 (oXD25 and oXD26), HOS1 (oXD79 and oXD80) and HOS2 (oXD83 and oXD84). The single knockout strains were 48 Table 2-1. Yeast strains used in this study Strain Relevant genotype MATa ade2-1 bar1∆ can1-100 his3-11, 15::pGAL- MCD1::HIS3 leu2-3, 112 trp1-1::PDS1-Myc13::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2- HHF2] MATa/α his3∆1 leu2∆0 met15∆0 ura3∆0 hht1-hhf1::KAN hht2-hhf2::Nat hta1-htb1::HPH hta2-htb2::Nat pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S-HHF2] Source or reference 27 This study yJL171 yJL340 yJL343 yJL475 yJL479 yJL486 yJL487 yJL492 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] 27 MATa/α his3∆1 leu2∆0 met15∆0 ura3∆0 hht1-hhf1::KAN hht2-hhf2::Nat hta1-htb1::HPH hta2-htb2::Nat pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A-HHF2] This study MATa/α his3∆1 leu2∆0 met15∆0 ura3∆0 hht1-hhf1::KAN hht2-hhf2::Nat hta1-htb1::HPH hta2-htb2::Nat pMK439T45A [ARS CEN LEU2 HTA1-HTB1 hht2-T45A-HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3- 1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pJH33 [ARS CEN URA3 HTA1-HTB1 HHT2- HHF2] This study MATa ade2-1 bar1∆ can1-100 his3-11, 15::pGAL- MCD1::HIS3 leu2-3, 112 trp1-1::PDS1-Myc13::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A-HHF2] MATa ade2-1 bar1∆ can1-100 his3-11, 15::pGAL- MCD1::HIS3 leu2-3, 112 trp1-1::PDS1-Myc13::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439T45A [ARS CEN LEU2 HTA1-HTB1 hht2- T45A-HHF2] This study This study 49 Table 2-1 (cont’d) yJL506 yJL507 yJL508 yJL509 yJL510 yJL540 yJL543 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3- 1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2- HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3- 1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S-HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3- 1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439G44A [ARS CEN LEU2 HTA1-HTB1 hht2-G44A-HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3- 1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A-HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3- 1::gcn5 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439T45A [ARS CEN LEU2 HTA1-HTB1 hht2- T45A-HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439K42A [ARS CEN LEU2 HTA1-HTB1 hht2-K42A-HHF2] This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439T45A [ARS CEN LEU2 HTA1-HTB1 hht2-T45A-HHF2] This study yMK1141 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 27 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK440 [ARS CEN URA3 HTA1-HTB1 HHT2- HHF2] yMK1174 MATa/α his3∆1 leu2∆0 met15∆0 ura3∆0 hht1-hhf1::KAN This study hht2-hhf2::Nat hta1-htb1::HPH hta2-htb2::Nat pJH33 [ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] 50 Table 2-1 (cont’d) yMK1243 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 27 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2- HHF2] yXD24 yXD26 yXD27 yXD29 yXD87 yXD88 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH hda1Δ::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH sin3Δ::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH rpd3Δ::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH RPD3-3HA::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH hos1Δ::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH hos2Δ::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study yXD100 MATa ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH GCN5-13MYC::TRP1 /pMK440[ARS CEN URA3 HTA1-HTB1 HHT2-HHF2] This study 51 Table 2-2. Plasmid constructs used in this study Plasmid Main features Source or reference pJH33/pMK440 pRS316-HTA1-HTB1 HHT2-HHF2 27 pJL51 pJL52 pJL53 pJL55 pJL74 2μm URA3 pADH1-3xHA-SGO1-tADH1 This study ARS1 CEN4 URA3 pADH1-3xHA-tADH1 This study ARS1 CEN4 URA3 pADH1-3xHA-SGO1-tADH1 This study pGEX-4T-1 3xHA-SGO1 pRS315-HTA1-HTB1 pMK120 2μm URA3 vector with CUP1 promoter pMK144 2μm URA3 pCUP1-GCN5 pMK144E173H 2μm URA3 pCUP1-gcn5E173H pMK144F221A 2μm URA3 pCUP1-gcn5F221A pMK572 pMK573 2μm URA3 vector with ADH1 promoter and terminator 2μm URA3 SGO1 pQQ18/pMK439 pRS315-HTA1-HTB1 HHT2-HHF2 This study This study Kuo et al. 1998 Kuo et al. 1998 Kuo et al. 1998 Kuo et al. 1998 27 27 27 pXD32 pXD33 2μm URA3 RPD3 with ADH1 promoter and terminator 2μm URA3 rpd3H150A with ADH1 promoter and terminator This study This study 52 Table 2-3. Oligos used in this study Oligo Sequence CEN16 S ATGCAAAGGTTGAAGCCGTTA CEN16 AS TTTGCCGATTTCGCTTTAGAAC CEN16-0.3kb S GAAGCACTCCGACCTTTC CEN16-0.3kb AS CTTGCCTTTTCTGGATCAG CEN16-1.7 GATGAGCACATATGCATG CEN16-1.7as CTTAATCCATCAATTCTGG CEN16+4.0 GCCCTGATAAAGTCGACC CEN16+4.0as GAACTCTTGCAAGTTGAAG CEN16+6.5 CCGATGATGGTTGTTATG CEN16+6.5as CTCTAATAGTGGCAATGTTG CEN16+9.1 AAACTCAATGATGACCTTG CEN16+9.1as TATGTTACTCTTACGATGTG mk93 GGC AAG TGG TAT TCC GTA AG mk93as CTT GGT TTT CCT CTT AAG TG OJL19 OJL20 TGTCATCATGCGTATTAGAG CGTATAGGGAATTTAACGTC OJL100 GACTAAAGTAGAGCAACA OJL101 AGTGGAGTAATGCCACAT OJL102 ACGTCTAGCTGAGCATGT OJL103 GAACTGTCGAAACTGAGT oXD15 oXD16 ATCCATATGACGTTCCAGATTACGCTGCTCAGTGCGTATATGA AGCAACACCTTTTGATC TATCGGGGGGATCCACTAGTTCTAGCTAGAGCGGCCTCAATAG AATTCATTGTCATGCTC 53 Table 2-3 (cont’d) oXD25 oXD26 oXD33 oXD34 oXD37 oXD38 oXD59 oXD60 oXD79 oXD80 oXD83 oXD84 AGCATGGATTCTGTAATGGTTAAGAAAGAAGTACTGGAAAATT GAAGCTTGATATCGAAT TTCTTCATCACTCCATTCTTCAAACGAATCCAGTATAAAGTCTA CGACTCACTATAGGGC AAAATGTCACAGGTTTGGCATAATTCGAATTCGCAATCAAACTG AAGCTTGATATCGAAT TTGAATCTTAGCCCCCTTGTCTGAAGATTCAGTATTCCCAGTTA CGACTCACTATAGGGC CAGATGGTATATGAAGCAACACCTTTTGATCCGATCACGGTCT GAAGCTTGATATCGAAT ATAGAATTCATTGTCATGCTCAACATGTAGGTCCCTCGCATATA CGACTCACTATAGGGC TCAACTATGCGGGTGGTTTGGCTCATGCAAAAAAATCGGAGGC TTCTGGGTTTTGTTATT AATAACAAAACCCAGAAGCCTCCGATTTTTTTGCATGAGCCAAA CCACCCGCATAGTTGA TAATATGAATTAATAAACACCTGTCCATTTTAGAAAAACGCTTG AAGCTTGATATCGAAT TCGCATTATTAATTTGTATTCAAACGACTAATTAAAACTATCTAC GACTCACTATAGGGC AGTACGTTAAAATCAGGTATCAAGTGAATAACAACACGCAACT GAAGCTTGATATCGAAT AAAAAAAAAAACGGGAGATTAACCGAATAGCAAACTCTTAAATA CGACTCACTATAGGGC 54 then subjected to plasmid shuffling and 5-FOA selection to obtain either WT or G44A H3. To create pXD32, a 2µm URA3 RPD3 plasmid, primers oXD15 and oXD16 bearing 42 bp of homology to the vector pMK595 at the 5' end were used to amplify RPD3 ORF from yeast genome. The PCR product was co-transformed with Not I- digested pMK595 into yMK839. Ura+ transformants were subjected to DNA extraction for bacterial transformation. Miniprep DNA was then verified by sequencing. To introduce the H150A catalytically dead mutation to Rpd3p, primers oXD59 and oXD60 were used for site-directed mutagenesis PCR with pXD32 as template, generating pXD33. The mutagenesis was confirmed by sequencing as well. Myc-tagged Gcn5p and HA-tagged Rpd3p strains were created as previously described (38). Yeast methods. Yeast growth media, conditions, and transformation were based on standard procedures (39). When appropriate, 5% casamino acids (CAA) were used to substitute for synthetic amino acid mixtures as selective medium for uracil, tryptophan, or adenine prototroph. Yeast transformation was done with the lithium acetate method (40). Chromosome stability assays were conducted by measuring the mating behavior of diploid strains bearing wild-type or selective tension sensing mutants. Homozygous diploid cells created by the transformation of YCp50-HO (41) were grown overnight in YPD, and then patched onto YPD plates and incubated at 30° for 2 to 3 days until saturation. Cell plates were replica plated to another fresh YPD plates covered with 55 approximately 5 x 107 MATa or MATα tester cells and allowed to mate at 30° for 10 hours, followed by further replica plating to minimum medium plates. Mating between the tester and the subject strains resulted in complete complementation of nutrient requirement and were able to survive in the minimal medium. Genomic PCR that examined the status of the MAT loci on chromosome III was conducted by using the primers OJL100, OJL101, OJL102, and OJL103 targeting the MAT locus but not either of the two silent loci. Tension sensing test using the PGAL1-MCD1 strains was according precisely to reference (13) using strains yJL171, yJL487 and yJL492. Western analyses of yeast proteins and benomyl washout assays were conducted as mentioned in reference (35). Chromatin immunoprecipitation (ChIP). ChIP was conducted as previously described (35, 42) using primers listed in Table 3. To quantify the ChIP results, PCR products were purified and resolved by 9% polyacrylamide gel electrophoresis, and stained by ethidium bromide. The captured gel images were then quantified by NIH ImageJ. Intensities of each CEN/pericentric fragment were compared to a common internal control (DED1, PGK1, or TEL). The ratio was further normalized to 0.1% input DNA for PCR amplification carried out in parallel of all reactions. The ChIP data were obtained from at least three independent yeast cultures. Sgo1p-H3 interaction. Histones were prepared according to reference (43). Recombinant Sgo1p was prepared as previously described (35). For pull-down assays, approximately 5 µg of soluble recombinant Sgo1p was incubated with about 3 µg of yeast histones in 150 µl of HEMGT buffer at 4°C for 1 hour. 6 µl of glutathione beads along with 150 µl of the HEMGT buffer were added to the reactions and rocked gently at 56 4°C for another hour. Beads were washed with 500 µl of the HEMGT buffer for 3 times, 5 min each, followed by boiling in 2x SDS-PAGE loading buffer for 5 minutes. Eluate was resolved by 15% SDS-PAGE and blotted for anti-H3 Western analyses (35). RESULTS Mutations of a cluster of amino acid residues of H3 cause mitotic chromosome instability Our initial discovery that Gly44 of H3 is important for Sgo1p interaction and tension sensing (35) suggests that this residue is part of a motif directly responsible for Sgo1p recruitment. Several scanning mutagenesis studies of histone H3 indeed identified residues, some are close to Gly44, to be important for maintaining mitotic integrity and chromatin stability (26, 36, 37). To more specifically map the functional domain integrating Gly44 for tension sensing, we first compared benomyl hypersensitivity of strains bearing histone H3 mutations from K36 (the end of the tail domain) through K56 (the end of the αN helix of the histone core). Benomyl depolymerizes mitotic spindles. Cellular hypersensitivity to benomyl is a trait shared by many mutants with defects in mitotic regulation. Figure 2-1A shows that K42A, G44A and T45A mutations caused strong hypersensitivity to benomyl, and that a milder phenotype was caused by the adjacent H39A, R40A, Y41A, and R49A mutations. Intriguingly, P43A, flanked by K42 and G44, apparently is phenotypically neutral. 