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1!” WM“

IDENTIFICATION AND CHARACTERIZATION OF PROMOTER REGIONS OF
THE GENE FOR RAT TYPE I HEXOKINASE

By

Wenjing Liu

A DISSERTATION

Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1997

ABSTRACT

IDENTIFICATION AND CHARACTERIZATION OF PROMOTER REGIONS OF
THE GENE FOR RAT TYPE I HEXOKINASE

By

Wenjing Liu

The 5’-flanking region of the gene for rat type I hexolcinase has previously been
isolated from genomic clones and sequenced. 5’-RACE, RT-PCR and RNase protection assays
indicated that there are multiple transcriptional start sites clustered in three regions at positions
approximately 460, -300, and -100 relative to the translational start codon. These regions lack
classical TATA sequence and are located in a GC-rich segment, a “CpG island”. These
characteristics are frequently associated with “housekeeping genes”. The goal of this
dissertation is to identify the promoter regions of type I hexokinase gene and to characterize
important cis-elements and corresponding trans-factors regulating the promoter activity.

PC12 cells and H9c2 cells were transfected with luciferase reporter constructs
containing genomic sequence between positions -3366 and -171. Marked (85%) decrease in
promoter activity was associated with deletion of sequence between -742 and -516. In DNase I
footprinting experiments, two regions, called P1 (-5 52 to -529) and P2 (-480 to -458) boxes,
were protected by proteins present in nuclear extracts from PC 12 cells. The P2 box overlaps
with the most upstream cluster of transcriptional start sites, which is about 80 bp downstream

from the P1 box.

Mutations or deletions in the P2 box had no effect on promoter activity. In contrast,
mutations or deletions in the P1 box had markedly detrimental effects on promoter activity. A
second Spl site (-570), just upstream from the P1 box, was also shown to be fimctionally
important although not protected in footprinting experiments. F urtherrnore, the Pl box could
be fimctionally replaced by the ~57O Spl site.

Two DNA-protein complexes were observed in gel-shift experiments with P1 box
sequence and PC12 nuclear extract. Maintenance of a consensus Spl binding site centrally
located in the Pl box was critical for the formation of both complexes. Supershifi experiments
demonstrated the involvement of Spl, Sp3, and Sp4 in formation of these complexes, and
implicate these transcription factors in regulating promoter activity associated with this region.

Another series of reporter constructs, including sequence between -171 and - l,
permitted detection of an additional promoter activity downstream from -364. While not yet
extensively characterized, it is already evident that the cis-elements influencing the downstream
promoter activity are distinct from the Sp factors determined to be important in expression

from the upstream promoter region.

To my family

ACKNOWLEDGMENTS

I am grateful to my mentor, Dr. John E. Wilson for his guidance and
encouragement over the years. I learned so much from his discipline in science and
generosity in social life. I would like to express my deep appreciation to my committee
members, Dr. Paul Coussens, Dr. Don Jump, Dr. Steve Triezenberg and Dr. John Wang
for their criticisms and advice on my work. I also thank Dr. Zach Burton for his discussion
and attendance at my dissertation defense.

I would like to thank my colleagues, past and present Wilson lab. members. They
were always friendly and willing to help. Whether attending a scientific meeting or just a
lunch out together, I enjoyed their company and friendship. Special thank goes to Dr. Joe
White who taught me molecular biology techniques and cooperated with me at early stage
of my research project.

I feel extremely lucky to make friends with some Chinese women here in the
Department of Biochemistry at MSU. They are Yin Tang, Jie Qian, Yue Li and Tong Hao.
Because of our common cultural and academic background, we shared many things
personal and professional. I thank them for being always available when I need comfort
and support in difficult times.

I want to thank my parents, sister and brother back in China. Their unconditional
love and firm faith in me pushed me go this far. Last, but not least, I thank my husband,

Yijun Guo, for everything.

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... x
LIST OF FIGURES ........................................................................................................ xi
LIST OF ABBREVIATIONS ........................................................................................ xii
CHAPTER I INTRODUCTION ........................................................................ l
l. Isozymes of mammalian hexokinase ...................................................................... 2
Background and kinetic properties ............................................................ 2

cDNA sequences and evolution ................................................................. 4

Tissue distribution ..................................................................................... 7

Subcellular association .............................................................................. 8

2. Regulation of hexokinase gene expression and promoters of

hexokinase genes ................................................................................................ 10
Type I hexokinase ................................................................................... 11

Type II hexokinase ....................................................... , .......................... 14

Type IV hexokinase (Glucokinase, GK) .................................................. 17

Type III hexokinase ................................................................................ 20

3. Spl family of transcription factors ...................................................................... 20
Spl ......................................................................................................... 20

Sp3 and Sp4 ........................................................................................... 22

Roles of Spl in transcriptional regulation ................................................ 24

Roles of multiple Sp factors in transcriptional regulation ......................... 25

4. Thesis overview .................................................................................................. 26
CHAPTER II MATERIALS AND METHODS ................................................. 27
1. General methods .......................................................................................................... 28
2. Generation of promoter test constmcts ........................................................................ 28

vi

3. Transfection of PC12 and H9c2 cells with reporter constructs ................................... 3O
4. Reporter assay .................................................................................................... 31
S. Non-radioactive labeling of RNA probe and RNase

protection assay .................................................................................................. 31
6. Preparation of nuclear extracts from PC 12 cells .......................................................... 32
7. Electrophoretic mobility shift (" gel shift") and "supershifi"

experiments .................................................................................................................. 33
8. DNase I footprinting .................................................................................................... 33
9. Mutation of promoter reporter constructs ................................................................... 34
10. Hexokinase assay and western blot ..................................................................... 35

CHAPTER III RESULTS ................................................................................... 37

1. Promoter activity associated with Sac I fi'agment in

HKI upstream region .......................................................................................... 38
2. Sequence between -742 and -516 is important for

promoter activity .......................................................................................................... 42
3. DNase I footprinting reveals two protected regions ..................................................... 45
4. The P1 box and Spl site at -570 are functionally important

cis-elements for promoter activity but the P2 box is not .............................................. 50
5. Gel shift and supershift experiments demonstrate binding

of Sp family members to the Pl box ............................................................................ 57
6. Effect of overexpression of Sp transcription factors on

HKI expression in PC 12 cells ...................................................................................... 69
7. Reporter constructs including sequence between positions

-171 and +1 reveal another region with promoter activity ........................................... 69
8. Effect of downstream sequence (+1 to +77) on promoter

activity .......................................................................................................................... 76

vii

CHAPTER IV DISCUSSION ............................................................................. 79

1. The upstream promoter: P1 box and -570 Spl site ...................................................... 80
2. Multiple members of Sp family of transcription factors
regulate the activity of upstream promoter ................................................................... 81
3. The P2 box and transcription initiation ......................................................................... 82
4. The downstream promoter ........................................................................................... 83
5. Efl‘ect of +1 to +77 on the promoter activity ............................................................... 84
CHAPTER V FUTURE WORK ........................................................................ 85
1. To investigate roles of Sp factors in regulation of the
upstream promoter - Transfection into Dr050phila SL2 cells ............................... 86
2. To test downstream promoter activity in H9c2 cells ............................................ 86
3. To identify cis-elements in the downstream promoter .......................................... 87
4. To study transcriptional regulation of hexokinase I expression ............................ 87
5. To define transcriptional start site and promoter usage in
different tissues and cell lines .............................................................................. 88
LIST OF REFERENCES ............................................................................................... 9O
APPENDICES
APPENDIX A
DNA SEQUENCE AROUND HKI PROMOTER REGION ............................. 100
APPENDIX B
DNA SEQUENCE OF HKI UPSTREAM REGION ......................................... 101
APPENDIX C
SCHEMATIC REPRESENTATION OF RESTRICTION
SITES IN HKI UPSTREAM REGION ............................................................ 104

viii

APPENDIX D
LIST OF RESTRICTION SITES IN HKI UPSTREAM REGION ................... 109

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

LIST OF TABLES

Molecular weights and kinetic parameters of mammalian
hexokinases .............................................................................................. 3

Comparison of amino acid sequences of N- and C-
terminal halves of rat Type I, II and III hexokinases,

glucokinase, and yeast hexokinase A ......................................................... 5

Expression of luciferase in PC12 cells transfected with

reporter constructs .................................................................................. 41
Potential cis-elements in P1 box and P2 box sequences .............................. 51
Primers used for generation of substitution or deletion mutants ...................... 52
P2 box mutation data .............................................................................. 56
DNA Fragments Used in Gel shift Experiments ............................................. 58

Hexokinase activity in PC 12 cells overexpressing Sp

transcription factors ................................................................................ 72
Effect of downstream sequence on promoter activity ............................... 77

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

LIST OF FIGURES

Partial Restriction map of HKI upstream region ..................................... 39

Transfection of PC 12 and H9c2 cells with promoter-
reporter constructs ......................................................................................... 43

RNase Protection Assay .................................................................................. 46

DNase I footprinting analysis with a nuclear extract from
PC12 cells ....................................................................................................... 48

Effect of mutations in the P1 box and upstream (-570) Spl
site on promoter activity .................................................................................. 54

Gel shifi experiment showing complexes formed by proteins
in nuclear extracts from PC12 cells and an oligonucleotide
including the P1 box sequence ......................................................................... 59

Gel shift experiment showing the effect of mutations in the Spl
binding site within the P1 box sequence ......................................................... 62

Supershifi experiment showing involvement of Spl, Sp3,
and Sp4 in complexes formed with nuclear extracts from

PC12 cells ....................................................................................................... 65
Summary of gel-shift and super-shift experiments .......................................... 67
Overexpression of Spl, Sp3 and Sp4 in PC12 cells ......................................... 70

Transfection of PC12 cells with promoter-reporter constructs
including sequence between -l7l and -1 ........................................................ 74

xi

Glc-6-P

KDa

GK
RACE
PCR
RT-PCR
bp

Kb

nt

LIST OF ABBREVIATIONS

glucose-6-phosphate

kilodalton

molecular weight

hexokinase

glucokinase

rapid amplification of cDNA ends
polymerase chain reaction

reverse transcription followed by PCR
base pair(s)

kilobase pair(s)

nucleotide(s)

hour(s)

minute(s)

specificity protein

simian virus 40

cytomegalovirus

microgram(s)

microlitre(s)

xii

CAMP

dig
Hepes
PBS
EDTA
PMSF

DTT

SDS
Fig.
SD

mu

mg

luc

cyclic AMP

untranslated region
orthophosphate

digoxigenin
N—(2-hydroxyethyl)-1-piperazineethanesulfonic acid
phosphate buffered saline
(ethylenedinitrilo) tetraacetic acid
phenylmethylsulfonyl fluoride
dithiothreitol

count(s) per minute

volt(s)

sodium dodecyl sulfate

figure

standard deviation

milliunit(s)

milligram(s)

B-galactosidase

luciferase

xiii

CHAPTER I

INTRODUCTION

1. Isozymes of Mammalian Hexokinase

Backgmncgnd kinetic properties

Hexokinase (ATP: D-hexose 6 phosphotransferase, EC 2.7.1.1) catalyzes the
phosphorylation of glucose, using MgziATP as phosphoryl donor. The product, glucose-
6-phosphate, is a common substrate for phosphohexoisomerase, phosphoglucomutase, and
Glc-6-P dehydrogenase, which introduce Glc-6-P into glycolysis, glycogen synthesis and
the pentose monophosphate pathway, respectively.

Four distinct isozymes of hexokinase exist in mammalian tissues. They are
designated as type I, II, III and IV isozymes, based on their order of electrophoretic
mobility on starch gels (1). Alternatively, they can be separated by chromatographic
technique, and named isozymes A-D according to their order of elution from DEAE-
cellulose columns (2).

Although catalyzing the same reaction, the four isozymes can be distinguished from
one another by their different molecular weights and kinetic properties. As summarized in
Table 1, type I-III isozymes have molecular weights of about 100 kDa, low Kms for
glucose, in the submillimolar range, and are therefore often referred to as the “low K...”
isozymes. On the other hand, type IV isozyme (EC 2.7.1.2, commonly called glucokinase),
has a molecular weight of about 50 kDa and a much higher K... for glucose (4.5mM). All
four isozymes have similar Kms for ATP. The “low Km” isozymes are sensitive to

inhibition by their reaction product Glc-6-P at physiologically relevant concentrations, but

Table 1. Molecular weights and kinetic parameters of mammalian hexokinases

 

HEXOKINASE ISOZYME
PARAMETER I 11 111 IV
M.(kDa) 102.3 102.6 100.3 49
K... Glc (mM) 004 0.13 0.02 4.5
K... ATP (mM) 0.42 0.70 1.29 0.49
K; Glc-6-P vs. ATP (mM) 0.026 0.021 0.074 15

 

This table was adapted fiom Ureta (3), and the references therein. Indicated molecular

weights are based on amino acid sequences deduced from cloned cDNAs (4-9).

type IV is not (3).

cDNA seqyencewd evolution

In recent years, the cDNAs coding for all four isozymes have been cloned from rat
(4-9), and the respective amino acid sequences have been deduced. Additionally, cDNAs
coding for hexokinases and glucokinase from many other organisms have also been
cloned. By comparing amino acid sequences deduced from cDNAs, rat hexokinase I, II
and III are very similar. Table 2 shows the extensive sequence similarities of N- and C-
terminal halves of type I, II and III hexokinases, to one another, and to glucokinase (9)
and yeast hexokinase A (10).

Based on the fact that the molecular weight of mammalian hexokinase I-III is
twice that of yeast hexokinase and glucokinase, it has been proposed by many researchers
(3, 11-15) that mammalian 100 kDa hexokinases have evolved by gene duplication and
fusion of an ancestral 50 kDa hexokinase which is similar to present-day yeast hexokinase
and glucokinase. It has been suggested that one of the duplicated catalytic sites retained
the catalytic function, while the other evolved to acquire a regulatory role.

This theory is strongly supported by the internal repetition of amino acid sequence
between N- and C-temrinal halves of 100 kDa hexokinases, as well as the sequence
similarities among N- and C- terminal halves of hexokinases I-III, glucokinase and yeast

hexokinase. This gene duplication and fusion theory is further supported by the same

 

Table 2. Comparison of amino acid sequences of N- and C- terminal halves of
rat Type I, II and III hexokinases, glucokinase, and yeast hexokinase
A
NI NII NIII C1 C11 CIII
(1-475) (1-475) (1-488) (476-918) (476-917) (489-924)
N11 68 (14)
NIII 39 (16) 44 (14)
CI 46 (17) 54 (14) 38 (14)
C11 49 (17) 55 (14) 41 (13) 76 (11)
C111 45 (15) 48 (15) 40 (14) 62 (11) 66(9)
Iv 46 (18) 52 (15) 38 (15) 49 (15) 53 (14) 49 (15)
YHKA 27 (14) 22 (13) 27(15) 27(13)

 

a- This table was adapted from Schwab and Wilson (6), and Thelen and Wilson (7).

b- Abbreviations used: NI, N-terminal half of rat Type I isozyme; NII, N-terminal half
of rat Type II isozyme; NIII, N-terminal half of rat Type III isozyme; Cl, C-
terrninal half of rat Type I isozyme; CII, C-terrninal half of rat Type II isozyme;
CIII, C-terminal half of rat Type III isozyme; IV, rat Type IV hexokinase

(glucokinase); YHKA, yeast hexokinase A.

c- Percentage of identical residues is shown without parenthesis; Percentage of
conservative substitutions is shown in parenthesis.

intron-exon structure among hexokinases. The splicing sites and the exon sizes of type II
(16) and type I (17) hexokinase genes repeat directly between the N- and C-terminal
halves. The same splicing pattern is also observed in the gene for 50 kDa glucokinase (18),
and thus suggested that an ancestral 50 kDa hexokinase gene similar to glucokinase
underwent gene duplication and fusion to form the hexokinase I and 11 genes.

The gene duplication and fusion theory underwent some modification as more
information became available. White and Wilson (19) were able to digest hexokinase I into
a 52 kDa N-terminal fragment and a 48 kDa C-terminal domain. The N-terminal domain
was selectively protected by Glc-6-P from denaturation in guanidine hydrochloride (19);
the C-terminal half was protected by a glucose analog, N-acetylglucosamine (20). The
isolated C-terminal half of the enzyme retained the catalytic activity (20). Thus, they
concluded that the binding site for Glc-6-P resides in the N-terminal half of the intact
enzyme and is separate from the catalytic site which is associated with the C-terminal half.
Further study demonstrated, surprisingly, that the isolated C-terminal half of the enzyme is
inhibited by Glc-6-P and that both halves of the enzyme possessed binding sites for the
inhibitor Glc-6-P as well as the substrate glucose and ATP (19). This led to a modification
of gene duplication and fusion theory such as that the ancestral 50 kDa hexokinase would
have had both the glucose binding site and the Glc-6 P regulatory site before gene
duplication and fusion occurred.

Direct measurement of ligand binding on the intact hexokinase I enzyme showed

only one binding site for glucose (21, 22) and one for Glc-6-P (21, 23). Therefore, it was

suggested that the glucose site in the N-terminal half and the Glc-6-P site in the C-
terrninal half were masked in the intact enzyme molecule.

Studies using chimeric hexokinases consisting of the N-tenninal half of one
isozyme and the C-terminal half of another confirmed that catalytic activity is associated
with the C-terminal halves and regulatory function is coupled to the N-terminal halves of
the type I and type III isozymes (24, 25). However, in the type II isozyme, both halves
possess catalytic activities and both halves are sensitive to Glc-6-P inhibition (26). Thus,
type II hexokinase gene is suggested to be the immediate product of gene duplication and
fusion, which subsequently evolved into type I and III, in which N- and C-terminal halves

are fimctionally differentiated.

Tissue distribution

 

Each of the four isozymes has its distinct tissue distribution (reviewed in references
27, 28). Generally, more than one isozyme is found in most tissues. Type 1 isozyme is
present in virtually all tissues examined to date, at relatively high levels in most tissues
except for liver. In fact, in those tissues with a heavy reliance on blood-borne glucose, the
type I isozyme is the predominant form. Brain is totally dependent on blood-bome
glucose, through glycolysis, to provide energy and it contains exclusively the type I
isozyme. It is also the case with erythrocytes. Therefore, type I isozyme has been referred
to as' the “basic” hexokinase and is suggested to play an important role in introducing

glucose into glycolysis (27, 28) .

Type II isozyme, on the other hand, is the major form in insulin-sensitive tissues
such as skeletal muscle, diaphragm, adipose tissue, and mammary gland (27, 28). There is
an apparent relationship between the predominance of the type II hexokinase and insulin
sensitivity of the tissue. It has been speculated that type II isozyme is responsible for
directing glucose into energy storage forms such as lipids (in adipose or mammary tissue)
or glycogen (in skeletal muscle, diaphragm) (27, 28).

Type III, the least studied isozyme, has not been found to be the predominant form
in any tissue. The tissues which show the highest amount of activity attributable to type III
hexokinase are liver, spleen, and lung (27, 29). Type IV, or glucokinase, is known to be
present in the B -cells of the pancreas and in liver (3 0-3 2). Because its K... for glucose is in
the range of normal blood glucose levels, fluctuation of blood glucose greatly regulates its
activity. This in turn makes glucokinase a key enzyme to convert excess blood glucose
into glycogen in liver and to serve as a “glucose sensor” governing the release of insulin

in the pancreatic B-islets (31).

Subcellular association

Intracellular distribution of hexokinases is not homogeneous. The association of
hexokinases with particulate fractions of tissue homogenates has been well documented in
a number of tissues (27, 28). A large portion of hexokinase I and II has been found to be
associated with mitochondria in tissues such as brain, heart, diaphragm, skeletal muscle

and mammary gland (27, 33, 34).

Type I hexokinase is believed to be bound to the outer mitochondrial membrane
via both hydrophobic and electrostatic interactions between the N-terminal half of the
enzyme and moieties on or in the membrane. The electrostatic interaction between the
negative charge on the hexokinase surface and presumably mitochondrial membrane
phospholipids is bridged by divalent cations such as Mg” (35). The hydrophobic
interaction is dependent on a small hydrophobic N-terminal segment of type I isozyme
which targets hexokinase I to mitochondria. The importance of this hydrophobic segment
has been confirmed by several studies. First, cleavage of a 9 residue peptide from the N-
terrninus of type I hexokinase with chymotrypsin prevented this enzyme from binding to
mitochondria (36). Second, this essential N-terminal hydrophobic region of the intact
enzyme is inserted into the core of the lipid bilayer when hexokinase is bound to
mitochondria (3 7). Recently, another approach was employed to show that the chimeric
construct of the first 15 amino acid residues of hexokinase I coupled to reporter protein,
chloramphenicol acetyltransferase (CAT), was able to bind to liver and hepatoma
mitochondria, while the native CAT was not (38). Similar study with Green Fluorescence
Protein (GFP) as reporter protein showed that N-terminal fragment of type I or II
hexokinase was able to target the reporter protein to mitochondria (3 9).

The outer mitochondrial membrane protein to which type I hexokinase binds was
originally isolated as the “hexokinase binding protein” (HBP) (40), but is now known to
be identical with the pore forming protein (porin) (41, 42), through which molecules such
as ADP and ATP flow. This association between hexokinase and porin gives the enzyme

preferential access to rnitochondrially generated ATP (43, 44). Together with the fact that

10

mitochondrially bound hexokinase has slightly greater aflinity for ATP and considerably
less sensitivity to G-6-P inhibition (45-47), the binding of hexokinase to mitochondria
represents a mechanism for activation of the enzyme.

' Hexokinase III was thought to be “soluble” and hence cytoplasmic in location
(30), but recently it was found to be weakly associated with the nuclear periphery. This
was demonstrated via confocal microscopy after staining the isozyme through the use of a
monoclonal antibody (29).

It is generally accepted that hepatic type IV hexokinase is located in the cytosol

(48). However, Miwa et al. showed both nuclear and cytoplasmic localization of
glucokinase in liver (49) and translocation of glucokinase during fasting-refeeding (50)
and postnatal development (51). Both the type III and type IV isozymes lack the

hydrophobic N-terminal fragment critical for mitochondrial binding.

2. Regulation of Hexokinase Gene Expression and Promoters of Hexokinase Genes

Mammalian hexokinase is the key enzyme committing glucose into cellular
metabolic pathways, and it is not surprising that this enzyme is under complex regulation.
In short term, hexokinase activity can be regulated in response to altered metabolic status
via product Glc-6-P inhibition (for type I-III), antagonism (for type I) or supplementation
(for type II and III) of this inhibition by P5, substrate glucose inhibition (for type III) and
altered interaction with mitochondria (reviewed in 28). In the long term, levels of

hexokinase protein change during development, in response to changes in hormone and

ll

nutritional status, or under chronic alteration in metabolic status. In principle, these long-
term changes in level of the enzyme could be the results of transcriptional regulation,
posttranscriptional regulation (e. g., mRNA stability or translational rate) or

posttranslational modification (e. g., phosphorylation or glycosylation) (28).