57 Figure 2-1. Identification of the tension sensing motif of histone H3. A. Scanning mutagenesis of histone H3 residues from K36 to K56. Yeast cells bearing a sole copy of H3 containing the indicated mutations were tested for their sensitivity to benomyl. B and C. More detailed analyses of 42KPGT tension sensing motif. D. Mutations in and near the 42KPGT tension sensing motif are suppressed by a multicopy plasmid bearing the SGO1 gene. 58 Crystal structures of nucleosomal particles show that 42KPGT region adapts a unique b turn structure transitioning from the flexible tail domain to the well-structured histone-fold domain (44) (Figure 1-4). Curiously, both K42 and T45 have been shown to be modified post-translationally (45, 46) for the control of gene expression and replication, respectively. Their role in mitosis, if any, is unclear. To examine the potential involvement of K42 and T45 modifications in mitosis, we introduced additional mimetic mutations and tested the cellular response to benomyl treatment. Figure 2-1B shows that the K42R non-acetylatable mutant behaved similarly to the wildtype but K42Q cells were hypersensitive to benomyl. The T45E phosphomimetic mutation caused cell death, due possibly to dysregulation of replication (45). The differential effects of alanine, glutamine, and arginine substitutions at K42 suggest that maintaining a positive charge at this position is critical but the actual side chain, that is, lysine or arginine, is not as critical. On the other hand, the K42R mutation alone could not rescue the benomyl hypersensitivity phenotype of G44S (which shared comparable traits with G44A) (fourth row, Figure 2-1B), indicating that glycine at this position is essential and cannot be rescued by a constitutive, positive charge at position 42. The original G44S mutation that impairs tension sensing can be suppressed by overexpressing Sgo1p, the key factor for this checkpoint function ensuring error-free segregation (13, 35). If the newly identified mutant alleles also perturbed the function of tension sensing, we predicted that they would be suppressed by Sgo1p overexpression as well. Results confirm this hypothesis (Figure 2-1C). In the presence of a multicopy plasmid bearing a constitutively expressed SGO1 gene, all mutant alleles tested showed apparent restoration of robust growth in the presence of benomyl. Neighboring 59 residues replaced by alanine (i.e., His39, Arg40, Tyr41, and Arg49) were all rescued by 2µ SGO1 (Figure 2-1D). These data suggest that K42, G44, and T45 form the core of a tension sensing motif that also require several nearby residues for full function. Chromosome instability conferred by K42, G44, and T45 mutants Mitotic defects frequently trigger chromosome instability and aneuploidy, a phenotype that can be realized by the acquisition of the ability of diploid cells to mate. In budding yeast, only haploid can mate. Normal diploid cells inherit the transcriptionally active MATa and MATa mating loci on chromosome III from the two haploid parents. Concomitant expression of these two loci in diploid cells represses genes essential for mating. The a/a diploid cells thus are non-mater. If there is an increase in chromosome instability, sporadic loss of one of the two chromosome III homologues from a population will enable the underlying strain to mate as both MATa and MATa mating types, regardless of the ploidy for the rest of the genome. The emergence of this bi- mater phenotype thus manifests chromosome instability and aneuploidy (47). To take advantage of this method to examine the effects of tension sensing motif mutations on chromosome stability, we first generated diploid strains bearing homozygous K42A, G44S, or T45A mutation. As an additional control, we included another novel histone mutant allele, H4 R35S, which was also hypersensitive to benomyl but was not suppressed by Sgo1p overexpression (Luo and Kuo, unpublished data). Diploid cells from single colonies were grown on solid medium as patches before replica plating to haploid mating tester strains. Successful mating to either tester strain complemented the nutrient marker gene defects, hence allowing cells to grow on the SD minimal plate. Figure 2-2A shows that a substantial portion of diploid cells bearing any of these H3 60 mutant alleles were able to mate with both tester strains, while wide-type or H4 R35S mutant cells maintained their diploid non-mater character. Genomic PCR using primers differentiating MATa and MATa loci demonstrated that the non-mater isolates maintained both copies of chromosome III, that is, the two PCR fragments were of roughly equal intensities. On the other hand, those capable of mating, wildtype haploid or mutant diploid, showed substantial bias toward one of the two copies. The mating type correlated well with the intensities of the two bands (Figure 2-2B), strongly suggesting that the bi-mater phenotype was due to imbalance of the two chromosome III homologues. To further rule out the possibility that these maters were a result of meiosis before or during experiments that would have generated mating-capable haploids, we did fluorescence activated cell sorting (FACS) analysis to examine the overall DNA content of cells from the patches on YPD (before mating) and SD minimal plates (after mating). Figure 2-2C shows that prior to mating with the tester, all starting diploid cells contained 2N or slightly higher DNA content. After mating with the haploid tester cells, the DNA content further increased (marked by the arrows of the G1 peaks), confirming the fusion of two sets of genome that resulted in triploidy. In addition to high frequencies of aneuploidy (i.e., bi-mater phenotype), the K42A, G44A, G44S, and T45A homozygous diploid cells either had a very low sporulation efficiency, or, when tetrads were formed, low germination rate (data not shown). Finally, in contrast to the histone H3 tension sensing mutants, the H4 R35S mutant behaved like the wild-type cells in mating tests (Figure 2-2A), arguing against the possibility that all benomyl hypersensitive mutants suffer from chromosome instability. We surmise that K42, G44, and T45 are important for cells to maintain mitotic chromosome stability. 61 Figure 2-2. Mutations introduced to the tension sensing motif cause chromosome instability. A. Diploid cells bearing homozygous mutant alleles of histone H3 or H4 were tested for their mating behavior. Cells mate with a tester strain (227a or 70a , see Strain List) acquire the ability to grow in SD minimal medium. Cells containing the R35S 62 Figure 2-2 (cont’d) mutation in histone H4 are hypersensitive to benomyl and do not affect the tension sensing function. The three wildtype control strains on the bottom roll are, from left to right, MATa , MATa and MATa/a . B. Genomic PCR reveals the stochastic loss of one of the two copies of chromosome III. Cells scraped from the YPD (before mating) patches were subjected to DNA isolation and PCR, using primers amplifying both silent mating loci concomitantly. The corresponding mating behaviors of each patch on YPD and SD plate are shown below each lane of the DNA gel. The relative intensity of the two mating loci PCR products correlates with the mating ability. C. FACS analysis of the DNA content of asynchronous diploid strains before and after mating. Successful mating increases the DNA content. The G1 peak of the wildtype and each mutant diploid is marked by a broken and a solid vertical line, respectively. The right-shift of the G1 peak after mating is indicated by arrows. 63 K42A and T45A mutations compromise tension sensing function and Sgo1p recruitment To more firmly link 42KPGT to the tension sensing function, we tested the spindle assembly checkpoint activation in response to a tensionless crisis. Benomyl treatment depolymerizes microtubule, eliminating both attachment and tension, and consequently activates the spindle assembly checkpoint. Both K42A and T45A mutant cells were able to stabilize the securing protein Pds1p in response to benomyl insult (Supplemental Figure S2). We then tested the spindle checkpoint activation induced specifically by the lack of tension. To this end, we placed the MCD1 gene under the control of the galactose-repressible GAL1,10 promoter (13). MCD1 (a.k.a SCC1) encodes an integral component of the cohesin complex that holds sister chromatids together. Wildtype and mutant strains were synchronized at, and released from G1 phase in a galactose-containing medium. Normal mitotic progression is manifested by the fluctuation of the securin protein Pds1p (Figure 2-3, solid lines). If cells were released from G1 arrest into a glucose-containing medium which repressed MCD1 transcription, Mcd1p deficiency disrupted cohesion and prevented tension buildup. Such a tension-less crisis activated the spindle assembly checkpoint by stabilizing Pds1p (broken line, top row, Figure 2-3). On the other hand, K42A and T45A mutant cells showed Pds1p fluctuation in both galactose- and glucose-containing media (middle and bottom row). The inability of cells to respond to the lack of tension is in excellent agreement of the documented tension sensing defects caused by the G44S mutation (35). We therefore conclude that K42, G44, and T45 together form a tension sensing motif (TSM). 64 Yeast and other eukaryotic cells monitor the tension status via the Shugoshin family proteins (48). Recruitment of the yeast Sgo1p to the pericentromeres is essential for the tension sensing function (19, 35). Given that overexpressing SGO1 suppresses the benomyl hypersensitivity phenotype of both K42A and T45A mutants (Figure 2-1C), we suspected that these two alleles also crippled the ability of H3 to retain Sgo1p at the pericentric region, hence compromising the tension sensing activity. To test this hypothesis, we conducted ChIP experiments to check the localization of Sgo1p in K42A or T45A mutant strains. Indeed, Sgo1p was recruited efficiently to CEN16 in all strains tested (Figures 2-4A, B). However, the pericentric Sgo1p was significantly reduced in both mutants, despite that the total levels of Sgo1p were comparable in all three strains tested (Figure 2-4C). The abrupt descent of the Sgo1p signal at as close as 0.3 kb to CEN16 in these two mutants strongly suggested that the recruitment of Sgo1p to the centromere, in which the canonical H3 is replaced by a centromere-specific H3 variant Cse4p, is mediated by a mechanism different from that for pericentric Sgo1p enrichment. This notion was further confirmed by in vitro pulldown assays that examined the physical interaction between H3 and Sgo1p. Figure 2-4D shows that the wild-type H3 purified from yeast was able to interact with a GST-tagged Sgo1p. K42A and T45A mutations weakened, but did not eliminate, the affinity for Sgo1p (Supplemental Figure S4). Consistent with the observation that the P43A mutation did not cause benomyl hypersensitivity (Figure 2-1A), histone H3 bearing the P43A mutation bound Sgo1p similarly as wild-type. We thus conclude that both K42A and T45A mutations compromise severely the ability of H3 to interact with Sgo1p in vitro and in vivo, and that the tension sensing motif of H3 functions through physically recruiting 65 or retaining Sgo1p at the pericentromeres. Figure 2-3. K42A and T45A mutations diminish the ability to activate spindle assembly checkpoint when tension is absent. A cohesin complex component Mcd1p is under the control of the GAL promoter. Yeast cells were released from G1 arrest into galactose (Gal) or glucose (Glc) medium. The abundance of Pds1p securin was examined by immunoblotting, and quantified by comparing with an internal control glucose-6-phosphate dehydrogenase (G-6-PDH) (see Supplemental Figure S3). 