Type I hexokinase

Type I isozyme is ubiquitously expressed in mammalian tissues and appears to play a
general role in mammalian glucose metabolism. Thus, type I hexokinase may be considered a
”housekeeping enzyme". The type I isozyme is found at particularly high levels in brain (52),
consistent with the importance of glycolytic metabolism of glucose for sustaining a highly
active energy metabolism in this tissue (53). However, distribution of hexokinase activity
shows marked variations in difl‘erent brain regions (54) and in different layers of retina (55) and
cerebellum (56). Subcellular fractionation suggested that major hexokinase activity is located in
the nerve endings (57). The variation in hexokinase activity in different neural structural
elements, measured histochemically, are correlated with the amount of the enzyme protein
itself, measured by irnmunofluorescent procedure (58). This is consistent with the fact that no
posttranslational modification has been found to affect the specific activity of hexokinasel
(28). In situ hybridization with hexokinase I-specific oligonucleotide probe also demonstrated
extensive neuronal distribution of hexokinase I mRNA with regional differences in the

expression pattern (59).

12

Changes in hexokinase levels during development have been particularly well studied in
brain. Rat brain hexokinase levels are relatively low prenatally, and increase several fold within
the first three weeks postnatally to attain the adult levels (54, 60). Developmental increases in
hexokinase activity in neural tissues are correlated with the amount of the protein found in
those tissues, measured by irnmunofluorescent staining (61, 62). Griffin et al. examined the
levels of mRNA for type I hexokinase (relative to that for phosphoglycerate kinase, PGK) in
developing brain and other tissues of rat (63). They found that mRN A levels for type I
hexokinase in brain were relatively high during all developmental stages, compared to those in
other tissues. The relative level of hexokinase I mRN A increased to a maximum at about one
week postnatally before declining to adult levels by about 3-4 weeks postnatally. However,
close examination of the data revealed that the mRNA levels of PGK were not constant during
development, so the absolute levels of HKI mRNA actually peaked at 2 to 4 weeks postnatally.
Nevertheless, based on the lack of correlation between relative levels of mRNA and activity of
hexokinase in brain and other tissues during development, it is proposed that both
transcriptional and posttranscriptional regulation processes are involved in developmental
stage- and tissue- specific regulation of hexokinase I (63).

Yokomori et al. reported that levels of mRNA for type I hexokinase were increased 2.5
fold in response to treatment of cultured rat thyroid FRTLS cells with thyroid stimulating
hormone (T SH) (64). This effect was due to an increase in the rate of gene transcription,
shown in nuclear run-on transcriptional assays, but not due to the change in RNA stability.
(Bu)chMP and forskolin had a similar effect, suggesting that TSH stimulates hexokinase gene

expression via the CAMP-dependent pathway (64). Thyroid hormone is known to have a major

l3

influence on development of brain (65). Hypothyroidism delays the normally observed
postnatal increase in hexokinase activity, whereas hyperthyroidism accelerates the increase
(62).

Using quantitative immunofluorescence techniques, the levels of type I hexokinase (66,
67) in various regions of rat brain have been correlated with previously reported basal rates of
glucose utilization in these regions (68, 69), except for several regions (referred to as group II)
in which hexokinase content exceeded that expected from basal glucose utilization. It is
suggested that group H regions may be adapted to sustain a large range of changes in glucose
utilization rates. Hexokinase activity in various brain regions has been reported to be influenced
by several physiological perturbations which cause persistently altered metabolic activity,
including water deprivation (70), hypertension (71, 72), streptozotocin-induced diabetes (73 ),
and surgically-induced heart failure (74).

The 5’-flanking region for the rat type I hexokinase gene has been studied in our
laboratory (75, 76 and this thesis). My early work in cooperation with Dr. White was focused
on isolation of the promoter region and identification of transcriptional start sites. A genomic
clone containing sequence identical to the 5’ region of the cDNA for rat type I hexokinase was
isolated. A 5.4-kb EcoR I fi'agment from this clone, containing the matching sequence, was
sequenced in its entirety (75). 5’-RACE, RT-PCR and RNase protection assays indicated that
there are multiple transcriptional start sites clustered in three regions at positions approximately
-460, -300, and -100 relative to the translational start codon (75). These regions lack classical
TATA sequences and are located in a GC-rich segment, a "CpG island" (77, 78),

approximately 1 kb in length. These characteristics are frequently associated with

l4

“housekeeping genes". In this thesis, the promoter region for type I hexokinase is analyzed;
important cis-elements and corresponding trans-factors regulating promoter activity are

identified.

Type II hexokinase

Type II hexokinase is distributed in insulin-sensitive tissues such as skeletal
muscle, diaphragm, adipose tissue, and mammary gland (27). It has long been known that
the levels of type H hexokinase are regulated by insulin, with decreased type II hexokinase
observed in insulin-sensitive tissues of diabetic animals (33, 79). Frank and Fromm
reported that the rate of hexokinase II degradation increases by a factor of 3 in the skeletal
muscle of diabetic rats as compared with that of normal animals (80). Furthermore, the
relative rate of synthesis of hexokinase II is approximately 1.9 times higher in the normal
than in the diabetic rat (81). Insulin treatment of diabetic animals restores the degradation
and synthesis of hexokinase II to normal levels (80, 81). Printz et al. (16) reported that
hexokinase H mRNA was decreased in adipose tissue from diabetic rats, but was restored
by insulin treatment. Insulin also induced hexokinase II mRNA in adipose and skeletal
muscle cell lines. In one of the skeletal muscle cell lines, the increase in mRN A is
accounted for by a corresponding increase of gene transcription (16).

The activity of the type II hexokinase in muscle is regulated by contractile activity.
Increases in HK activity have been demonstrated in exercising muscle (82) and muscles

subjected to chronic, low-frequency stimulation (83, 84). Up to a 14-fold increase in total

15

hexokinase activity and the hexokinase II isoform was observed in rat fast-twitch muscle
after 2 weeks of chronic, low-frequency stimulation. This increase in enzyme protein
content was related to an approximately 30 fold increase in protein synthesis rate (85, 86).
Cessation of stimulation resulted in a normalization of hexokinase activity associated with
decreased rate of synthesis of type II hexokinase (86). The same stimulation also evoked
an immediate increase in the ratio between structure (mitochondria)-bound and free
hexokinase (87), and presumably represents an early response to increased energy
demand. The observed transient increase in hexokinase II content represents an additional
increase in glucose phosphorylation capacity under these stimulation conditions. After
prolonged stimulation (3 weeks), hexokinase II activity declined, consistent with the
previously observed switch from a carbohydrate-based to a fatty-acid-based energy
metabolism (87). It was observed that under the same condition, hexokinase II mRNA
was elevated significantly after one hour of stimulation and 30-fold after 12 hour, and the
rate of HKII protein synthesis increased 20-fold after 24 hours (88). Another group also
reported an increase in the levels 'of mRN A for type II hexokinase after a single brief
period of exercise (89). Those experiments suggest that the increase in hexokinase II
activity in response to contractile activity is at least partly at the transcriptional level.

The 5’-flanking region of hexokinase II has been isolated from a rat liver genomic
library and analyzed in the rat skeletal muscle cell line, L6. The rate of hexokinase II gene
transcription in L6 cells is increased by insulin, catecholamine, and CAMP, resulting in
increased hexokinase II mRNA, protein synthesis, and glucose phosphorylation (16, 90,

91). The 5’ untranslated region of hexokinase II mRNA is 462 bp long. The basal

16

promoter consists of about 160 base pairs of S’-flanking sequence that includes a classical
TATA box, an inverted CCAAT box (referred to as a'Y box), a CCAAT box, and a
CAMP response element (CRE). The CCAAT box and the CRE are both involved in
CAMP responsiveness. The Y box contributes to basal promoter activity. Several known
transcription factors bind to these sequences, notably CREB and ATF-l to the CRE and
NF-Y to both the Y and the CCAAT boxes (91).

Tumor cells exhibit increased glycolytic rates, and increased levels of key enzymes
like hexokinase. The fast growing tumor cells have increased hexokinase activity (92) and
higher percentage of hexokinase associated with mitochondria (93). It has been shown
that various tumor cell lines have relatively high levels of mRNAs, particularly for the type
II isozyme (7, 94, 95). The 4.3 kb proximal promoter region of the hexokinase II gene has
been isolated and characterized in a rapidly growing hepatoma cell line, AS-30D (96). The
DNA sequence of this promoter region is the same as that of the hexokinase 11 promoter
in normal rat liver cells (91). However, in the AS-3OD cells transfected with promoter-
reporter construct, the promoter activity was enhanced by glucose, phorbol 12-myristate
l3-acetate (a phorbol ester), insulin, CAMP, and glucagon, whereas these same agents
produced little or no effect on promoter activity in transfected hepatocytes (96). The
differences in the transcriptional regulation of MCI between normal cells and tumor cells
suggested that transcription of the type 11 tumor gene may occur independent of metabolic
state. The HKII promoter also contains two functional p53 response elements (96a). The
highly abundant mutant form of p53, which lacks the ability to suppress or control cell

cycle progression as wild type p53 does, activted the HKII promoter, thus providing a

l7

linkage between the loss of cell cycle control and the high glycolytic rate in fast growing

cancer cells (96a).

Type I V hexokinase (Glucokinase. GK)

Type IV hexokinase, or glucokinase, is expressed in the B—CCllS of the pancreas and
in liver, and is important in glucose metabolism and homeostasis (30-3 2).

In liver, glucokinase is involved in the utilization of excess circulatory glucose. The
levels of glucokinase activity in rat liver vary with the nutritional status of the animal.
Hepatic glucokinase activity falls during fasting and is restored by glucose refeeding (97,
98). The glucokinase mRNA is undetectable in liver from rats fasted for 24-72 h. Oral
glucose administration causes a rapid, massive and transient accumulation of the hepatic
glucokinase mRNA (9). The hepatic glucokinase is also up-regulated by insulin. Both
enzyme protein and enzyme mRNA are absent from the livers of streptozotocin-induced
diabetic rats. Insulin treatment causes a prompt transient build-up of mRNA and enzyme,
resulting fi'om a burst in the transcriptional activity of the glucokinase gene, as evidenced
by run-on assays with isolated nuclei from liver (99) as well as from primary culture of rat
hepatocytes (100). On the other hand, cyclic AMP exerts dominant negative control over
glucokinase gene expression. Glucagon or derivatives of CAMP have suppressor effects on
induction by insulin; this effect is primarily at the transcriptional level (100).

In contrast, the major function of glucokinase in B-Cells is to “sense” the

Circulating glucose level and to allow flux through glycolysis which controls the synthesis

and secretion of insulin. Levels of islet glucokinase mRNA and protein are relatively
constant during the fasting-refeeding cycle (101). Hormones like insulin do not regulate
glucokinase mRNA or protein in islet cells (92). The major determinant of glucokinase
expression in islets is glucose (102). Transfection experiments suggested that glucose
phosphorylation by glucokinase is the rate limiting step of glucose catabolism in B-cells
and the key step for activation of the insulin promoter by glucose. The glycolytic
intermediates between fructose 1,6-diphosphate and phosphoenolpyruvate are essential for
B-cell glucose sensing (103).

Recent reports on the differences in glucokinase gene products in liver and
pancreatic B-cells provide a mechanism for tissue-specific regulation of this enzyme (101,
104; reviewed in 48, 105). Although glucokinase proteins in liver and pancreatic B-cells
display similar kinetic properties, they actually arise from alternative splicing of a single
gene. The first exon encoding the 5’ end of the hepatic mRN A is contiguous to the body
of the structural gene. But the first exon for the 5’ end of the insulinoma mRNA is more
than 12 kb further upstream. The tissue specific splicing specifies not only the difference in
5’ untranslated region of the islet and liver mRNA, but also their initial 15 amino acids
(104). Also, the usage of alternative promoters presumably allows different regulation of
glucokinase expression in two tissues, e.g., insulin regulation in liver and glucose
regulation in B-cells.

The hepatic glucokinase promoter, or downstream promoter, has been studied in
primary culture of rat hepatocytes (106). The sequence between -123 to -34 (relative to

the transcription start site) is the minimal promoter driving reporter expression in

l9

hepatocytes, as well as in insulinoma and in hepatoma cells, which do not express the
endogenous glucokinase gene. The fragment between -1003 to -707, however, is a
hepatocyte-specific enhancer, which. stimulates reporter expression in hepatocytes when
linked to the SV40 promoter or the glucokinase promoter regardless of orientation or
position. The same sequence is a silencer in hepatoma and is neutral in insolinoma cells
(106).

The B—cell glucokinase promoter, or upstream promoter, has no TATA box. Thus,
transcription initiates over a region of 62 bases (104). Multiple cis-elements in the region
between -280 to -1 (with the most proximal initiation sites designated as +1) contribute to
transcription in insulinoma cells (107). The first element is three binding sites with a
consensus sequence of CAT(T/C)A(C/G), designated as upstream promoter elements
(UPEs). The factor binding to these sites is expressed preferentially in pancreatic islet B-
cells and is 50 kDa in size. The same factor also binds to similar elements, termed CT
boxes, in the insulin promoter, suggesting a common control mechanism for pancreatic
islet B-cells specific gene expression (107). The second element is two copies of a pair of
perfect palindromic repeats separated by a single base, TGGTCACCA, that have been
termed Pall and Pal2 (107). A factor specific to neuroendocrine (NE) cell types, including
the pancreatic B—CCll and pituitary corticotrope, binds to Pal elements, in addition to many
other factors. The presence of the NE-specific factor in certain NE cell lines correlates
with transcription of GK promoter-reporter constructs, suggesting a key role of this factor

in determining NE-specific expression of GK (108).

20

T me 111 hexokinase

The regulation of expression of the gene for type III isozyme has not been studied.

3. Spl Family of Transcription Factors

l‘é’

Spl (Specificity protein 1) was first identified as a factor required for the efficient
transcription of the SV40 early promoter (109, reviewed in 110). In the SV40 promoter,
Spl binds to proximal promoter elements, GC boxes, and contributes to the basal
promoter activity. Alternatively, Spl can bind to a distal site (an enhancer) to activate
gene expression. Moreover, when combined, the distal and proximal GC boxes act
synergistically to give a strong Spl response through cooperative protein-protein
interactions between Spl proteins, a phenomenon called superactivation (111). However,
Spl molecules bound to adjacent sites are apparently unable to make such favorable
protein-protein contacts. Spl is unable to bind simultaneously to adjacent two sites if the
center-to-center distance between the two sites is less than 10 bp (1 10).

Spl contains three Cys-2His-2 zinc finger motifs at the carboxy-terminal end,
serving as DNA binding domains. The activation domain is comprised of alternating
serine/threonine-rich and glutamine-rich regions that constitute much of the amino-

terrninal two-thirds of the protein (112,113). The two domains can be fimctionally

21

uncoupled. The binding domain binds to DNA even when the activation domain is deleted
(114). The activation domain, when attached to the transcriptionally inactive GAL-4 DNA
binding domain, activates promoters containing GAL-4 binding sites. Even the fingerless
Spl, which lacks the DNA binding domain, could act with native Spl to reach
superactivation, suggesting that superactivation is a result of protein-protein interaction
between Spl factors (111). Spl also interacts and synergizes with other transcription
factors, such as C/EBP beta, to activate gene expression (115).

Spl activates transcription by contacting components of the basal transcription
machinery. For example, a glutamine-rich hydrophobic patch in Spl contacts the
dTAFIIl 10, a component of the Drosophila TFIID complex, and mediates transcription
activation (116). A study with synthetic promoters containing TATA and/or Inr (initiator)
showed that the Spl activation domain stimulates Inr-containing and TATA-containing
core promoters equally well, while the VP16 activation domain activates the TATA-
containing core promoter only (117). The lack of preference for TATA-containing core
promoters might explain the frequent involvement of Spl in activation of TATA-less ,
housekeeping genes.

Two posttranslational modifications, glycosylation and phosphorylation, regulate
activity of Spl factor. A recent report showed that in cell culture under glucose
deprivation, Spl protein becomes hypoglycosylated and more susceptible to proteasome
degradation. This process could potentially reduce general transcription under conditions

of inadequate nutrients (1 18).

22

Spl is a preferred substrate for a double-stranded DNA-dependent protein kinase.
Infection of cells with SV40 virus results in a significant increase in the extent of Spl
phosphorylation (119). A recent study showed the importance of Spl phosphorylation in
the activation of HIV transcription, induced by okadaic acid (OKA), a selective inhibitor
of the serine-threonine phosphatase (120). Another study showed that glucose-induced
Spl dephosphorylation resulted in enhanced binding of Spl to promoter II of the acetyl-
CoA carboxylase gene and transcriptional activation of this gene (121). The regulation of
Spl by phosphorylation makes Spl a potential linkage between signal transduction

pathways and transcriptional regulation of gene expression (110).

Sp3 and S24

Other members from the Spl family of transcription factors have been recently
discovered. Sp2 and Sp3 have been cloned by screening a human HUT 78 (0113 T cells)
cDNA library using the Spl zinc finger domain as a probe (122). Sp4 and Sp3 (designated
as SPR-l and SPR-2 originally) have been Cloned by screening an Ishikawa (a human
endometrial cell line) cDNA expression library for proteins that bind to an Spl recognition
site (123). Spl, Sp3 and Sp4 are Closely related members of a gene family encoding
proteins with very similar structural features, including a zinc finger containing DNA
binding domain as well as glutamine and serine/threonine-rich stretches. They bind to GC

boxes and GT boxes with comparable specificity and affinity (123).

23

Spl is ubiquitously expressed, but expression levels vary considerably; how
expression of Spl is regulated has not been determined (110). Sp3 is also ubiquitously
expressed in various cell lines and organs, with the relative mRNA amount varying
moderately. Sp4 transcripts, on the other hand, are abundant in brain and barely detectable
in other organs (123).

In contrast to the structural similarities, Sp3 and Sp4 are functionally quite
different from Spl. Sp3 generally plays a repressive role in transcriptional regulation. It is
suggested that Sp3 inhibits transcription by competing with Spl for binding sites in various
viral and cellular promoters (124-126). Sp3 also acts as an activator on some promoters in
some cell lines, depending on the presence of other factors in the same cells (127). A recent
study showed that the glutamine-rich domain of Sp3 alone activates transcription; in intact Sp3
protein, an inhibitory domain can silence the glutamine-rich activation domain and completely
suppress transcriptional activation (128). Sp4 is an activator like Spl (125), but is unable to act
synergistically through multiple binding sites. However, Sp4 mediated activation can be
enhanced in the presence of fingerless Spl, suggesting the direct interaction between Spl and
Sp4 (129).

Recently, other transcription factors are also identified as GT/GC box binding
proteins. BTEB was isolated from rat liver by binding to a GC box found in the P-4501Al
gene promoter and was capable of activating other GC box-containing promoters (130).
BTEB2 was isolated from human placenta by a similar procedure and had a similar

activation fiinction (131). These factors bind to the same DNA elements as the Sp family

24

of transcription factors, and potentially make the regulation through these Cis-elements

more complex.

Roles of Spl in trapscriptional regulation

It is now known that Spl binds to a 9-bp consensus recognition sequence
G/TG/AGGCG/TG/AG/AG/T in promoters of many viral and cellular genes; many of
them are TATA-less housekeeping genes (110). The roles of Spl in transcriptional
regulation are diverse.

Spl activates transcription of many viral genes, such as SV40 early gene (109). It
mediates the activation of the hepatitis B virus pregenomic promoter by retinoblastoma
susceptibility gene product (Rb) (132). In addition, Spl activates expression of many
cellular genes by interacting with other transcription factors. For example, a distance-
dependent cooperative interaction between transcription factors Spl and Oct-1 is critical
for full activity of human U2 snRNA gene promoter (133).

Spl has been shown to be important in maintaining the basal, constitutive
expression of many housekeeping genes, such as endothelial prostaglandin H synthase-l
gene (134). On the other hand, it is an essential element in directing tissue-specific
expression of human CD14 in monocytes (135), as well as human insulin-like growth
factor H in adult liver (136). It is also critical in start site selection for the TATA-less

human Ha-ras promoter(137). Another recent study showed that a developmental

25

activation of an episomic hsp70 gene promoter in two-cell mouse embryos is mediated by Spl

(138).
Roles Qfmgltiple .Sjpfactors in ”@scriptional regulation

Since the discovery of other members of the Sp family of transcription factors,
regulation of many promoters has been shown to involve multiple members bound to Spl
binding sites. One example is the U5 repressive element of the long terminal repeat of
human T cell leukemia virus type I, with a Spl binding core CACCC motif (139). Another
report showed that expression of the SIS/PDGF-B gene in human osteosarcoma cells, U2-
OS, requires both Spl and Sp3. Cotransfection of U2-OS cells with Sp expression
plasmids and PDGF-B promoter/reporter constructs demonstrated that Spl and Sp3 can
independently and additively activate the PDGF-B promoter (140). In a third study,
transcription from the uteroglobin promoter was shown to be controlled by Spl and Sp3
through a non-classical Sp binding site. Gene transfer experiments into Drosophila SL2
cells that do not contain endogenous Sp factors revealed that expressed Spl activates the
uteroglobin promoter, while Sp3 suppresses the activation by Spl (141). Finally, the
pyruvate kinase M gene promoter is activated by expressed Spl in Drosophila SL2 cells.

Sp3 has a synergistic effect on this Spl activation (142).

26

4. Thesis Overview

The goal of this thesis work was to identify the Cis-elements and trans-factors
governing the transcriptional regulation of the hexokinase I gene. This is the first step toward
the understanding of transcriptional regulation of hexokinase I in different tissues, in response
to hormone changes, such as that of CAMP, and ultimately, in response to changes in energy
demand. Identification and characterization of Cis-elements and trans-factors is the initial step
that provides valuable information about how the promoter activity is maintained and
regulated.

In this thesis, the PC 12 cell line is used to study the promoter of hexokinase I gene.
These cells are frequently used as a neuronal model (143) and, as with brain itself(144, 52),
express a high level of the type I isozyme (145). Various techniques were employed, including
transfection of PC12 cells with nested deletions of the 5’-flanking region of hexokinase I gene
linked to a luciferase reporter gene, footprinting and gel-shift experiments, and mutagenesis
made in important cis-element sequences. Most of the work in this thesis has been published in

Archives of Biochemistry and Biophysics (7 5, 76).