66 The HAT activity of Gcn5p regulates TSM function While it is highly likely that Sgo1p was first recruited to the centromeres and then spreads to pericentromeres [Figure 2-4 and (19, 24, 35)], it is enigmatic as to how Sgo1p is confined at such loci when H3 is nearly ubiquitous in chromosomes. One possibility is that the H3-Sgo1p interaction is subjected to direct or indirect regulation at or surrounding centromeres such that only the pericentric H3 is amenable to Sgo1p association. Because bacterially expressed Sgo1p and H3 interact well (35), it is possible that the H3-Sgo1p interaction might be negatively regulated in vivo by a reversible modification. Of the many chromatin modifying activities, we were particularly interested in the potential involvement of Gcn5p, a prototypical histone acetyltransferase well-known for its role in transcriptional regulation. Additionally, Gcn5p controls the chromatin structure at the centromeric region, an activity shown to be important for ensuring timely mitotic progression (49, 50). We suspected that Gcn5p might also be involved in the tension sensing function of H3, and therefore examined the effect of deleting GCN5 from strains bearing mutations within the tension sensing motif. Figure 2-5A shows that deleting GCN5 effectively suppressed the benomyl hypersensitivity of G44S, G44A and K42A strains. Intriguingly, the T45A mutant was much less susceptible to this suppression, arguing against that deleting GCN5 resulted in a global change in benomyl flux or metabolism. To further delineate whether the suppression was related to the HAT (or lysine acetyltransferase, KAT) activity of Gcn5p, we overexpressed two dominant negative alleles of GCN5, gcn5 F221A and gcn5 E173H (51, 52), that were devoid of the catalytic activity. Essentially identical results as those from GCN5 ORF deletion were obtained (Figure 2-5B). The HAT activity of 67 Figure 2-4. Defective tension sensing motif fails to retain Sgo1p in pericentromeres. A and B. Chromatin immunoprecipitation of Sgo1p epitope-tagged with HA. Cells were arrested at G2/M by benomyl before ChIP. Each PCR reaction contained a pair of locus-specific and another common internal control primers corresponding to the PGK1 gene. Quantification results of PCR reactions are shown in panel B. C. The steady state abundance of Sgo1p is not affected by mutations of the 68 Figure 2-4 (cont’d) tension sensing motif. Yeast whole cell lysates were probed with anti-HA antibodies to detect the HA-tagged Sgo1p D. Sgo1p interaction with H3 is weakened by K42A, G44A, and T45A, but not by P43A mutation. Bacterially expressed GST-Sgo1p was incubated with core histones purified from yeast. H3 retained by GST-Sgo1p on the glutathione beads was eluted and resolved by SDS-PAGE. Results of Western blotting with and anti-H3 antibodies are shown. Error bars were calculated from at least three biological replica. 69 Gcn5p was thus concluded to be elemental to the mitotic function of Gcn5p. In addition to benomyl hypersensitivity, mutations at K42, G44, and T45 also caused cellular hypersensitivity to a nucleoside analog, hydroxyurea (HU) (Figure 2-5B). However, overexpression of the GCN5 HAT inactive mutants had no effect on HU hypersensitivity, indicating that the genetic interaction between Gcn5p and H3 is specific to mitotic functions. The notion that Gcn5p acted as a negative regulator for the H3 mitotic function was further supported by the enhanced benomyl sensitivity when the wild-type Gcn5p was overexpressed in the K42A, G44A, and G44S mutants (Figure 2-5B). Moreover, the suppression brought about by gcn5 F221A and gcn5 E173H alleles was reverted upon SGO1 deletion (Figure 2-5C), suggesting that Gcn5p regulates H3 function in the same pathway as Sgo1p. The E173H gcn5- suppressor for mitotic defects caused by G44S was further examined by two additional methods (Figure 2-6). Firstly, the cellular viability decline caused by progressively longer incubation with benomyl was assessed. Benomyl treatment depolymerizes microtubules and arrests cells at the G2/M phase. After returning to a benomyl-free medium, cells re-establish spindle-kinetochore attachment for metaphase-to-anaphase transition. Cell cycle progression with uncorrected attachment mistakes leads to aneuploidy and cell death. Defects in tension sensing thus causes elevated death rates (35) (Figure 2-6A). Overexpressing the dominant negative mutant Gcn5p restored viability of the G44S mutant to nearly the wildtype level (black broken line, Figure 2-6A). Secondly, the chromosome missegregation rate of the G44S mutant with or without the E173H allele of Gcn5p was further quantified by examining haploid cells bearing a GFP-marked TRP1 locus 12 kb from CEN4 (53). Haploid G1 70 Figure 2-5. Gcn5p histone acetyltransferase is a negative regulator of the histone H3 tension sensing motif. A. Deleting GCN5 rescued the benomyl hypersensitivity 71 Figure 2-5 (cont’d) phenotype of mutations at K42 and G44, but to a lesser extent for T45A allele. B. The HAT activity of Gcn5p is linked specifically to the mitotic defect of tension sensing motif mutations. Dominant negative, catalytically inactive mutant Gcn5p bearing the E173H or F221A mutation (KUO et al. 1998) was overexpressed in different tension sensing motif mutant strains. The cellular sensitivity to benomyl and hydroxyurea (HU) was tested. Only benomyl hypersensitivity was rescued. C. The E173H and F221A Gcn5p suppressors for tension sensing motif mutations require Sgo1p. SGO1 was deleted in WT and different tension sensing motif mutants expressing different GCN5 alleles. The cellular tolerance to benomyl was assessed. 72 Figure 2-6. Gcn5p and Rpd3p affect chromosome stability via genetic interaction with the tension sensing motif. A. Wildtype and G44S strains bearing a gcn5 E173H overexpressing plasmid were treated with benomyl for the indicated time before plating to a benomyl-free medium to assess cellular viability. G44S cells lost viability fast but could be rescued by the dominant negative allele of Gcn5p. B. Segregation of a GFP- marked chromosome was assessed by fluorescent microscopy. G1 phase cells bearing 73 Figure 2-6 (cont’d) two GFP dots, indicating co-segregation of the indicator chromosome, were scored and expressed as percent of missegregation. C. Deleting RPD3 augments chromosome instability caused by a tension sensing mutation. The indicated yeast strains bearing an artificial chromosome that rescues the ade2- red colony phenotype were plated to YPD medium after overnight growth. Retention of the artificial chromosome gives rises white colonies, loss of which renders colonies exhibiting red color. The relative size of the red sector is dictated by the time of the chromosome loss during colonization. Total red colonies result from chromosome loss before inoculation to the plate. G44S rpd3∆ double mutant cells produce practically only all-red colonies. 74 phase cells with two GFP dots indicated missegregation from the previous round of mitosis. Figure 2-6B shows that the percentage of two-dotted G44S cells was significantly reduced by the gcn5– E173H allele. From Figures 2-5 and 2-6, we concluded that the histone acetyltransferase Gcn5p functions as a negative regulator for the mitotic regulatory function of the histone H3 tension sensing motif. Histone deacetylases Rpd3p and Hos2 interact genetically with tension sensing motif of H3 If the HAT activity of Gcn5p plays a negative role in tension sensing, a lysine deacetylase(s) (KDAC) would likely be involved as well. If true, deleting such a deacetylase gene (equivalent to upregulating the HAT activity of Gcn5p) was predicted to cause a phenotype opposite to deleting GCN5, e.g., elevated benomyl intolerance. Indeed, when RPD3 or HOS2 was deleted, the G44A cells exhibited benomyl hypersensitivity more severe than G44A, rpd3∆, and hos2∆ single mutants. Knocking out SIN3, which encodes a partner of Rpd3p for protein deacetylation (54), caused the same phenotype as that of G44A rpd3∆ (Figure 2-7A), strongly suggesting that the Rpd3p/Sin3p deacetylase complex was involved. In contrast, the null alleles of two other KDAC genes HDA1 and HOS1 failed to cause a similar synthetic phenotype, indicating functional differentiation among these enzymes in mitosis. Besides benomyl hypersensitivity, the combination of rpd3∆ and H3 G44S allele caused significantly frequency of chromosome loss, as revealed by the sectoring assays using a non- essential artificial chromosome (Figure 2-7C) (55). Deleting RPD3 alone did not cause a discernible effect on chromosome stability. Moreover, similar to the observation that a single catalytic dead mutation of Gcn5p was sufficient to elicit the suppression 75 Figure 2-7. Histone deacetylases Rpd3p and Hos2p functionally interact with the H3 tension sensing motif. A. Different HDAC genes were deleted in the G44A 76 Figure 2-7 (cont’d) background to assess the effect on the benomyl hypersensitivity of the G44A cells. B. The HDAC activity of Rpd3 underlies the genetic interaction between RPD3 and H3 G44A allele. Wildtype and G44A cells with an rpd3∆ null allele were transformed with an empty vector, wildtype RPD3, or rpd3 bearing an inactivating H150A mutation. The cellular sensitivity to benomyl was examined. C. Sgo1p multicopy suppressor for the G44A mutant allele of the tension sensing motif requires Rpd3p. 77 Figure 2-8. Both Gcn5p and Rpd3p are present in centromeres and pericentromeres. Myc-tagged Gcn5p and HA-tagged Rpd3p were ChIP’ed with the 78 Figure 2-8 (cont’d) cognate antibodies in benomyl-arrested cells. Quantitative PCR was conducted with primers corresponding to a selective chromosomal locus and a common telomeric region as the internal control. The ratio of selective vs. common region were calculated and normalized to that of input. Quantification results of PCR reactions are shown in panel B, n=3. 79 phenotype (see Figure 2-5 above), a H150A point mutation introduced to the active center of Rpd3p KDAC (56) was sufficient to cause synthetic benomyl hypersensitivity in the H3 G44A background (Figure 2-7B), indicating that the KDAC activity of Rpd3p was responsible for the observed mitotic defects. Western blotting of whole cell lysates demonstrated comparable abundance of the wildtype and the H150A species of Rpd3p (Supplemental Figure S5). Moreover, all tension sensing motif mutants are suppressed by Sgo1p overexpression ((26, 35) and Figure 2-1D above). This suppression was prevented in rpd3∆ cells (Figure 2-7C), indicating that Rpd3p is essential for Sgo1p to function in the surveillance of biorientation when the tension sensing motif is crippled. A role of Rpd3p and Gcn5p in tension sensing was further supported by chromatin IP that showed enrichment of both enzymes at centromeres and pericentromeres (Figure 2-8). When these two proteins were epitope-tagged (13xMyc for Gcn5p and 3xHA for Rpd3p) and ChIP’ed from benomyl-arrested G2/M phase cells, both CEN and pericentromeres harbored significant levels of Gcn5p and Rpd3p when compared with a transcriptionally silenced subtelomeric region. From genetic and biochemical data presented in Figures 2-5 – 2-8, we conclude that the tension sensing motif of histone H3 is subjected to the regulation by Gcn5p and Rpd3p protein acetylation and deacetylation enzymes. Overexpression of gcn5E173H does not restore pericentric localiztion of Sgo1p in G44S mutant To further investigate the mechanism of how HAT-null gcn5p suppresses TSM mutants, we tested if Sgo1p level is restored at the pericentromeres of G44S cells when gcn5E173H is overexpressed. Interestingly, with overexpression of gcn5E173H, we 80 could only observe a similar level of Sgo1p at the pericentromeres when compared to G44S alone. However, another suppressor, high-copy Sgo1p could restore its pericentric localization up to WT level in G44S mutant (Figure 2-9). This data suggests that the suppression of HAT-null gcn5p works through different pathway, instead of restoring Sgo1p. 81 Figure 2-9. Overexpression of HAT-null gcn5p-E173H doesn’t restore pericentric localization of Sgo1 in benomyl-arrested G44S mutant. HA-tagged Sgo1p were ChIP’ed in benomyl-arrested cells. Quantitative PCR was conducted with primers corresponding to a selective chromosomal locus and a common telomeric region as the internal control. Quantification results of PCR reactions are shown in panel B, n=2. 