CHAPTER H

MATERIALS AND METHODS

27

28

I. General method;

Standard methods (146) were used for routine procedures of molecular Cloning. DNA
sequencing was done by the method of Sanger (147) using Sequenase v.2.0 kits fiom U.S.
Biochemicals. PCR methods were as described by Innis et al. (148). Oligonucleotides for PCR
and electrophoretic mobility shift (gel shift) experiments were synthesized in the
Macromolecular Structure Facility, Michigan State University. Spl and Ap2 consensus
Oligonucleotides were from Promega, with sequences of
AT’TCGATCGGGGCGGGGCGAGC and GATCGAACTGACCGCCCGCGGCCCGT,
respectively. Protein was determined with the BCA Protein Assay reagent and bovine
serum albumin standard (Pierce Chemical Co.). Human Spl expression vector was kindly
provided by Dr. R. Tjian from Howard Hughes Medical Institute, Department of
Molecular and Cell Biology, University of California, Berkeley; rat Sp3 and Sp4
expression vectors were by Dr. G. Suske from Institut filr Molekularbiologie find

Tumorforschung, Philipps-Universitat, Marburg, Germany.
2. Generation of promoter test constructs
Promoter test constructs were prepared in the vector pGL2-basic (Promega), which

carries the Coding sequence for firefly luciferase. Various fragments fiom previously described

(75) genomic Clone, pBS39.5, were inserted into pGL-2 basic, as described below:

29

The constructs pGL2 SS+ (inserted in correct orientation) and pGL2 SS- (inserted in
reverse orientation) were obtained by subcloning a 572-bp Sac I fragment (positions -742 to -
171, relative to translational start codon, ATG, where A= +1) into the Sac I site of pGL2-
basic. pGL2 SM3 79 was constructed by subcloning SM fragment (position -742 to -516) into
pGL2-basic. Another luciferase reporter construct, designated pGL2 I-IKII H26, contained 2.6
kb of sequence upstream fi'om the transcriptional start site and included the promoter region of
rat type H hexokinase; this was prepared by Cloning a 2.9 kb Hind HI fiagment from the
previously described genomic Clone 5G3A (149) for Type II hexokinase into similar digested
pGL2 basic.

The first set of luciferase reporter constructs was generated as follows. An Eco RI-Sac
I fiagment (-3366 to -171) fiom pBS39.5 was inserted into pGL2-basic to give the reporter
construct designated pGL2 ES. A series of 5' deletions were then generated from pGL2 ES by
conventional restriction digestion and religation. These constructs contained only the upstream
transcriptional start sites (approximately -460 and -3 00) identified in the previous study (75).

A second set of constmcts, with 3' end at -1 and thus including the downstream
transcriptional start sites (75) at approximately -100, were generated from the set of constructs
described above. A PCR fragment corresponding to sequence fiom an Mlu I site at -3 64 to
position -1, with a Hind HI site included in the downstream primer, was used to replace the
Mu I to Sac I region in the parent constructs.

All plasmids were purified using Qiagen plasmid kits, and Checked for purity by

agarose gel electrophoresis.

30

3. T ransfection of PC 1 2 and H9c2 cells with reporter constmcts

Rat pheochromocytoma PC 12 cells were cultured, as described by Tischler and Green
(143), in RPMI medium (HyClone Laboratories) containing 10% horse serum (HyClone
Laboratories), 5% Cosmic calf senim (HyClone Laboratories) and antibiotics (Sigma). Rat
myoblast H9c2 cells (150) were grown in Dulbecco’s modified Eagle’s medium (DMEM,
HyClone Laboratories) containing 10% Cosmic calf serum and antibiotics. Nearly confluent
cultures of PC12 cells and H9c2 cells were replated at 1:3 dilution onto collagen coated and
regular 6-well tissue culture plates, respectively. Twenty four hours later, at which time cells
had attained 50-60% confluency, transfection was performed.

Initially, PC12 cells were transfected by incubation for 18 hours with 1.5 ug of test
construct DNA plus 5 ug Lipofectin (Gibco-BRL), using the protocol described by the
manufacturer. The medium was then exchanged for fiesh medium and extracts were prepared
after a further 48 h incubation. Cells were washed twice with phosphate-buffered saline (0.15
M NaCl, 0.015 M sodium phosphate, pH 7.4) and lysed in 200111 Lysis Buffer (Promega). The
plates were placed at -80°C for 30 min, then at 23°C for 15 min before collecting the lysate.
Lysates were centrifuged at 15,000g for 5 min at 4°C, and supematants were either assayed
immediately or frozen (-80°C) for later assay. In these experiments, luciferase activities were
expressed on a per milligram protein basis.

In later experiments, cells were co-transfected with a control vector, pCMV B-gal
(Clontech), fiom which B-galatosidase expression is driven by the CMV promoter and

enhancer. Transfection conditions were as described above except that PC12 cells were

31

transfected with 8 pg Lipofectin, 1.5 ug test constmct DNA, and 0.3 ug pCMV B-gal DNA,
while H9c2 cells were transfected with 10 pg Lipofectin, 2 ug test construct DNA, and 0.4 ug

pCMV B-gal DNA. Luciferase activities were then expressed relative to B-galactosidase

activities.

4. Reporter assay

Luciferase activities (arbitrary light units) in cell extract were assayed with the reagent
kit and protocol from Promega, using a Turner Model 20 Luminometer. B-galactosidase
activities in the same extract were determined using the assay kit fiom Promega and following
the manufacturer’s instruction on microtiter plate format. Luciferase activities were then
normalized to B-galactosidase activities, and were expressed as percentage of the normalized
luciferase activity seen with the pGL3 -control vector (Promega) in which luciferase expression
is driven by the SV 40 promoter. In mutant constructs, the activities were expressed as a

percentage of that of the corresponding wild type construct.

5. Non-radioactive labeling QLRNAfiprobe and RNase protection assay

To synthesize an RNA probe corresponding to the 3’ UTR region of HKI mRNA, a
BamH I/ Pst I fiagment (2855 to 3160) of HKI cDNA was subcloned into pBluescript H SK+
vector with same digestion. After digestion with BamH I, a 369 nt long anti-sense RNA probe

was synthesized by MAXIscript In Vitro Transcription Kits (from Ambion), using T7 RNA

32

polymerase. This probe was labeled with dig-UTP (from BMB) in the reaction containing dig-
UTP and normal UTP at the ratio of 1256. Full length probe was excised and eluted after
electrophoresis in 6% denaturing polyacrylamide gel.

RNase protection assays were done with RPA-II kits (from Ambion), following
manufacturer’s instruction, except that 10 units of RNase One (from Promega) was used to
digest RNA at 37°C for 60 min. Protected probes (306 at) were then separated in 6%
denaturing gel, transferred onto Amersham’s Hybond-N membrane, using Bio-Rad Trans-blot
SD Semi-Dry Transfer Cell. Signals on the membrane were then detected with Genius System
for Filter Hybridization (from BMB), following Cherniluminescent Detection protocol. The

membrane was then exposed to X-ray film.

6. Premation Qf nuclear extractsfiom PC 1 2 cells

Preparation of nuclear extracts followed the procedure of Ausubel et al. (151).
Specifically, PC12 cells fi'om confluent cultures were collected by centrifugation, washed with
PBS, and suspended in hypotonic buffer containing 10 mM Hepes, 1.5 mM MgC12, 20 mM
KCl, 0.2 mM EDTA, 0.2 mM PMSF, and 0.5 mM DTT, pH 7.9. After incubation for 10 min
on ice, the swollen cells were homogenized with a glass Dounce homogenizer and nuclei
pelleted by centrifiigation at 3300 x g for 15 min. Nuclei were resuspended in low salt buffer
(20 mM Hepes, 25% (v/v) glycerol, 1.5 mM MgC12, 20 mM KCl, 0.2 mM EDTA, 0.2 mM
PMSF, and 0.5 mM DTT, pH 7.9). Addition of an equal volume of "high salt" buffer, which

had the same composition as above except with 1.2 M KCl instead of 20 mM KCl, resulted in

33

release of soluble protein from nuclei. After centrifugation at 25,000 x g for 30 min, the
supematant was dialyzed against 20 mM Hepes, 20% (v/v) glycerol, 100 mM KCl, 0.2 mM
EDTA, 0.2 rnM PMSF, and 0.5 rnM DTT, pH 7 .9. The nuclear extract was divided into

aliquots and stored in liquid nitrogen.

7. Electrophoretic mobility shifl (”gel shift") and 'impershi " experiments

Nuclear extract (20ug protein) was incubated at room temperature for 30 min with 10-
50 x 103 Cpm of the 32P-labeled DNA fiagment, and 1.5 ug poly (dI-dC) as nonspecific
competitor in a buffer containing 10mM Tris-HC 1 (pH7.5), 50mM NaCl, 2.5mM MgC 1 2,
0.5mM DTT, 4% (v/v) glycerol and 0.05% (v/v) NP-40; the total volume was 15 ul. Samples
were then applied to 5% native acrylamide gel and electrophoresis n1n at 150 V. For supershift
experiments, 2 u] anti-Spl, anti-Sp3, or anti-Sp4 antibody (Santa Cruz Biotechnology) was
added after the initial incubation, and incubation continued for another 30 min before loading
onto the gel. Each anti-Sp antibody is specific and does not cross-react with other members in
the Sp family. Gels were dried and exposed to Kodak Biomax MR film or analyzed on a

phosphoirnager (Molecular Dynamics Model 4008).

8. DNase I footprinting

DNase I footprinting was done with the SureTraCk Footprinting Kit (Pharmacia),

following the protocol provided by the manufacturer. A Sac I-Mlu I fragment (-742 to -3 64),

34

labeled with Klenow fiagment (146) to a specific activity of approximately 200Cpm/bp, was
incubated at room temperature with indicated amounts of PC 12 extract and poly(dI-dC) in the
same buffer used for electrophoretic mobility shift assays (see above). After 30 min incubation,
DNase I was added; after a further one min incubation, digestion was stopped by addition of
the "DNase stop solution". Proteinase K (2 pg) was added, followed by incubation at 42° for
30 min. Samples were then extracted with phenol-chloroform, DNA collected by precipitation
with ethanol, and samples loaded onto 5% sequencing gel. The same DNA fragment, treated
with formic acid and piperidine to Cleave at G and A residues, was loaded on this same gel to

provide a sequencing ladder.

9. Mutation of; promoter reporter constructs

Deletion or substitution mutations were made by the two step PCR method of Higuchi
(148), using a Sac I fragment (-742 to -17 l) cloned in the pGL2-basic vector as template.
Description of the exact nature of these modified constructs will become relevant only after
presentation of results below. However, primers used for their generation are given in Table 1.
Outside primer JW 86 (equivalent to the GLprimerl sequencing primer from Promega) is
located 10 nt upstream from the multiple cloning site in the pGL2-basic vector, while outside
primer IW93 (equivalent to the GLprimer2 sequencing primer from Promega) corresponds to
sequence just downstream from the multiple Cloning site. Outside primer JW 63 corresponds to
hexokinase sequence at positions -361 to -332. Underlined regions in sequences for the inside

primers correspond to hexokinase sequence located at positions appropriate for generating

35

specific mutations or deletions described below. The position of deleted sequence generated
with primers JW115 and JW116 is indicated by double slashes within the primer sequences.
Mutated sequences are shown in italics. All mutations and deletions were confirmed by direct

sequencing of the entire region corresponding to the PCR fragment.

10. Hexola'nase asng and western blot

PC12 cells were transfected with human Spl and rat Sp3, Sp4 expression
plasmids, in which cDNA of Sp transcription factors is driven by CMV promoter.
Transfection procedures were same as described for luciferase reporter constructs, except
that 211g of expression plasmid was used in each transfection. Cells were lysed 48 hours
after transfection, in 200111 (per well) of ice-cold BTGE buffer (50 mM Bicine, 10 mM
thioglycerol, 10mM Glc, 0.5 mM EDTA, 0.1% (v/v)TritonX-100, pH 8.2) and went
through two freeze-thaw cycles. The lysate was then centrifuged at 800xg for 5 min at 4°C.
Hexokinase activity in the supernatant was determined immediately, and then stored at -80°C
for future use. Hexokinase activity was determined spectrophotometrically as described
previously (152). Protein was detennined with the BCA Protein Assay system. Samples were
pretreated with excess iodoacetamide (153) to avoid interference by thioglycerol present in the
PTGE bufl‘er.

For western blot experiments, 100 1.1g extract was loaded onto 8% SDS acrylamide
gel, then transferred onto nitrocellulose membrane. The Sp transcription factors were

detected by specific anti-Sp antibodies (at 1:3000 dilution, Santa Cruz Biotechnology),

36

which do not cross-react with other members of Sp family. The secondary antibody was
goat anti-rabbit IgG conjugated to horseradish peroxidase (from Bio-Rad), and detected
with SuperSignal Chemiluminescent Substrate (from Pierce). The membrane was then
either exposed to X-ray film or quantitatively analyzed on Molecular Imager System GS-
505 (Bio-Rad). Protein contents in the samples were assessed by densitometric analysis of
Coomassie Blue stained gels and used to adjust expression level calculated from western

blot.

CHAPTER HI

RESULTS

37

38

1. Promoter activity associated with Sac I fragment in HKI upstream region

Figure 1 shows a schematic representation of a partial restriction map of hexokinase I
upstream region (-3366 to +77), which is related to this work. As previously identified,
transcriptional start sites are clustered in three regions approximately located at -460, -300 and
-100, with A in start codon ATG designated as +1 (75). Complete DNA sequence and
restriction maps of the same region are shown in Appendices A-D.

A 572-bp Sac I fiagment (nt -742 to -171) was inserted into the multiple cloning site
upstream from the luciferase coding region of the reporter vector pGL-2 basic; plasmids with
the insert in either the correct (pSS+) or reverse (pSS-) orientation were prepared. The pSS+
plasmid contains transcriptional start sites located near the -460 and -300 positions. Also
constructed was pSM, containing only the upstream transcriptional start site (nt -737 to -359).

The various reporter constructs or the unmodified pGL-2 basic vector were used to
transfect PC12 cells. These cells express a high level of the type I isozyme (145). Thus we
anticipated that the promoter for type I hexokinase would be quite effective in PC 12 cells and
indeed, luciferase expression driven by the pSM and pSS+ constructs was well above that seen
with the promoter-less pGL-2 basic vector (Table 3). Reversing the orientation of the inserted
promoter region (pSS-) resulted in a marked reduction in expression of luciferase activity. The
previous experiment (75) indicated that PC12 cells favored the upstream (-460) start sites;
hence, deletion of the downstream (-3 00) transcriptional start site, in construct pSM, might
have been expected to have only a marginal effect on expression. Thus, the substantial decrease

(relative to pSS+) in expression from pSM, was somewhat surprising.

39

Figure 1. Partial restriction map and schematic organization of HKI promoter
region. Genomic DNA of the hexokinase I gene from the Eco RI site (-3366) to the Nar I
site (+77) is shown as a horizontal line. The positions of the restriction sites related to this
work are represented by the vertical lines. The three vertical bars represent transcriptional
start points (TSP) clustered at -460, -300 and -100. The horizontal bar represents the first
exon (+1 to +66). Refer to Appendix B, C and D for complete DNA sequence and

restriction map.

Fig. 1

4O

 

in b -742 -520 -359 -248 -l71 +77
p 1 1 I 1 I 1 1 I
SaCI BstXI MluI PstI SacI NarI
\ \ TSP TSP TSP
\ \ 4. \ -460 -300 -100 l
‘\\N 1
‘ \ \ 1
F1 Pv P K P S B M PS N
M
in kb
-3.4 -2.5 . -2.l -l.8 -1.0 -0.7
R = EcoR 1
PV = Pvu II
P = Pst l
K = Kpn l
S = Sac I
B = Bst XI
M = Mlu I
N = Nar I

Table 3. Expression of luciferase in PC 12 cells transfected with reporter constructs

 

Construct Sequence in HKI Luciferase Fold
upstream region activity' increase
pGL2-basic --- 4.3 :t 1.2 1
pGL2 SS+ -742 to -171 1100 3; 96 260
pGL2 SS- -742 to -171 (inverse) 56 :1: 23 13
pGL2 SM -737 to -359 300 :1: 150 70
pGL2 HKII H2.6 --- 50 d: 16 12

 

' Luciferase activity expressed as units/mg protein (mean 3: SD for four transfections).

42

Nonetheless, expression of luciferase activity remained 70-fold increased over the basal
level. The type H isozyme of hexokinase is expressed at low level in PC 12 cells (J .E. Wilson,
unpublished observation). In agreement with this, low level of luciferase were expressed in
PC12 cells transfected with a reporter construct (pI-IKII H26) containing the promoter and

transcriptional start site for type H hexokinase.

2. Sequence between -742gfi -51 6 is importanL for promoter activity

Results of experiments with reporter constructs containing Type I hexokinase genomic
sequence between -3366 and -171 are shown in Fig. 2. Deletions of upstream sequence
between -3366 and the Sac I site at -742 had no significant efi‘ect on expression of luciferase
activity in PC12 cells (Fig. 2B). However, deletion of sequence between -742 and the Bst XI
site at -516 resulted in 85% loss of promoter strength. Another significant decrease occurred
after deletion of sequence between the Mlu I site at -364 and the Pst I site at -245.

Very similar results were seen with these reporter constructs transfected into H9c2
cells (Fig. 2C). The one qualitative difference between results with PC 12 cells and those with
H9c2 cells (Fig. 2B, C) was in expression driven by the PvS construct. In H9c2 cells, there was
a modest but significant decrease in expression relative to that seen with longer and shorter
constructs, ES and KS, respectively. This suggests the possible existence of positive and
negative regulatory elements in sequence from -3366 to -2511 and fiom -2511 to -1833,
respectively. If these indeed exist, they do not appear to be generally fiinctional since their

effect is not seen with PC12 cells. This observation has not been pursued firrther at this time.

43

Figure 2. Transfection of PC12 and H9c2 cells with promoter-reporter constructs. A,
schematic representation of reporter constmcts. All constmcts included only sequence
upstream from position -171 in the 5' flanking region of the gene encoding rat Type I
hexokinase (4); see text for more detailed comments. B and C, normalized (to B-galactosidase
activity) luciferase activities after transfection of PC12 (B) or H9c2 (C) cells with indicated
construct or basal control vector, pGL2-basic; results are the mean i SD for at least eight
independent transfections, and are given as percentage of normalized luciferase activity
expressed from the pGL3-control plasmid. An asterisk means that the activity of a constnict is

significantly, according to statistic analysis, different from that of the next longer construct.

44

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45

As noted previously (75), the overall expression was much lower in H9c2 cells than in
PC12 cells. Thus, expression of luciferase from the longer constructs in PC 12 cells was 10-
15% of the pGL3-control, while in H9c2 cells, these same constructs were expressed at 0.4-
0.5% of the pGL3-control, a level of more than 20 fold lower than that in PC12 cells.

The promoter activities are consistent with the observed levels of HKI mRNA by
RNase protection assays (Fig. 3). The level of HKI mRNA seen with long PC 12 total RNA
(lane 5) was comparable to that seen with long rat brain total RNA (lane 4). The level of HKI
mRNA in 100ug H9c2 total RNA (lane 7), on the other hand, was a little lower than those in
long total RNA from either PC12 cells or rat brain, indicating that the level of HKI mRNA in
H9c2 is at least 10 fold lower than that in PC12 cells and rat brain.

The major point emerging from these transfection experiments was the importance of
sequence fiom -742 to -516 for promoter activity. Thus, attention was focused on this

segment.

3. DNase Liootprinting revea_ls two protected regions

DNase I footprinting analysis was done using a Sac I-Mlu I fiagment containing
sequence between -742 and -359. Using nuclear extracts from PC 12 cells, two protected
regions were observed (Fig. 4). These were designated as the Pl box (24 nt in length, positions
-552 to -529) and the P2 box (23 nt in length, positions -480 to -458). Densitometric analysis
revealed that, with the maximum amount of nuclear extract used, densities in the P1 and P2

regions were reduced to approximately 10% and 25%, respectively, of that in the unprotected

46

Figure 3. RNase Protection Assay. The same amount of the dig-labeled anti-sense RNA
probe corresponding to BamH I/ Pst I fragment (2855 to 3160) in the 3 ’-untranslated region of
HKI cDNA, was incubated with total RNA fiom yeast (lanes 2,3) or various tissue or cell lines
(lanes 4-7), then subjected to RNase I digestion (lanes 3-7). Lane 1 is RNA marker. In lane 2,
one tenth of the sample was loaded onto the gel to give a signal comparable to that in lanes 3

to 7, where all the samples were loaded onto each lane.

47

 

       

H +

 

 

.32 +.
2.: £3. .

Fig. 3

48

Figure 4. DNase I footprinting analysis with a nuclear extract from PC12 cells. Protected
regions in a 32P-labeled Sac I-Mlu I fiagment (positions -742 to -3 59), designated as the P1 and
P2 boxes, are noted. The location of an additional Spl site upstream (position -570) fiom the
Pl box (see text) is also indicated, although no protection was noted in this region. Lane 1 is a
G+A chemical sequencing reaction of the same labeled fragment to provide a sequence ladder.
The sequence of the P1 box is CT’ITI'I‘CCACGCCCAC'I‘TGCGTGC, and the P2 box is

GAGGAAGGGGTGTGGCCCCGTTC.

Fig. 4

49

02.55102040
0.811.216 2.44
2112 24

 

Nuclear extract (pg)

DNase I (u)
Poly (dl-dC) (119)

    

fl 1] Spt (.570)

P1 BOX

P2' BOX

50

control (no nuclear extract). The P2 box is more weakly protected, includes the upstream
transcriptional start sites identified in previous work (75), and does not appear to be important
for promoter activity (Fig. 2). In contrast, the P1 box lies within the region identified as being
of major importance for promoter activity. The sequence of the P1 box,
CT'ITTTCCACGCCCAC'ITGCGTGC, includes a consensus Spl binding site (154) in its
central region. We also noticed another potential Spl binding site, CCGCCCA, just upstream
from the P1 box (positions -574 to -568). The possible significance of these elements for
promoter activity was further examined using a series of mutated or deletion constructs
(results below). Although these experiments demonstrated the fiinctional importance of the
upstream (-570) Spl site, we were unable to detect significant protection of this region in
DNase I footprinting experiments.

Compared to consensus sequences of binding sites for known transcription factors by
searching the GCG database, the P1 box and P2 box contain some potential Cis-elements

shown in Table 4, notably GC-rich Spl and AP2 binding sites in the P1 box.