82 DISCUSSION This work characterizes a tension sensing motif in histone H3 that is important for mitotic checkpoint control. The core residues of this motif, Lys42, Gly44, and Thr45 are important for interactions with Sgo1p both in vitro and in vivo. Alanine substitutions of these three residues share similar mitotic phenotypes, and are all susceptible to the overexpression of SGO1, suggesting that they function in the same pathway for monitoring tension between sister chromatids. These results are consistent with the scanning mutagenesis experiments reported by others (26, 36). Chromatin IP assays showed that mutating residues of the tension sensing motif selectively diminished the pericentric, but not the centromeric recruitment of Sgo1p. We suggest that Sgo1p is first recruited to the centromeres by factors such as the kinase Bub1p and phosphorylated histone H2A (19, 25, 57), and then relocates to pericentromeres where histone H3 is part of the canonical nucleosomes (35). Retention of pericentric Sgo1p is mediated through the tension sensing motif of H3. The loss of pericentric domains of Sgo1p results in malfunction in tension sensing and chromosome missegregation. Intriguingly, the mitotic function of the tension sensing motif appears to be regulated by the histone acetyltransferase Gcn5p, and at least two deacetylases, Rpd3p and Hos2p. The involvement of these two opposing protein modifying activities suggests a new dimension of the critical tension sensing function of mitosis. The benomyl hypersensitivity phenotype caused by K42A, G44A, and G44S alleles is suppressed by deleting GCN5 or inactivating its HAT activity, and is augmented by the loss of RPD3, SIN3, or HOS2 deacetylase genes. Chromosome instability is also rescued by the E173H catalytically inactive mutant of Gcn5p (Figures 83 2-6A, B), but drastically augmented by the deletion of RPD3 (Figure 2-6C). Importantly, the suppressor activity of gcn5– alleles depends critically on Sgo1p (Figure 2-5C). The SGO1 high-copy suppressor for G44A is practically eliminated in the absence of RPD3 (Figure 2-5C), whereas the chromosome loss rate of G44S cells rises greatly (Figure 2- 6C). Together, these results strongly suggest that protein acetylation is a critical component of tension sensing executed by H3 and Sgo1p. Indeed, both Gcn5p and Rpd3p are found to be enriched at centromeres and pericentromeres during mitosis (Figure 2-8). But interestingly, Sgo1p is not restored at the pericentromeres in hat-null gcn5p strain (Figure 2-9). One explanation for the absence of Sgo1p is that hat-null gcn5p may bypass the requirement of proper Sgo1p localization through an unknown mechanism. This possibility needs to be tested in the future. At the same time, it is tempting to speculate that one or more centromeric and pericentric proteins are acetylated by Gcn5p, antagonizing H3 and Sgo1p functions. While the tension sensing motif includes a potentially acetylatable lysine residue, it is unlikely that Lys42 is a functional acetylation target herein because the K42R mutation, which mimics a constitutively un-acetylated state, does not suppress the concomitant G44S mutant (Figure 2-1B). Instead, one can envision that acetylation of Sgo1p by Gcn5p diminishes the affinity for the tension sensing motif. The biochemical test of this hypothesis is currently being pursued. In addition to Sgo1p, cohesin may also be targeted by Gcn5p and Rpd3p. Cohesin plays an important role in localizing Sgo1p to the pericentromeres (29, 58, 59). The recruitment of mammalian cohesion complex to centromeres and pericentromeres requires the acetyltransferase activity of San (59) and Esco1 (60). However, our genetic data indicate that the acetylation is a negative regulator of tension 84 sensing. If one of the cohesin subunits is acetylated by Gcn5p, this action may inhibit either the pericentric loading of cohesin or its interaction with Sgo1p. Compared with other mutations in the tension sensing motif, the T45A allele responds poorly to the suppression by gcn5– and less well to multicopy SGO1 overexpression (Figures 2-5B and 2-1C, respectively). Because K42A, G44A, G44S, and T45A alleles all show weakened interactions with Sgo1p (Figure 2-4D and (35)), it seems likely that Thr45 plays a qualitatively distinct role in mediating H3-Sgo1p interaction. One possibility is that the H3-Sgo1p interaction requires the side chain of Thr45, whereas Lys42 and Gly44 together provide an environment (e.g., the sharp b - turn structure, Figure 2-1E) that facilitates the Thr45-Sgo1p contact. If true, this hypothesis suggests that a post-translational modification at this residue would impose a significant impact on Sgo1p interaction. Indeed, Thr45 phosphorylation has been reported in both yeast and humans. The yeast Dbf4p-Cdc7p complex catalyzes Thr45 phosphorylation for DNA replication (45), whereas the mammalian Thr45 phosphorylation event is linked to apoptosis (61) and to DNA damage (62). The phosphomimetic T45E mutation causes cell death [(45) and data not shown], preventing detailed genetic experiments for the possible involvement of Thr45 phosphorylation in mitosis. However, our preliminary biochemical experiments showed that phosphorylation at Thr45 blocked Sgo1 interaction in vitro (data not shown), suggesting that Dbf4p-Cdc7p or another kinase may control the H3-Sgo1p interaction via Thr45 phosphorylation. Additionally, given the potential contribution of acetylation in the regulation of the tension sensing motif, it is worth noting that serine and threonine can also be acetylated (63-65). Whether Gcn5p could use Thr45 as an acetylation target for 85 mitotic control awaits to be examined. Sgo1p undergoes biorientation-dependent removal from chromatin (34), suggesting that Sgo1p and, in particular, its interaction with chromatin is tightly linked to the status of tension. Pericentric chromatin structural changes seem to be an obligatory outcome of bipolar attachment (8, 10, 66). In tension defects, sgo1∆ cells either cannot activate the spindle checkpoint (13) or cannot prevent the silencing of SAC (67). The latter model was supported by a recent report that Sgo1p, along with Ipl1p, Dam1p, and protein phosphatase 1 (PP1), is required to keep SAC on in the absence of tension (67). The data presented above demonstrate a causal role of the loss of pericentric Sgo1p in crippling the tension sensing function. We suggest that the tension sensing motif of histone H3 in the pericentromeres functions initially as a docking site for Sgo1p, which is recruited to the centromeres by Bub1 and phosphorylated H2A, while cells establish bipolar attachment. Biorientation generates tension, which in turn alters chromatin structure in the pericentromeres (8, 10, 66), resulting in conformational changes of the tension sensing motif that disrupt Sgo1p-H3 interaction. The eviction of Sgo1p molecules from the pericentromere of the last pair of chromosomes under tension silences the checkpoint, hence allowing cells to progress from the metaphase to anaphase. Whether the mere absence of Sgo1p or the exiting Sgo1p proactively shuts off SAC awaits to be delineated. 86 REFERENCES 87 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Nasmyth K. 2002. Segregating sister genomes: the molecular biology of chromosome separation. Science 297:559-565. Kschonsak M, Haering CH. 2015. Shaping mitotic chromosomes: From classical concepts to molecular mechanisms. Bioessays 37:755-766. Akera T, Watanabe Y. 2016. The spindle assembly checkpoint promotes chromosome bi-orientation: A novel Mad1 role in chromosome alignment. Cell Cycle 15:493-497. 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Proc Natl Acad Sci U S A 110:21036-21041. 93 CHAPTER III TRIPARTITE CHROMATIN LOCALIZATION OF BUDDING YEAST SHUGOSHIN INVOVLES HIGHER-ORDERED ARCHITECTURE OF MITOTIC CHROMOSOMES 94 Affiliation: Authors: Xiexiong Deng and Min-Hao Kuo* Department of Biochemistry and Molecular Biology, Michigan State University. East Lansing, MI 48824. *Correspondence to: Min-Hao Kuo, kuom@msu.edu. 95 ABSTRACT The spindle assembly checkpoint (SAC) is key to faithful segregation of chromosomes. One requirement that satisfies SAC is appropriate tension between sister chromatids at the metaphase-anaphase juncture. Proper tension generated by poleward pulling of mitotic spindles signals biorientation of the underlying chromosome. In the budding yeast, the tension status is monitored by the conserved Shugoshin protein, Sgo1p, and the tension sensing motif (TSM) of histone H3. ChIP-seq reveals a unique TSM-dependent, tripartite domain of Sgo1p in each mitotic chromosome. This domain consists of one centromeric and two flanking peaks 3 – 4 kb away, present exclusively in mitosis. Strikingly, this trident motif coincides with cohesin localization, but only at the centromere and the two immediate adjacent loci, despite that cohesin is enriched at numerous regions throughout mitotic chromosomes. Chromosome conformation capture assays reveal apparent looping at the centromeric and pericentric regions. The TSM-Sgo1p-cohesin triad is therefore at the center stage of higher-ordered chromatin architecture for error-free segregation. 96 INTRODUCTION Equal partition of the duplicated chromosomes is crucial for genome integrity and species perpetuation. Aneuploidy resulting from erroneous segregation causes developmental defects and tumorigenesis (1). The spindle assembly checkpoint (SAC) is a failsafe for faithful segregation. The SAC registers the kinetochore-microtubule attachment and the tension between sister chromatids (2). The tension generated by poleward pulling of the spindles signals bipolar attachment, after which cells irreversibly initiates events leading to the onset of anaphase. In Saccharomyces cerevisiae, each kinetochore attaches to a single microtubule spindle emanating from the spindle pole bodies (3). To the two sister kinetochores, three types of attachment may occur: monotelic, syntelic and amphitelic (2). While the amphitelic attachment signals biorientation, monotelic and syntelic attachment errors have to be corrected before anaphase onset. Monotelic attachment refers to the situation when only one of the two sister kinetochores is attached to the microtubule. The presence of an unoccupied kinetochore triggers the formation of the Mitotic Checkpoint Complex (MCC) (4) that halts cell cycle progression by trapping Cdc20p, the E3 ligase subunit of Anaphase Promoting Complex (APC). In syntelic attachment, both sister kinetochores are occupied by spindles, but these two spindles originate from the same spindle pole body. Even though the attachment requirement is met, there may be no tension between syntelic sister chromatids as they are pulled toward the same pole. Left uncorrected, monotelic and syntelic attachment results in aneuploidy. In what form tension is perceived by the mitotic machinery remains elusive. In prometaphase, transient sister chromatid separation without cohesin proteolysis is 97 caused by kinetochore-microtubule attachment (5, 6). Conformational changes of centromeric chromatin (DNA, nucleosomal arrays, and selective proteins) thus are suggested to be the “tensiometer” or “spring” that reflects the tension status (7). Among these candidates, Shugoshin proteins