4. The P1 box and the Spl site at -5 70 arevfimctionally important cis-elements jar; promoter

activity hurt the P2 box is not

The locations of the P1 and P2 boxes, as well as the Spl site at -570, within the
sequence of the parental pGL2SS are shown in Fig. 5. The construct pGL2SS- was identical to
pGL2SS except that the insert was in the reverse orientation (The reporter constmcts pGLZSS

and pGLZSS- were referred to as pS572+ and pS572-, respectively, in Table 3). Several

51

Table 4. Potential cis-elements in P1 box and P2 box sequences

 

P1 BOX CTTTTTCCACGCCCAC'I'I‘GCGTGC

cis- Consensus Matching Sequence’ Degree
Elements Sequence in P1 box of Matching
XRE ' TTGCGTG CTTTTTCCACGCCCACTTGCGTGC 7/7

Spl C/AC/TC/TC/AGCCC/TC/A
Myc CACGTG

AP2 CCCA/CNG/CG/CG/C

CTTTTTCCA CGCCCACTTGCGTGC 8/9
CTTTTTCCACGCCCACZIGCGTGC 4/6, 5/6

CTTTTTCCA CGCCCACTTGCGTGC 7/8

 

 

P2 BOX GAGGAAGGGGTGTGGCCCCG’ITC
Cis- Consensus Matching Sequence’ Degree
Elements Sequence in P2 box of Matching

B-globin GGGTGTGGC
PEAB-RS AGGAAG
TEF-Z-GT-I occrorco

Pu box GAGGAA

GAGGAAGGGGTGTGGCCCCGTTC 9/9
GAGGAAGGGGTGTGGCCCCGTTC 6/6
GAGGAAGGGGTGTGGCCCCGTTC 8/ 8

GAGGAAGGGGTGTGGCCCCGTTC 6/6

 

’ Bold letters in underlined sequence represent nucleotides identical to consensus sequence
of known cis-element; italic letters represent mismatching nucleotides.

52

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53

modified constructs were prepared by the two step PCR method of Higuchi (148), using
primers shown in Table 5. These were: pGLSSde1P1(24), in which a 24 nt sequence
corresponding to the P1 box was deleted; pGLSSsubPl, in which the entire 24 nt sequence
was modified, converting C to A, T to G, A to C, and G to T; pGL2SS(Nde I), in which the -
570 Spl site CCGCCC was substituted with an Nde I restriction site, CATATG;
pGL28SsubP2, in which the P2 box (—480 to -458), GAGGAAGGGGTGTGGCCCCGTTC,
was replaced by the permuted sequence, GTTTCCTGTCGTG'ITACCATGGC. Additional
constructs were then generated from those just described: pGL2NdeS was prepared by
digestion of pGLSSCNde I) with Kpn I (cutting upstream within the multiple Cloning site) and
Nde I, and religation; pGL2SSdelP1(55), in which a 55 nt segment (-572 to -518) containing
the P1 box and the upstream Spl site was deleted, was obtained by digestion of pGLZSS(Nde
I) with Nde I and Bst XI, followed by polishing the ends and religation; pGL2BSsubP2 was
generated by deleting sequence between -742 and -561 as a result of digesting pGLSSsubP2
with Kpn I and Bst XI, followed by religation.

Results of transfection experiments with several of these constructs are compared in
Fig. 5. Deletion of the P1 box and upstream Spl site (in pGL2delP1(55)) resulted in expression
of normalized luciferase activity that was only about 16% of that seen with the parental
pGLZSS. This reduced activity was virtually identical to that seen with pGL2SS-, in which the
promoter region had been inserted in reverse orientation. Similar reduction in promoter activity
was seen with pGL2SSsubP1, in which the sequence of the P1 box had been permuted as
described above. Thus, the Pl box was Clearly a critical cis-element for promoter activity. The

adjacent Spl site, at -570, also had a significant effect on promoter activity, with either

54

Figure 5. Effect of mutations in the Pl box and upstream (-570) Spl site on promoter
activity. Normalized luciferase expression from the parental pGL2-SS (top of figure),
containing sequence between -742 and -171, was taken as 100%. pGL2-SS- (bottom of figure)
corresponds to the same sequence, but inserted in reverse orientation. Other constructs contain
mutations or deletions as described in the text. Filled boxes represent wild type sequence while
open boxes represent mutated sequence; bent lines represent deletions. The positions of the P1
box (-552 to -529) and upstream (-570) Spl site are indicated. The angled arrow indicates the
location of P2 box (-480 to -458), which encompasses the multiple transcriptional start sites

identified in the -460 region (4). Results given are mean :t SD for eight transfections.

Constructs
in pGL2-basic
SS

SSdelPl(55)

SSsubPl

SS(NdeI)

NdeS

SSdelP1(24)

SS-

Fig. 5

55

Activity

 

 

 

 

 

 

 

 

 

 

 

 

_ (% of pGLZSS)
-742 -570 Spl Pl 171 , meanzl: 3D
l—p lucrferase
1 I_- I 1 100111
SacI SaCI
1 \f I I 15.8:l:l.4
l—D
I‘D
1..- l—‘l 52.4i4.6
-568 Ndcl Sacl
1 I I", r——'] 43.8i3.2
1 -_- l—l 14.3115
SaCI SacI
-171 -742

“also.“ unfits't Mum Baal-1W

56

Table 6. P2 box mutation data j

P2BOX sequence” GAGGAAGGGGTGTGGCCCCGTIC 1.

 

P2 BOX mutant sequence GTTTCCTGTCGTGTTACCATGGC

 

Construct Wild type construct Activity '
pGL2 SSsubP2 pGL2 SS 142 at 14
pGL2 BSsubP2 pGL2 BS 108 i 16

 

' Activities were expressed as percentage of the activities of corresponding wild type
constructs. Data were mean i SD of six transfections.

bUnderlined G and T in P2 box sequence represents the most upstream transcriptional start
sites for HKI gene.

57

mutation of the sequence in pGL28S(Nde I) or deletion of this site in pGL2NdeS resulting in
50-60% decrease in expressed luciferase activity. Interestingly, when the P1 box was deleted in
pGL2SSdelP1(24), bringing the upstream -570 Spl site into the position previously occupied
by the Pl box, expressed luciferase was about 2-fold higher than that seen with the mutated Pl
box of pGLSSsubPl and was similar to that seen with the mutated Spl site of pGLSS(NdeI),
suggesting that PI box can be functionally replaces by the Spl site.

In contrast to the importance of sequence within the P1 box for promoter activity,
extensive modification of the sequence in the P2 box did not have any detrimental efi‘ect on
promoter activity (Table 6). Thus, normalized luciferase activity expressed from pGL2SSsubP2
was 142 d: 14% (mean :1: SD for 8 transfections) of that seen with the parental pGL2SS
construct. Similarly, luciferase expression from pGLZBSsubPZ was 108 :1: 16% of that seen

with the corresponding construct, pGLZBS, containing the wild type P2 box sequence.

5. Gel shift and wershift experiments demonstrate binding QfSpfamily members to the P1

Ear.

Gel shift experiments were done with a 33 bp oligonucleotide having the sequence
GAT- [Pl box]-TAGATC, where "[Pl box]" represents the 24 nt sequence (positions -5 52 to
-529) given above, annealed with its complement. Sequences for other DNA fragments in gel
shift experiments are shown in Table 7. After incubation of the 32P-labeled oligonucleotide with

nuclear extracts fiom PC12 cells, two major complexes (Fig. 6), referred to here as the "upper"

58

Table 7. DNA Fragments Used in Gel shift Experiments

Name Primers DNA sequence

 

Pl BOX SEQUENCE CTTTTTCCACGCCCACTTGCGTGC

Probe

Pl BOX

P1 #1

P1 #2

P1 #3

P1#4

P1 (79)

P1(10-11)

Pl (12-13)

P1 (14-15)

P1(16-18)

127/130

113/114

182/183

184/185

186/187

188/189

195/196

201/202

191/192

203/204

197/198

GATCTITTTCCACGCCCACTTGCGTGCTA
AAAAAGGTGCGGGTGAACGCACGATCTAG

TC'I‘TTTTCCACGCCCACTTGCGTGCT
AGAAAAAGGTGCGGGTGAACGCACGA

ATAGGGGGCCACGCCCACTTGCGTGC
TCCCCCGGTGCGGGTGAACGCACGTA

ATCT'I'I'ITAACATACCACTTGCGTGC
GAAAAATTGTATGGTGAACGCACGTA

ATC I I I I I CCACGCAACAGGGCGTGC
GAAAAAGGTGCGTTGTCCCGCACGTA

ATCTTFTTCCACGCCCACTFTATGTA
GAAAAAGGTGCGGGTGAAATACATTA

CI I I I IAACCGCCCAC'I'I‘GCGTGC
GAAAAATTGGCGGGTGAACGCACG

C'I'ITITCCAATCCCAC'ITGCGTGC
GAAAAAGGTIAGGGTGAACGCACG

CI I I I ICCACGMCACTTGCGTGC
GAAAAAGGTGCEGTGAACGCACG

CI I I I ICCACGCCA_CCTTGCGTGC
GAAAAAGGTGCGGEGAACGCACG

C'ITTT'I‘CCACGCCCAAGGGCGTGC
GAAAAAGGTGCGGGTTCCCGCACG

 

Note: Underlined sequences represent mutations in the P1 box.

 

59

Figure 6. Gel shift experiment showing complexes formed by proteins in nuclear
extracts from PC12 cells and an oligonucleotide including the Pl box sequence. In Lane
1, the oligonucleotide and no nuclear extract were added. In Lane 2, nuclear extract from
PC12 cells and no competing oligonucleotide were present. In Lanes 3 and 4, same as in Lane
2, but the indicated molar excess of unlabeled Pl box oligonucleotide was added as
competitor. In Lanes 5-12, same as in Lane 2, but a molar excess of unlabeled mutant
Oligonucleotides containing the indicated modifications within the P1 box was used as
competitor, the 24 nt P1 box sequence was Changed progressively, 6 nt at a time, as indicated
schematically at top of figure (see text for details). Lanes 13 and 14 show the lack of
competition by added molar excess of AP2 consensus oligonucleotide. Lanes 15 and 16 show

the competition by added molar excess of Spl consensus oligonucleotide.

60

Catapult: .. or
hinder exam

 

1.- Iree P1box

Fig. 6

61

and "lower" complexes, were observed. As expected, the unlabeled oligonucleotide was
competitive with the labeled nucleotide (Fig. 6, Lanes 3 and 4) for formation of both
complexes. An oligonucleotide containing a consensus Spl binding sequence was also
competitive with the 32P-labeled P1 box oligonucleotide, but an oligonucleotide containing the
somewhat similar (GC-rich) consensus AP2 binding sequence was not (Fig. 6, Lanes 13-16). A
series of oligonucleotides in which the 24 nt sequence of the P1 box was modified (A and C
interchanged, and G and T interchanged), six nt at a time, were then examined for their ability
to compete in complex formation (Fig. 6, Lanes 5-12). Sequence Changes in the first and last
six nt of this 24 nt sequence had little, if any, effect on the ability to compete with the
oligonucleotide having the intact P1 box sequence. However, modifications within the central
twelve nt in this sequence abolished the ability to compete. This region contained the consensus
Spl binding sequence (154).