are of particular interest. Shugoshin is a family of conserved proteins playing critical roles in ensuring appropriate chromatid cohesion during cell division (8). The budding yeast Shugoshin, Sgo1p, was first identified as a protector of meiotic cohesin against precocious cleavage (9), and later found to be also crucial for cells to activate the SAC in coping with tensionless conditions in mitosis (10). Expressed in S and M phases of the cell cycle (10, 11), Sgo1p is localized to centromeres and pericentromere (12-14) without stashing a significant extrachromosomal pool (15). Shugoshin is recruited to centromeres by binding to histone H2A phosphorylated by the Bub1 kinase (16, 17). The centromeric recruitment of budding yeast Sgo1p may also involve the interaction with the centromere-specific histone H3 variant Cse4p (18). In human mitotic cells, Sgo1 recruited to the outer kinetochore nucleosomes is then driven by RNA polymerase II to the inner centromere where it is retained by cohesin (19). Besides cohesin, the fission yeast meiosis-specific Shugoshin Sgo1 interacts with the heterochromatin protein 1 (HP1) homologue Swi6 that docks on the heterochromatic mark H3K9me3 in pericentromere (20, 21). Unlike other eukaryotes where heterochromatic marks decorate pericentromere to create a footing for Shugoshin, budding yeast lacks such heterochromatic features in the region immediately next to centromeres (3). The geographic pericentromere recruitment of Sgo1p in budding yeast, instead, is accomplished by the association with the tension sensing motif (TSM) of histone H3 in pericentric regions (22, 23). TSM (42KPGT) is a 98 conserved b -turn that connects the flexible N’ tail to the rigid histone-fold domain of H3 (24). Mutations at K43, G44, or T45 diminish the pericentric localization of Sgo1p and obliterate the cellular response to defects in tension. Restoring pericentric association of Sgo1p by overexpression, via Sgo1p-bromodomain fusion (22), or by mutating the inhibitory residues K14 or K23 of the H3 tail (15) rescues the mitotic defects of these TSM mutations, thus manifesting the pivotal role of Sgo1p retention at the pericentromere. Sgo1p is removed from chromatin after tension is built up in the metaphase (25). The inverse correlation between Sgo1p retention and amphitelic attachment suggests that Sgo1p is an integral part of the gauge by which cells use to monitor the tension status. In addition to the TSM, another factor important for targeting Sgo1p to the pericentromere is the cohesin complex. Mutations that impair cohesin loading ablate pericentric localization of Sgo1p, while leaving the centromeric Sgo1p largely unaffected (13). A similar contribution of cohesin to Sgo1 localization has been observed in human systems as well (19). Cohesin performs its tension sensing-related function by facilitating the formation of the “C” loop of chromatin near the centromeres in mitosis (26, 27). Direct interaction between cohesin and the human Sgo1 has been reported (28). The triad of Sgo1, H3 TSM, and cohesin thus likely constitute the core of the tension sensing device. The present work presents evidence for a cohesin- and TSM- dependent tripartite chromatin localization domain of Sgo1p that also involves high- ordered chromatin architecture. 99 RESULTS Sgo1p displays unique tripartite localization in each mitotic chromosome Sgo1p is critical for the tension sensing branch of the SAC function in mitosis (8). We and others have previously used chromatin immunoprecipitation (ChIP) to demonstrate that Sgo1p is enriched at centromeres and several kb on either side of the centromere in mitosis (12, 13, 22, 25). However, the range of the Sgo1p enrichment on each mitotic chromosome has not been carefully delineated. To better understand Sgo1p retention pertaining to its checkpoint function, we used ChIP-seq to map the Sgo1p distribution on mitotic chromosomes at a higher resolution. Cells bearing a C- terminally HA-tagged Sgo1p expressed from its native locus were arrested by benomyl for ChIP-seq. At a lower resolution scale, Sgo1p is detectable in one area per mitotic chromosome (Figure 3-1A), consistent with the anticipation of centromeric and pericentric enrichment (13). However, more rigorous inspection revealed that each chromosomal domain of Sgo1p is actually composed of discrete peaks of Sgo1p that form a trident-like structure, not a continuous motif covering several kb of a centromeric and pericentric area (Figure 3-2). Each of the trident motif consists of a middle centromere (CEN) and typically one pericentromere (PC) peak on each side of the CEN enrichment. By aligning all sixteen chromosomes at the centromeres, the average counts plot for Sgo1p enrichment as a function of distance to CEN shows that the average distance between the PC and CEN peaks is approximately 4 kb (Figure 3-3A, magenta line). Additional outward peaks may be seen in some chromosomes, but the overall peak height drops quickly. 100 Figure 3-1. Sgo1p is localized only to the centromeric area in each chromosome. A. ChIP-seq data of Sgo1p expressed from its native locus or from a high-copy plasmid (o/p), in a wildtype or a tension sensing motif G44S mutant background, are presented as one single linear DNA. The range of each chromosome is shown on the top. B. Expression of Sgo1p is not affected by the status of the tension sensing motif. Sgo1p is tagged with HA at N’ or C’ terminus. The 3xHA-Sgo1p is expressed from a plasmid. The Sgo1p-6xHA is expressed from the native SGO1 locus. 101 Figure 3-2. Sgo1p is recruited to centromeres and pericentromere to form a tripartite localization domain on each mitotic chromosome. The 100-kb region centering on the centromere of all 16 chromosome is aligned. Sgo1p expressed from its 102 Figure 3-2 (cont’d) native locus (magenta), or from a multi-copy episomal plasmid (green) are compared with the Mcd1p distribution (dataset from Verzijlbergen et al., 2014). The two peaks labelled “a” and “b” close to CEN2 and CEN5 correspond to ARS209 and URA3 respectively. These loci were from two plasmids in the strains used for experiments. 103 Figure 3-3. Sgo1p enrichment overlaps with cohesin domains at the centromeres and pericentromere. Average counts (per million reads) plot comparing the distribution of Sgo1p expressed in different backgrounds (panel A) or between Sgo1p and cohesin (panel B). The Mcd1p ChIP-seq data were from Verzijlbergen et al., 2014. 104 Chromosomal retention of Sgo1p depends critically on the tension sensing motif (TSM) of histone H3 (22), and the cohesin complex (29). H3 is a ubiquitous component of chromatin, yet it controls the pericentric localization of Sgo1p (22), despite that no discernible epigenetic marks have been found specifically in budding yeast pericentromere that are relevant to mitotic regulation. Mutations introduced to the tension sensing motif (42KGPT45) cause defects in detecting and/or responding to tension defects (23). These mutations diminish the affinity for Sgo1p, a molecular defect that can be suppressed by overproduction of Sgo1p (22, 23). Indeed, ChIP-seq data show that the overall chromatin association of Sgo1p is significantly reduced in a tension sensing motif mutant, G44S (Figure 3-1A, orange curve). Expressing Sgo1p from a multi-copy plasmid and the ADH1 promoter restored the tripartite chromatin association (green curves, Figure 3-2, and brown curve, Figure 3-1A). In addition to re- establishing the original enrichment pattern, a small number of new peaks distal to the CEN/PC peaks were seen. Intriguingly, these still are discrete peaks with clear valleys in between (see, for example, chromosome XVI, Figure 3-2). The emergence of these new enrichments is consistent with our original model that Sgo1p is recruited to the centromeres and then spills over to the nearby chromatin region (22). However, the non-continuous nature of Sgo1p distribution suggests the involvement of at least one other factor (see below). While histone H3 and its tension sensing motif are ubiquitously distributed throughout the genome, another Sgo1p recruitment factor, the cohesin complex, localizes at specific loci of chromosomes. Besides centromeres and pericentric regions, the majority of cohesin-associated regions are the intergenic area between two 105 convergent transcription units throughout the genome (30, 31). By comparing with the chromosomal distribution of Mcd1p (the kleisin subunit of cohesin) (29), we observed that Sgo1p co-localizes with cohesin at and immediately adjacent to centromeres (compare magenta and blue peaks, Figure 3-2 and Figure 3-4A). The plot of average count reads (Figure 3-3B) clearly shows the highly significant co-localization of Sgo1p- and Mcd1p at the centromeric and pericentric region. It is also noteworthy that most additional Sgo1p peaks resulting from overexpression are at the loci where cohesin is also enriched (Figure 3-2). These results strongly suggest that Sgo1p targets existing cohesin enrichment sites for interaction with the tension sensing motif of histone H3. In addition to comparing our Sgo1p ChIP-seq data with a published Mcd1p dataset (29), we conducted another set of ChIP assays and used quantitative PCR to examine the localization of Mcd1p and Sgo1p in the same genetic background. To this end, Sgo1p-HA and Mcd1p-Myc expressed from their native loci were subjected to ChIP. DNA products were then examined by quantitative PCR for 21 amplicons that spanned 11 kb of the centromeric region on chromosome XVI, including the three CEN and PC peaks (shaded boxes, Figure 3-4A top panel). Discrete peaks and valleys are readily visible and show a high degree of overlapping between Sgo1p and Mcd1p with the ChIP-qPCR data. Additional qPCR analysis of chromosome I amplicons equivalent to those of chromosome XVI also verifies the ChIP-seq observations (Figure 3-5). In addition, parallel ChIP reactions were conducted in the G44S tsm- background. While the Sgo1p signals diminish significantly in this region (orange bars, Figure 3-4C), the Mcd1p-Myc enrichment is not significantly affected, which demonstrates that TSM is required for the retention of Sgo1p, not Mcd1p, at pericentromere. 106 Figure 3-4. The histone H3 tension sensing motif is essential for pericentric Sgo1p localization but not Mcd1p. A. Distribution of Sgo1p (magenta) and Mcd1p 107 Figure 3-4 (cont’d) (blue) across chromosome XVI as revealed by ChIP-seq. The centromeric region is blown up to show the detail distribution of these two proteins. PCR amplicons are enumerated and shown in light pink bars below the Mcd1 peaks. The open reading frames and their transcription directions are shown at the bottom. B and C. Quantitative real-time PCR analysis of separate ChIP experiments. Sgo1p-HA and Mcd1p-Myc (both expressed from their native loci) were ChIP’ed from cells bearing the wildtype or a mutant TSM (G44S). The three enrichment sites are marked with the shaded boxes. 108 Figure 3-5. ChIP-qPCR to verify the ChIP-Seq findings. Shown are 25 amplicons spanning CEN1. 109 The exceptional selectivity of Sgo1p for a subset of cohesin localization motifs prompted us to compare its genome-wide distribution to that of Gcn5p in mitotic chromosomes. Gcn5p is a critical transcription regulatory histone acetyltransferase. In mitosis, Gcn5p negatively regulates the tension sensing motif (23), and is important for maintaining the normal centromere chromatin structure (32). Consistently, Gcn5p is present at mitotic centromeres (23). To see whether Gcn5p exhibits a mitotic chromosome localization pattern similar to that of Sgo1p, ChIP-seq was conducted on a Myc-tagged Gcn5p. The results show that, while Gcn5p is found enriched at all centromeres, its pericentric presence is practically negligible (shaded boxes showing CEN/PC peaks of Sgo1p, Figure 3-6). Importantly, throughout the genome, there is very little overlapping between Gcn5p and Mcd1p enrichment. This is not unexpected for Gcn5p is recruited to the 5’ region of many genes for transcriptional regulation, but Mcd1p and the rest of the cohesin complex are enriched at the intergenic region of convergent genes. There appears to be an enrichment of Gcn5p at RNA polymerase III- controlled targets, such as tRNA genes. These ChIP-seq results are consistent with the canonical roles of Gcn5p in transcription (33), although we do not exclude the possibility that at least part of the mitotic distribution pattern of Gcn5p might be for chromatin metabolism during mitosis. Together, ChIP-seq data presented above reveal unique association between Sgo1p and Mcd1p at and near the centromeres. However, this connection does not apply to the recruitment of Gcn5p, indicating a specific functional interplay between Sgo1p and the cohesin complex. 110 Figure 3-6. Gcn5p is enriched in centromeres but shows no overlap with cohesin elsewhere. Genome-wide distribution of Gcn5p is compared with that of Sgop1 (magenta) and Mcd1p (blue). The trident Sgo1p localization domain in each chromosome is marked with the shaded boxes. 111 The cohesin complex is required for chromatin association of Sgo1p in both budding yeast and human (Kiburz et al., 2005, Liu et al., 2015). To further confirm that the highly specific centromeric and pericentric localization of Sgo1p requires the local cohesin populations, we took two approaches. Firstly, we deleted IML3 that encodes a subunit of the Ctf19 kinetochore subcomplex. ChIP and quantitative PCR analysis shows that this manipulation disrupts only the pericentric, but not chromosome arm recruitment of cohesin (Kibruz et al., 2005) (Figure 3-7A). As predicted, the pericentric Sgo1p enrichment in chromosome XVI is completely lost in iml3∆ cells (Figure 3-7B). In the second approach, we targeted a specific cohesin associated region (CAR) on chromosome IV for inducible disruption. Active transcription can dislodge cohesin enrichment (Glynn et al., 2004). Accordingly, we replaced the pericentric CAR between YDR004W and YDR005C to a galactose-inducible promoter GAL1 (pGAL1, Figure 3-8). Changing from a non-inducing (raffinose) to an inducing (galactose) condition caused transcription-driven removal of both cohesin and Sgo1p (Figure 3-8). From data presented in Figure 3-4, 3-7 and 3-8, we conclude that the tripartite localization of Sgo1p in each chromosome depends on an intact tension sensing motif and likely is established at pre-existing or concomitantly with cohesin localization domains. 112 Figure 3-7. Sgo1p recruitment requires cohesin. IML3+ and iml3 cells were arrested in G2/M before ChIP for the localization of Mcd1p (panel A) and Sgo1p (panel B). The enrichment of these two proteins was quantified by qPCR. Amplicons are same as those in Figure 3-4. Shown are average of two biological replicas. 113 Figure 3-8. Pericentric localization of Sgo1p and Mcd1p is susceptible to ectopic transcription through the region. A. Blow-up of the CEN4 area showing the major Sgo1p and Mcd1p peaks, amplicons for quantitative PCR in light pink, and the position of the ectopic GAL1 promoter (pGAL1, in red). pGAL1 drives galactose-induced transcription toward yDR004W. Amplicons 7 – 9 are highlighted by a shaded box. B 114 and C. ChIP and quantitative PCR results of Sgo1p and Mcd1p localization in the absence (Raf., raffinose) or presence (Gal., galactose) of transcription from the pGAL1 promoter. Fold-enrichment of each amplicon was obtained by comparing with the control corresponding to the subtelomeric region of chromosome VI (TEL06R). Error bars are standard deviations from three biological replicas * indicates p-value < 0.05. D. The left panel shows the DNA gel images of reverse-transcription quantitative PCR of the indicated regions. ChrIV_Amp7 and 8 are within the YDR004W gene. GAL1 and ACT1 are positive and internal control for galactose induction. The right panel shows the quantification data, using ACT1 expression difference (Raf. vs. Gal.) for normalization. YDR004W is induced 2-fold by galactose. 115 Pericentric Sgo1p domain formation does not involve intervening valley regions Sgo1p docks on centromeres via direct association with Bub1p-phosphorylated Ser121 of histone H2A (phos.H2A) within the single centromeric nucleosome (16). Sgo1p also binds the N’ tail of the centromere-specific histone H3 variant, Cse4p (18). It is likely that phos.H2A and Cse4p provide the docking site for Sgo1p that nucleates outward spread toward the pericentric regions. The establishment of PC enrichment of Sgo1p may be accomplished by one of two mechanisms. In the rippling mode, a wave of Sgo1p spreads along the nucleosomal path before it stops and accumulates at the first cohesin block. Alternatively, Sgo1p “leaps” directly from centromeres to the PC region where it is retained by the tension sensing motif. In both modes, Sgo1p is underrepresented at the region between the CEN and PC peaks, resulting in the “valleys” seen in the two-dimensional presentation of the ChIP-seq results. These two modes of Sgo1p recruitment can be differentiated by examining the dynamics of CEN and PC peaks emergence when cells progress through mitosis. An intermediate stage where a significant elevation of Sgo1p signals at the valley region before they move outward to generate the final PC peaks would support the rippling mode. To test these two models, we tagged Sgo1p and Mcd1p in the same strain to avoid any variation between cells with different genotypes. Cells expressing Sgo1-6HA and Mcd1-13Myc were arrested in G1 phase by a factor. They were then released into the division cycle before collection at 30, 37.5, 45, 52.5, 60, 75, and 90 minutes after the release. Budding index revealed the timing of the progression through mitosis during the course of experiments (Figure 3-9A). ChIP results (Figure 3-9B and Figure 3-10) show that Sgo1p was first detectable at CEN16 37.5 minutes after release from G1 arrest, when 116 Figure 3-9. Dynamic recruitment of Sgo1p and cohesin at centromere and pericentromere through cell cycle. A. Budding index of cells collected from the indicated time points. B and C. Sgo1p-HA and Mcd1p-Myc co-expressed in the same cells were examined by ChIP-qPCR. PCR amplicons correspond to CEN16 and nearby regions. See Figure 3-4 for positions of these amplicons. We have noticed that the ChIP efficiency (%IP) of both Sgo1-6HA and Mcd1-13Myc in cells synchronously progressing through cell cycle was consistently 5- to 10-fold higher than in those benomyl-arrested cells (Figure 3-4). 117 Figure 3-10. Dynamics of Sgo1p (A) and Mcd1p (B) localization at CEN1 region. See Figure 3-9 legends for description. 118 cells were at the juncture of G1 and S phases. This coincides with the time when Sgo1p expression starts (10). While Sgo1p centromeric abundance continued to rise, the adjacent PC peaks started to surface in the next 7.5 minutes (amplicons 3, 16, and 21). These signals culminated at T60’ (green bars, Figure 3-9B) and progressively diminished at T75’ T90’. Between T60’ and T75’, approximately 20% of cells entered the anaphase (green sector, Figure 3-9A), indicating that biorientation had been established in this population of cells. The concomitant reduction of Sgo1p signals is in excellent agreement with the tension-dependent removal of Sgo1p from the chromatin (25). The kinetics of Mcd1p association with CEN and PC exhibited several important distinctions. Firstly, while Mcd1p signals jumped at T30’, the three subsequent time points (T37.5’, T45’ and T52.5’) saw a reduction of the overall Mcd1p signals, which then climbed up again, and peaked at T75’ before abrupt disappearance by T90’, when the majority of cells passed the metaphase-to-anaphase transition (Figure 3-9A). The dynamic changes before T60’ probably resulted from transcriptional activities in S and G2 phases. The abrupt increase of Mcd1p signal at T60’ agreed well with the budding index that 80% of the cells were in the metaphase when cohesion of sister chromatids was most critical. Lastly, the highest levels of the Mcd1p abundance were found to be at T75’ before its quick disappearance by T90’, both were 15’ later than Sgo1p. The different kinetics of Sgo1p and Mcd1p dissolution concurs with the anticipated sequence of biorientation, Sgo1p removal, and Mcd1p cleavage that marks anaphase onset. One critical observation from results in Figure 3-9 is that during the formation of the Sgo1p CEN and PC tripartite motif, the two valleys flanking the CEN peak never rose to the levels of PC at any given time. Given the stochastic nature of cellular 119 physiology even in a synchronized population (see budding index, Figure 3-9A), the lack of Sgo1p signal at these valley region is significant, and strongly favors the notion that Sgo1p spreads from CEN by a “hopping” mechanism to PC, or is recruited simultaneously to CEN and PC to generate the tripartite motif. Chromosome conformation capture reveals correlation between Sgo1p enrichment and chromatin architecture If Sgo1p targets its pericentric destination immediately after or concomitantly with the centromeric recruitment, it is likely that the PC regions are rendered accessible to Sgo1p whereas the intervening regions are somehow hidden from Sgo1p. Because the interaction between Sgo1p and TSM does not require any posttranslational modification (22, 23), a non-epigenetic feature may distinguish the PC Sgo1p targets from other areas nearby. We felt that chromatin architecture would be a good candidate that dictates the (in)accessibility of the CEN/PC region to Sgo1p. Compaction of chromatin in mitosis involves condensin and cohesin complexes (34, 35). Both complexes are also shown to be critical for organizing pericentromere in prometaphase (26, 27, 36). Cohesin facilitates the formation of intrachromosomal centromeric loops for mitotic segregation and resides near the summits of these loops. On the other hand, the condensin complex holds and organizes the bottom of these loops along the spindle axis (26). Taking together these models and our results shown above, we suspect that higher-ordered chromosomal architecture, e.g., chromosome looping, might be part of the mechanism underlining the highly selective pericentric localization for Sgo1p. 120 Figure 3-11. Sgo1p tripartite localization domain is associated with high-ordered chromatin architecture in mitosis. Chromosome conformation capture (3C) assay was used to examine chromatin looping near CEN1 and CEN16. Cells arrested in G1 or G2/M phase were fixed with formaldehyde, and the isolated nuclei treated with Eco RI before DNA ligation. An identical amount of final ligated DNA library was amplified by PCR using one of two common anchor primers (oXD159 and oXD162 for chromosomes I and XVI, respectively; red arrows) against different locus-specific primers (black arrows; named for their distance to the centromere, L = left; R = right) 3 – 50 kb away. All primers face toward the same direction. PCR products were resolved by gel electrophoresis and quantified with the NIH Image J software. Shown are the signals relative to the same amplicons without formaldehyde crosslinking. Error bars are standard deviations from three biological replicas. 121 If Sgo1p recruitment is linked to chromosome looping in mitosis, we predicted that PC and CEN peaks of Sgo1p were spatially near each other owing to the action of such complexes as cohesin and condensin. This hypothesis was tested by chromosome conformation capture (3C) (37). Yeast nuclei were harvested from G1 and G2/M arrest and were subjected to EcoR I digestion with or without formaldehyde fixation, followed by ligation under a condition that favored intramolecular ligation. The resultant DNA libraries were analyzed by PCR using one of two centromere-proximal anchor primers, oXD159 for CEN1 and oXD162 for CEN16. In each quantitative PCR reaction, these anchor primers were paired with a distal primer that is 3 – 50 kb away (black arrows, Figure 3-11A). All primers hybridized to the same strand of DNA, hence should not produce any PCR product without the 3C treatment. On the other hand, ligation at the anticipated EcoR I sites after formaldehyde fixation would generate templates amplifiable by the anchor and the locus-specific primers. Comparing the intensity of PCR products amplified from samples with or without formaldehyde treatment yielded “crosslinking frequency” that is indicative of the propensity for the two primer target regions to be spatially brought together by chromatin-associating factors. The 3C assays indeed show that, after crosslinking, the centromeric primers oXD159 and oXD162 could amplify with primers hybridizing to Mcd1p peaks that were 3 to 15 kb away (e.g., oXD159 + CEN1L 5kb or CEN1R 5kb, and oXD162 + CEN16L 8kb or CEN16R 3kb; Figure 3-11B). Some of the amplification products spanned a region with a conspicuous Mcd1p signal without Sgo1p (e.g., oXD159 + CEN1L 20kb, oXD162 + CEN16L 15kb), consistent with the idea that chromosomal loops generated by the cohesin complex is upstream to and a prerequisite for Sgo1p localization (26, 29). The 122 crosslinking frequency from G2/M nuclei was in general higher than G1 (orange vs. blue bars), which indicates that the nuclear architecture climaxes during mitosis, but may be partially preserved after exiting from M phase. This notion is consistent with the weak but readily recognizable Mcd1p peaks in cells arrested at G1 (Figure 3-9). DISCUSSION This work captures high-resolution genome-wide localization of Sgo1p in mitotic S. cerevisiae cells. On each chromosome, Sgo1p displays a tripartite localization domain consisting of a middle centromeric and typically two flanking pericentric peaks. Sgo1p co-localizes with the cohesin complex. However, despite that cohesin is recruited to numerous loci across the genome, Sgo1p only rendezvouses with the centromeric and the adjacent pericentric cohesin. This confined localization of Sgo1p requires an intact tension sensing motif of histone H3. Ectopic transcription that disrupts pericentric cohesin localization also dislodges Sgo1p in situ. Overexpression causes Sgo1p to expand its presence, but the new Sgo1p peaks have high propensity to co-localize with cohesin. This unique trident shape of Sgo1p domain on each chromosome appears to be associated with chromatin looping in mitosis, thus linking higher-ordered chromatin architecture to positioning Sgo1p for the crucial tension sensing function of segregation. Studies of yeast and human cells have demonstrated the importance of cohesin in Sgo1p recruitment to pericentromere (13, 17). However, cohesin alone is not sufficient for the pericentric retention of Sgo1p. The tension sensing motif of H3 is also required for keeping Sgo1p in this region to ensure error-free segregation. While a Gly- 123 to-Ser mutation in the TSM has no effect on cohesin localization, both pericentric and centromeric (though to a lesser extent) enrichment of Sgo1p is compromised ((22) and Figure 3-4C). The establishment of the centromeric and pericentric domain of Sgo1p likely follows a spillover model in that Sgo1p is first recruited to the centromeres via direct association with Cse4p (18) and histone H2A phosphorylated at Ser121 by kinase Bub1p (12, 16). Congregation of Sgo1p molecules at centromeres permits its spread to the adjacent pericentric nucleosomes where cohesin has already been loaded. This spread may result from the turnover of a transient complex involving Sgo1p and centromeric proteins. Alternatively, the homodimerization activity of Sgo1p, evidenced by yeast two-hybrid tests (18), may facilitate the growth of the Sgo1p domain from centromeres to pericentric regions where the cohesin complex resides. By binding to nucleosomes, cohesin may also help to make the tension sensing motif more accessible for Sgo1p before biorientation is established (12, 16, 22, 23). Due possibly to the total pool size of Sgo1p, it only spreads to the first and nearest cohesin cluster. Overexpression of Sgo1p can further its spread primarily to adjacent pre-existing cohesin conglomerates (Figure 3-3). The distinct kinetics of engaging Sgo1p and cohesin (Mcd1p) at CEN16 (Figure 3-9) and CEN1 (Figure 3-10) is consistent with the notion that cohesin organizes chromatin into a platform for mitotic machinery to execute error-free segregation. Mcd1p appears earlier than Sgo1p but fluctuates in abundance before metaphase. In the meantime, Sgo1p continues to accumulate at CEN and PC peaks until it reaches the maximum. When cells enter anaphase, Sgo1p dissipates. It is critical that before Mcd1p levels climb to the highest, Sgo1p already starts disappearing from CEN and PC 124 regions (compare T60’ and T75’, Figure 3-9 and Figure 3-10). This time difference echoes the report of tension-dependent removal of Sgo1p from chromatin at the juncture of metaphase and anaphase (25), and is consistent with the model that the removal of Sgo1p from chromatin is registered by cells as achieving biorientation. The centromeric and pericentric clusters of Sgo1p appear almost simultaneously, leaving the intervening relatively free of Sgo1p throughout the lifespan of these peaks. Given that the histone H3 tension sensing motif decorates the whole genome and functions without a post-translational modification, the non-continuous nature of the confined Sgo1p peaks on each chromosome strongly suggests physical hindrance in these Sgo1p-free intervening sections. Our recent findings that Gcn5p acts as a negative regulator for tension sensing motif and Sgo1p functional interaction (15, 23) alludes to an intriguing possibility that Gcn5p, acetylated H3, or a downstream effector may prevent Sgo1p from binding to the chromosome arms. ChIP-seq data show a lack of correlation between Gcn5p and these Sgo1p-free valleys in mitosis (Figure 3-4), arguing against a direct, physical role of Gcn5p. Rather, we favor the possibility that a structural feature dictates the accessibility of pericentric chromatin to Sgo1p. Indeed, the chromosome conformation capture results (Figure 3-11) show that the DNA around the centromere loops into a higher-ordered structure that includes centromere and the adjacent Sgo1p and cohesin clusters, a scenario reminiscent of the C-loop model put forth by Bloom and colleagues (7, 27). The C-loop conformation posits that pericentric chromatin harbors alternating cohesin and condensin complex clusters. Condensin and the associated chromatin in pericentromere are restricted to the microtubule axis between spindle pole bodies, whereas cohesin and the cognate CARs are radially 125 positioned, forming the wall of a barrel. In this model, multiple layers of chromatin loops distribute axially, with the top and bottom of this barrel being the clustered centromeres from all 16 chromosomes. Poleward pulling from biorientation stretches the length of this barrel and narrows its diameter. How does Sgo1p fit into the tension sensing function? Taking together the ChIP- seq and 3C results, we suggest that cohesin is responsible for creating and joining multiple loops in pericentromere. With centromeres clustering in the center (38), these cohesin-capped loops (Figure 3-12A) can be viewed as a series of concentric circles (Figure 3-12B). Sgo1p is recruited to the centromere cluster, from which it encroaches radially to the first pericentric cohesin circle (red circles, Figure 3-12B). Biorientation instigates both intra- and inter-chromosomal tension (7). The increased space between individual nucleosomes causes a conformational change of the tension sensing motif (22, 23) or even nucleosome dissociation from pericentromere (39). In either case, Sgo1p loses its footings and dissipates from chromatin (Figure 3-12B, green circles). Tension-induced clearance of Sgo1p in pericentromere signals biorientation to the spindle assembly checkpoint (25). Anaphase thus ensues. This model provides a mechanistic explanation for the mitotic delay caused by Sgo1p overexpression (40). Biochemical fractionation experiments demonstrated that yeast cells do not seem to have a soluble pool of Sgo1p, but rather keep all Sgo1p molecules in the CEN/PC region (15). If true, the overall size of the Sgo1p motif on chromosomes (red circles, Figure 3-12B) would be dictated by the number of Sgo1p molecules. Overexpression raises Sgo1p levels and expands the range of Sgo1p occupancy to the next cohesin circle farther from the centromere cluster. Consequently, more extended axial 126 separation of kinetochores is required in order to evict the outermost Sgo1p molecules. Assuming that the quantitative removal of Sgo1p from centromeric and pericentric regions signals biorientation, Sgo1p overdose would require more time to clear Sgo1p before anaphase onset, resulting in mitotic delay. On the contrary, deleting Sgo1p or preventing the formation of the pericentric Sgo1p domain by mutating the tension sensing motif would be interpreted erroneously as biorientation by cells, thus triggering precocious anaphase onset and aneuploidy (10, 22). 127 Figure 3-12. Model for the formation and dynamics of Sgo1p chromatin domain. A. Sgo1p is first recruited to the centromeres via association with phosphorylated histone H2A (Pi). Centromere-bound Sgo1p then spreads to the nearby cohesin- occupied region. B. At the whole genome level, congregation of centromeres aligns the adjacent chromatin loops to form concentric rings (gradient yellow circle) that become the two terminals of the chromatin column. Prior to biorientation, Sgo1p (gradient red circle) resides on the centromere cluster and the first ring of chromatin loops. Poleward pulling from bipolar attachment stretches the centromeric and pericentric chromatin, resulting in a conformational change (gradient green circle) and evicting Sgo1p. 128 MATERIALS AND METHODS Yeast strains and plasmid constructs The yeast strains, plasmids, and primers used in this work are listed in Tables 3- 1 to 3-3. To study the genome wide localization of Sgo1p, the 6HA epitope-tagged Sgo1p strains, yJL345 (H3WT) and yJL346 (H3G44S) were constructed as previous described (22). The Sgo1p overexpression strains, yJL322 (H3WT) and yJL324 (H3G44S) were generated by transforming pJL51 (a URA3 plasmid with pADH1-3HA-SGO1-tADH1) into yMK1361 and yJL170, whose endogenous SGO1 gene was deleted using TRP1 marker. To ChIP Mcd1p, a 13Myc tag was introduced to the C terminus of MCD1 locus in yJL347 using pFA6a-13Myc-His3MX6 plasmid as described (41). The resultant strain yXD225 was transformed with either pMK439H3WT or pMK439H3G44S (a LEU2 plasmid bearing all four core histone genes) and followed by 5-FOA selection to select against pMK440 (a URA3 plasmid bearing all four core histone genes) containing cells, generating yXD233 (H3WT) and yXD234 (H3G44S). BAR1 was deleted in yXD233 and yXD234 to yield yXD237 and yXD238 respectively, using homologous recombination approach with URA3 marker. Another version of bar1 deletion was made in yXD233 to yield yXD282, using URA3 recycling approach as described previously (42). An adapted URA3 recycling method was used to replace the CAR sequence between RAD57 and MAF1 with GAL1 promoter. There were 4 steps PCR to attain the recombinant fragment. Step 1, primers oXD236 and oXD237 were used to amplify 3’ end of RAD57 from genomic DNA. Step 2, amplified pGAL1 from plasmid pFA6a-TRP1-pGAL1-3HA with primers oXD252 and oXD253. Step 3, PCR the URA3 from plasmid pMK440 using 129 primers oXD254, oXD255 and oXD240. Step 4, combined PCR products from the previous three steps and used primers oXD236 and oXD240 to amplify the final fragment. The resultant DNA was transformed into yXD282 to attain Ura+ transformant, which was then subjected to 5-FOA selection to generate yXD286. Yeast methods Yeast growth media, conditions, and transformation were based on standard procedures (43). When appropriate, 5% casamino acids (CAA) were used to substitute for synthetic amino acid mixtures as selective medium for uracil, tryptophan, or adenine prototroph. Yeast transformation was done with the lithium acetate method (44). ChIP-qPCR and ChIP-seq ChIP was conducted as previously described (22, 45). To quantify the ChIP results, ChIP DNAs were analyzed with quantitative PCR using primers from Table 3-3. The libraries of Sgo1p ChIP-seq were prepared as described previously (46). 10 ng of ChIP DNA was used for each library preparation. Size selection of libraries was 300-500 bp. Libraries passed quality control were then subjected to Illumina HiSeq 2500 to get 50 bp single-end reads. Reads were mapped to S. cerevisiae genome (Saccer 3.0) by Bowtie2 (version 2.2.6) using -m 1 setting for unique matching reads. BEDgraph files of each ChIP-seq experiments were generated by HOMER (version 4.7.2) and were visualized by Intergrative Genomics Viewer (Broad Institute). Read analysis across centromeres was done by using code of Cen-boxplot_100kb.pl adapted from Verzijlbergen et al. (2014). All ChIP-seq data in this study are available at the Gene Expression Omnibus with accession number GSE110953. 130 Table 3-1. Yeast strains used in this study Strain Relevant genotype yJL345 yJL346 yJL322 yJL324 yJL566 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S-HHF2] MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1:: sgo1∆::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1- htb1::Nat hta2-htb2::HPH pMK440 [ARS CEN URA3 HTA1- HTB1 HHT2-HHF2] pJL51 [2μm URA3 pADH1-3xHA-SGO1- tADH1] MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1:: sgo1∆::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1- htb1::Nat hta2-htb2::HPH pMK440 [ARS CEN URA3 HTA1- HTB1 hht2-G44S-HHF2] pJL51 [2μm URA3 pADH1-3xHA- SGO1-tADH1] MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S-HHF2] pMK144E173H [2μm URA3 pCUP1-gcn5E173H] Source or reference Luo et al, 2010 Luo et al, 2010 This study This study This study yMK1141 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK440 [ARS CEN URA3 HTA1-HTB1 HHT2- HHF2] Luo et al, 2010 yMK1361 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1:: This study sgo1∆::TRP1 ura3-1 hht1-hhf1::KAN hht2-hhf2::KAN hta1- htb1::Nat hta2-htb2::HPH pMK440 [ARS CEN URA3 HTA1- HTB1 HHT2-HHF2] yXD143 MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1:: bar1∆::URA3 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] This study 131 Table 3-1 (cont’d) yXD144 yXD237 yXD238 yXD286 This study This study MATa ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1- 1::SGO1-6HA::TRP1 ura3-1:: bar1∆::URA3 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S- HHF2] MATa ade2-1 can1-100 his3-11::MCD1-13MYC::HIS3, 15 leu2-3, 112 trp1-1::SGO1-6HA::TRP1 ura3-1:: bar1∆::URA3 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2- HHF2] MATa ade2-1 can1-100 his3-11::MCD1-13MYC::HIS3, 15 leu2-3, 112 trp1-1::SGO1-6HA::TRP1 ura3-1:: bar1∆::URA3 hht1-hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2- htb2::HPH pMK439G44S [ARS CEN LEU2 HTA1-HTB1 hht2-G44S-HHF2] MATa ade2-1 can1-100 bar1∆ his3-11::MCD1-13MYC::HIS3, 15 leu2-3, 112 trp1-1::SGO1-6HA::TRP1 ura3-1 hht1- hhf1::KAN hht2-hhf2::KAN hta1-htb1::Nat hta2-htb2::HPH pQQ18 [ARS CEN LEU2 HTA1-HTB1 HHT2-HHF2] MAF1- PGAL1 This study This study 132 Table 3-2. Plasmid constructs used in this study Plasmid Main features pQQ18/pMK439 pRS315-HTA1-HTB1 HHT2-HHF2 pJH33/pMK440 pRS316-HTA1-HTB1 HHT2-HHF2 pJL51 2μm URA3 pADH1-3xHA-SGO1-tADH1 pMK390/pFA6a- 13Myc-HIS3MX6 pMK389/pFA6a- TRP1-pGAL1-3HA 13Myc-tADH1-pTEF-HIS3-tTEF TRP1-pGAL1-3HA Source or reference Luo et al, 2010 Luo et al, 2010 Luo et al, 2016 Longtine et al, 1998 Longtine et al, 1998 133 Table 3-3. Oligos used in this study Name CEN1L5kb oXD159 CEN1R5kb CEN1R10kb CEN16L8kb oXD162 CEN16R3kb CEN1L20kb CEN1L9kb CEN1R15kb CEN16L50kb CEN16L15kb CEN16R12kb CEN16R47kb oXD236 oXD237 oXD242 oXD243 oXD244 oXD244 extender oXD245-02 oXD246 oXD252 oXD253 oXD254 oXD255 ChrI-Amp1 S ChrI-Amp1 AS ChrI-Amp2 S ChrI-Amp2 AS ChrI-Amp3 S ChrI-Amp3 AS ChrI-Amp4 S ChrI-Amp4 AS ChrI-Amp5 S ChrI-Amp5 AS ChrI-Amp6 S ChrI-Amp6 AS Sequence AAGAAGCATTTTTGGGATTG AAGAAACGTTGAACCTACTG CCTCACGCGCTCTAATCCTA GTAAGGTTGTCCGTATTGG CTATGATGAGGAACGTGCAA GGGATCAATCCCAATAGATG TCCTACGTCATGGAAAACG GGTTGACCCACAGAGGTTTG TCTCAAGCTGACGTCACTGTCT TGCTAAGGAGAAGGGCATCA AGTCATACGATGCATACGTTCC TCAGGTCATTTCTTCAACACG ATCAATTTTTCGCTCCAAGG ACAAGTGGGTGACGTTTTCC CATTATCCGTTGTGGTGGCT TCAGGCTGTTTCTATTCCTCGCTTAGTAAT CAATCAGAAGACCCAATGGC TATATGACCCTTCTAGACACTCTTTTTATT AATAAAAAGAGTGTCTAGAAGGGTCATATAGAAATCTGGAGTACAATTTC GAAATCTGGAGTACAATTTCTTTATAGCATATAAATATCAAATATATAGTCATTTTTAAT AATATATAGTCATTTTTAATACATGGAAAGCATAATAAAAGTGCACCACGCTTTTCAATT TTAATATGTTGGTTTACAGACATTTATTAAGACTGTAGAAGAAGCTCTAATTTGTGAGTT ATTACTAAGCGAGGAATAGAAACAGCCTGACTCCTTGACGTTAAAGTATAGAGG GTAATAGATGATAAATCAGATCAAGAAGAATCCCTACAGTAGCGGATTAGAAGCCGCC GA CTACTGTAGGGATTCTTCTTGATCTGATTTATCATCTATTACATTCTCATCATAAGTGAA ATTCTCATCATAAGTGAAATCGTATTCTCCTTCGTATTCAGTGCACCACGCTTTTCAATT CAAGAGCAAAAGGGAAATGAT CCAGAATTTGTAAGCTCTCAGC ACAGCGCCACCAAGATATG GCCAAGTTTTCGAGGCAAG AGGTCGAACATTTCTCACCA AGCCGTCCGATATATCCTCT AAACTTACGAATTCTTTCAACTGATT ATATACTTCTTTGACCAAACGGAAA CATCACCACGGACAGTCTTT GTGGGGTAGCATACCTGAGA GCAGTGCTGACATGCTGCT CGGAAGAGGCAGGTTAAAA 134 Table 3-3 (cont’d) ChrI-Amp7 S ChrI-Amp7 AS ChrI-Amp8 S ChrI-Amp8 AS ChrI-Amp9 S ChrI-Amp9 AS ChrI-Amp10 S ChrI-Amp10 AS ChrI-Amp11 S ChrI-Amp11 AS ChrI-Amp12 S ChrI-Amp12 AS CEN1 S CEN1 AS ChrI-Amp13 S ChrI-Amp13 AS ChrI-Amp14 S ChrI-Amp14 AS ChrI-Amp15 S ChrI-Amp15 AS ChrI-Amp16 S ChrI-Amp16 AS ChrI-Amp17 S ChrI-Amp17 AS ChrI-Amp18 S ChrI-Amp18 AS ChrI-Amp19 S ChrI-Amp19 AS ChrI-Amp20 S ChrI-Amp20 AS ChrI-Amp21 S ChrI-Amp21 AS ChrI-Amp22 S ChrI-Amp22 AS ChrI-Amp23 S ChrI-Amp23 AS ChrI-Amp24 S ChrI-Amp24 AS ChrIV-Amp1 S ChrIV-Amp1 AS TGCGAAAAAGCCTATACCC TTTCAGCGAGCTTTTACCA AATCAGTCGCATTGAAATTATCT TGTATCAGATAGCAAGGCAGCTT TGATCGTTTTACCACCTCAAC TGACGGGGTGGATAGTAATC CGAGAAGTAGTTCAAATGCAGA TGAGGACAGCCTATGGACATT TCCCACAGCTGATTCAAAG TCGGCTCCGTTTAGGTGA TGTTTATTTCACTTTCGCGACT GCATTTTCAAATACCGCTTG GCATAAGTGTGCCTTAGTATG GCGCTTGAAATGAAAGCTCCG TGTAGAAATGGCGCCAGAA AAAACACCCGAGGCAGCA CAACCACGCAATGAGTCTT TGGGGATATCTCAGAATGGA TGAACAAGGCGAAGAACCA AATCTTTGCTTGGCGCAGA TTGAGGCTTTCAAGTCCCTAT CGTATTGAGTTGGGCTATACG CCACTTGCTGAACCTTTCTG TGCGCAAGATTTTGGTGTC CTTCCCCTGGGGTTCAAG CAGCGAATGGATCCTGTAA AGAGCATAAGTACCAGGATGTGA AACCATGTGTAGAAGCGACTAAG TGCAGTCATCATAGGTTTCTCTT GGCATGACTATACCTCTTCAGTG CCTCACGCGCTCTAATCC AGGAAGAAGACCCCAACGA CACCTTTCTCGCTTCTTCC TCAATCGGCTGCTAGCTTA TCTTCCCAGCCCTTGAAGT AACGTAGACGTCCTCGGTATT TATCAGCACGCGACTGGA TCGCGTGATAATTGCAGA AAAATAGGCATTATAGATCAGTTCG CTTATTTACTGTAAAACTGTGACG 135 Table 3-3 (cont’d) ChrIV-Amp2 S ChrIV-Amp2 AS ChrIV-Amp3 S ChrIV-Amp3 AS ChrIV-Amp4 S ChrIV-Amp4 AS ChrIV-Amp5 S ChrIV-Amp5 AS ChrIV-Amp6 S ChrIV-Amp6 AS ChrIV-Amp7 S ChrIV-Amp7 AS ChrIV-Amp8 S ChrIV-Amp8 AS ChrIV-Amp9 S ChrIV-Amp9 AS ChrIV-Amp10 S ChrIV-Amp10 AS ChrIV-Amp11 S ChrIV-Amp11 AS ChrXVI-Amp1 S ChrXVI-Amp1 AS ChrXVI-Amp2 S ChrXVI-Amp2 AS ChrXVI-Amp3 S ChrXVI-Amp3 AS ChrXVI-Amp4 S ChrXVI-Amp4 AS ChrXVI-Amp5 S ChrXVI-Amp5 AS ChrXVI-Amp6 S ChrXVI-Amp6 AS ChrXVI-Amp7 S ChrXVI-Amp7 AS ChrXVI-Amp8 S ChrXVI-Amp8 AS ChrXVI-Amp9 S ChrXVI-Amp9 AS ChrXVI-Amp10 S ChrXVI-Amp10 AS GGCATTGACCCATCCAAA CCTCATCGTGTTGAAGCAG AGCATCGGGATTACTCCT GATCACAGCCCCCATTCTT GCGATCTTTTCAGGCCAAC GAAACTTGCGCGATCACC TTATCTACTGTGAGGAAAAGTTGCT ATGTGATTTCACAATGCTACATAAG CGACGTCCAGAACAGTCAA TCACCGTCCGTATAAATCG GTGTCCAACTTTCCGAAC TGGTAAATCGCCCTCAGTG TCAAGGCTTCAACAATAATTCA GTCCTCTTAACTTGCCAGA TATTGAAAAAGGACGCAATCA CAACCAGAGGAATGGGTCT TGCTCCCAAAAAGAGTTGC CTGCTCCCGAAACAAACC TTCCTCCAGCTCTGTTGC CCTTCTAAAACCTCCACCACC ACCGATTTCCCGTAAGACG TGCGGTTTCAAGTTGTTCC TTTGGGTTGTATCCCCACT GGGTAGGTTTGTGGGCTTC CCGGAATATTGGCCGAAC TCACCAGCTCGTGTCTACC AGCAGCCTTTAATTTTTCACG CTAGTTCGTGCCGAAGCTGAA TGTCGGTTACGTCGGAAA AGTTGTTGCCAGCGAACG TGATTGGACCCGCTTCTC GGCGGAAACTTTGTTCCTC GGAAAGTTTGGCTCGCAGTAA TCCTGACCTCCCCGTAAT TCCCTGGTTTGCGTTGATG ACCGGTGATCTCTCGTTGT TTGGTCCCTTAGTTCGACAG CTGGCAGCCTTTGACGAGT GGCCACGGATCCTGTCTT TGCTTCGTTTGGATCCTC 136 Table 3-3 (cont’d) CEN16 S CEN16 AS ChrXVI-Amp11 S ChrXVI-Amp11 AS ChrXVI-Amp12 S ChrXVI-Amp12 AS ChrXVI-Amp13 S ChrXVI-Amp13 AS ChrXVI-Amp14 S ChrXVI-Amp14 AS ChrXVI-Amp15 S ChrXVI-Amp15 AS ChrXVI-Amp16 S ChrXVI-Amp16 AS ChrXVI-Amp17 S ChrXVI-Amp17 AS ChrXVI-Amp18 S ChrXVI-Amp18 AS ChrXVI-Amp19 S ChrXVI-Amp19 AS ChrXVI-Amp20 S ChrXVI-Amp20 AS Tel06R S Tel06R AS CEN5L95kb S CEN5L95kb AS CEN1L100kb S CEN1L100kb AS CEN4R595kb S CEN4R595kb AS CEN16L465kb S CEN16L465kb AS ATGCAAAGGTTGAAGCCGTTA GCTACCATGGTGTGTCACT CGGACCGATTACCATTTCA ATGCCATGTTCGGGATCT ACCCTATGACCCAACTTGC CCATCTTTCCTATGAGGCCCTT TGGGATTAGCGGGTCCTT TCTCGTGCAATGCTGGAA CATGGGACCCTTGACACAAC GTGCTTCCAGACCCTCCA TGCGCTGAGTACAGCGTCT CGCGATGTCCTTCAAAAC GTACATGATCGCGGTTCC TTTCTTGCGACCTCATCC GTAGAGTACCCTGTTGGTCAC GCGCAGATGCCTTTGAAAT CAACTTGCAAGAGTTCGTCA TCTGGAGGTCCTGTGTTCG CAATATCATCACGTGCGGTCT CCAATCCTTGCAATTAGCTTCC TCATTGTTCGGGACGTTG ATAGCATTGATGCGGCTCT CAACTTCACGGATAACTTTTTCAAC AAACCGACAACGCTTGATCTAT CCCATCCGATACGAGCAT GGGAAGCCTGTGCGAAAT GGCCAAGCGAATAATACTCC AGCAAAGTCATTGCCAAACA AAGATGGTCCAGAGCCAAAT TTGACATGCTCTGACACAGG GCTGCCCATTCCTAGTAAACC GCTGAGGGCTCTTGCTTATC 137 Chromosome Conformation Capture, 3C 3C was performed in 100 OD600 cells of G1 or G2M arrest cells as previously described (47). Instead of using mortar and pestle to lyses cells, 50 U/mL lyticase was used to digest the cell wall for 25 min at room temperature. Primers are designed around 50 bp upstream of the targeted EcoR I sites. The digestion efficiency of each libraries was evaluated by qPCR. Samples with at least 70% digestion were carried on for following assay. PCR products were resolved by 9% PAGE and stained by ethidium bromide. The intensity of band was analyzed by NIH ImageJ. ACKNOWLEDGMENTS We are grateful for the technical assistance from Monique Floer, Alison Gjidoda, Mohita Tagore, Michael McAndrew, Kurtus Kok, and Sandhya Payankaulam. We thank Christopher Buehl for his critical reading of this manuscript and frequent discussion of the project. This work was supported the National Science Foundation (MCB1050132) and partly by National Institutes of Health (AG051820) to M.-H. Kuo. 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