Sequence specificity in this central region was then examined with a series of
oligonucleotides in which the central twelve nt of the P1 box were changed, two or three at a
time (Fig. 7). Modification of the first or last three nt within this central 12 nt sequence
(corresponding to positions 7-9 or 16-18 in the 24 nt P1 box sequence) did not prevent
competition for complex formation (Fig. 7, Lanes 5 and 6, Lanes 13 and 14) but, at
comparable concentrations, the modified oligonucleotides were much less effective than the
intact P1 box sequence (Lanes 3 and 4). Changes within the central six nt, which represents the
core of the consensus Spl binding site (154), virtually abolished the ability of the

oligonucleotides to compete in complex formation (Fig. 7, Lanes 7-12). These results Clearly

62

Figure 7. Gel shift experiment showing the effect of mutations in the Spl binding site
within the Pl box sequence. Conditions were same as in Fig. 4, except for oligonucleotides
added as competitors. In Lane 1, free oligonucleotide and no nuclear extract were added. In
Lane 2, nuclear extract from PC12 cells and no competitor oligonucleotide were present. In
Lanes 3 and 4, as in Lane 2, but indicated molar excess of unlabeled P1 box oligonucleotide
was added as competitor. Lanes 5-14, as in Lane 2, but the indicated molar excess of unlabeled
oligonucleotides containing modifications within the central 12 nt segment of the Pl box was

added as the competitor, mutations were as shown at top of figure.

Fig. 7

   
   

63

Preoxcrrrr'rCCA C c c C C A crr cceroc
P1t7~9i 6.21.9
91110.11) 11 r

P1(12-13) A
Null-15) AJ
Pitts-18) A Q 9
no P1box ripen) P1(12-13)P1(14-15) Puts-18) Competitor
. 10x so . W191“ ' in molar excess

+ upper complex
d— tamer complex

' 41— free P1box

234 6 81991011121314

 

64

demonstrate that formation of both upper and lower complexes require the presence of an
intact Spl binding site within the P1 box.

Other members of the Sp family of transcription factors are also known to bind to this
sequence (122,123). To examine the possible role for these in binding to the P1 box, supershift
experiments were done (Fig. 8). Quantitative analysis of these results was not considered
reliable in most cases due to overlap in position of native complexes and supershifted bands.
Nonetheless, qualitative interpretation clearly indicated involvement of Spl, Sp3, and Sp4 in
formation of the observed complexes. Thus, addition of antibody against Spl caused a shift
(Fig. 8, Lane 2) of approximately 50% (determined by analysis on a phosphoimager) of the
upper band but had no effect on the lower complex. Anti-Sp3 virtually completely shifted the
lower complex, apparently into the region just below that occupied by the upper complex, and
also shifted part of the radioactivity associated with the upper complex (Fig. 8, Lane 3).
Virtually identical results were reported by Majello et al. (125), using nuclear extract from
HeLa cells, i.e., two complexes were seen, with relative positions comparable to our upper and
lower complexes, and supershift analysis indicated that upper complex involved both Spl and
Sp3 while the lower complex was due solely to binding of Sp3. Addition of antibody against
Sp4 (Fig. 6, Lane 4) had no effect on the lower complex, but shifted approximately 10% of the
radioactivity associated with the upper complex into a Clearly defined region well above the
residual upper complex. Combinations of these antibodies (Fig. 8, Lanes 5-8) had effects
consistent with those predicted from observations with the individual antibodies, including a

virtually complete shift of both complexes when all three antibodies were present.

65

Figure 8. Supershift experiment showing involvement of Spl, Sp3, and Sp4 in
complexes formed with nuclear extracts from PC 12 cells. Conditions were as described for
Fig. 4 except with addition of antibody or antibodies indicated at top of figure. Lane 1, no
added antibody. The positions of the original "upper" and "lower" complexes are indicated by

arrows at right of figure.

66

+ + + + anti-Spl
+ + + + anti-Sp3
+ + + + anti-Sp4

4— upper complex

<-— lower complex

 

Fig. 8

 

67

Figure 9. Summary of gel-shift and super-shift experiments. Shown on the top of the
figure is the wild type sequence of the P1 box, in which the sequence matching to the Spl
consensus is shown in bold and italic. All the subsequent lines also represent wild type
sequences in competitor primers. Black bars represent block mutations in which six of the 24 nt
sequence in P1 box are mutated. The mutant sequence in the central 12 nt are shown by the

letters on the lines. The stars represent the unchanged sequences in the central 12 nt.

68

CTI'I'I'I‘CCACGCCCA CTTGCGTGC

 

 

 

_
—-

 

——
-

 

SP1 OLIGO
AP2 OLIGO

AActteneu-e
*ttATttttttt
tttttMttttt
teeetetACtet

sesuunAGf;

ANTI SP1
ANTI SP3
ANTI SP4

SUPER = supershift
LA = lower affinity

Fig. 9

competitor for
upper complex lower complex

YES

YES
NO

NO

YES
YES
NO

YES (LA)
N O
NO
NO

YES (LA)

SUPER
SUPER
SUPER

YES

YES
NO

NO

YES

NO

YES

NO

NO

NO

YES

N O
SUPER
NO

69

Figure 9 summarizes the results of gel shift and supershift experiments discussed above.
It Clearly shows that the central region of P1 box, which contains a Spl binding site, is critical
for the formation of both complexes. The Spl consensus sequence competes for
complexforrnation; the upper complex contains Spl, Sp3 and Sp4, while the lower complex

contains Sp3 only.

6. Effect afoverexpression 018p transcriptionfactors on HKI expression in PC 12 cells

Human Spl and rat Sp3, Sp4 were overexpressed in PC 12 cells by transfecting cells
with plasmids containing cDNAs for Sp factors driven by CMV promoter. Western blot
experiment (Fig. 10) showed that protein levels of Spl (lane 2), Sp3 (lane 4) and Sp4 (lane 6)
in extracts of transfected PC12 cells were higher than those in control PC12 cells (lanes 1, 3,
5), which were transfected with pGL3 -control. Although some non-specific bands were also
detected, the ones indicated by arrows were the major ones in the expected positions according
to molecular weights; thus they were analyzed on Molecular Imager System, and the intensities
relative to controls were shown in Table 8. Spl, Sp3 and Sp4 in transfected cells were
overexpressed 2.9, 6.7 and 2.0 fold, respectively, over the control. However, hexokinase
activities in transfected PC 12 cells were not significantly Changed in transfected PC 1 2 cells.

This might be due to a saturating amount of endogenous Sp factors in PC12 cells.

7. Rgporter constructs including sequence between positions -1 71 and + 1 reveal another

region with promoter activity

70

Figure 10. Overexpression of Spl, Sp3 and Sp4 in PC12 cells. 100 ug protein was loaded
into each lane. In lanes 1, 3 and 5, PC12 cells were transfected with pGL3-control
plasmid; in lanes 2, 4 and 6, PC 12 cells were transfected with Spl, Sp3 and Sp4
expression plasmids, respectively. Membranes were probed with corresponding antibodies

shown on top of the figure. The arrows indicate the major bands for Sp factors.

71

 

Fig. 10

72

Table 8. Hexokinase activity in PC12 cells overexpressing Sp transcription factors

 

PC12 cells transfected Expression levelb Hexokinase activity’
with plasmid expressing (over control) (mu/mg)
control 1 14.1 3: 0.7
Spl 2.9 1453:15
Sp3 6.7 148124
Sp4 2.0 11.8i3.0

 

' Data for hexokinase activity were expressed as mean :t SD of six independent

transfection experiments.
I’Data for expression level were obtained from a representative experiment shown

in Figure 10.

73

Transfection studies were also done with a series of reporter constnicts that included
the most downstream transcriptional start sites, in the -100 region (75). The results are shown
in Fig. 11. Comparison with data in Fig. 2 immediately reveals very notable differences. In
contrast to the virtual lack of effect of deletions in the -251 1 to -742 region, seen with the
previous set of reporter constructs (Fig. 2), significant decreases in luciferase expression were
observed with the second set of constructs which included the -171 to -1 sequence (Fig. 11).
While an 85% decrease in promoter activity was seen with deletion of the -742 to -516 region
when the shorter constructs were used (Fig. 2), the corresponding deletion had only a modest
effect with the longer reporter constructs (Fig. 1 1). Progressive decreases in luciferase
expression were seen as the sequence between -364 and -l were deleted. The decrease with
loss of sequence between -364 and -245 was also seen with the shorter reporter constnicts
(Fig. 2), but this was, quantitatively, much less striking than the effects of deletions in the -742
to -516 region; this was, of course, why we focused on this latter region for the detailed
analysis presented above.

These results indicate the presence of an additional region of promoter activity, located
in the -364 to -1 region. The strength of this promoter is comparable to that identified in the -
742 to -516 region. Thus, luciferase expression driven by pGL2SS (which includes sequence
fiom -742 to -171) was 10.9 i 2.4% of that seen with the pGL3-Control (Fig. 2); this appears
to be largely associated with the upstream promoter, being markedly decreased by deletion of
this region (-742 to -516). Very similar expression, 8.8 d: 1.5% of that with the pGL3-Control

(Fig. 11), was seen with the construct including only the sequence between -364 and -1, and

74

Figure 11. Transfection of PC12 cells with promoter-reporter constructs including
sequence between -171 and -1. A, schematic representation of reporter constructs. All
constructs included sequence upstream from position -1 in the 5' flanking region of the gene
encoding rat Type I hexokinase (75); see text for more detailed comments. B, normalized (to
B-galactosidase activity) luciferase activities after transfection of PC 12 cells with indicated
construct or basal control vector, pGL2-basic; results are the mean :1: SD for at least six
independent transfections, and are given as percentage of normalized luciferase activity
expressed fi'om the pGL3-control plasmid. An asterisk means that the activity of a construct is

significantly, according to statistic analysis, different from that of the next longer construct.

75

A. Constructs in pGL2-basic

 

 

-2511 E
-1833 41:]
-1044 :1

 

-742 —————l::l

 

Luciferase o s to 15 20 25
Luciferase/gal (% of pGL3-control)

Fig. 11

76

thus completely lacking any promoter activity associated with the upstream region. Moreover,
the activity of these promoter regions was not additive since luciferase expression from the
reporter construct pGL2ST (Fig. 11), which includes sequence from -742 to -1 and thus
contains both promoter regions, was 10.5 i: 1.6% of the pGL3-Control; this is virtually
identical to the activity seen with the constructs containing either one of the promoter regions

alone.

8. Efiect of downstream sequence L+ 1 to + 7 7) on promoter activity

To investigate the possible influence of downstream sequence on the hexokinase I
promoter activity, two more promoter-reporter constnicts were made. The first one, pGL2-
SN, containing hexokinase I sequence from -742 to +77, was made by replacing the Mlu I (-
364) /Hind III (in multiple cloning site of vector) fragment in pGL2-SS construct with Mlu I (-
364) / Nar I (+77) fragment of hexokinase I genomic DNA (pbsS). pGL2-SN2 construct (-17l
to +77) was made by deleting Sac I (-742) / Sac I (-171) fiagment from the pGL2-SN
constnict. Transfection data are shown in Table 9. The promoter activity of ST construct, as
shown previously, was about 10.5% of that of pGL3-control. When sequence from +1 to +77
was included, as in SN construct, promoter activity was increased by about 60%, to 16.1% of
that of pGL3-control. On the other hand, when the same region (+1 to +77) was added to ST2
construct resulting in SN2 construct, the promoter activity remained at basal level, e. g., around

2% of that of pGL3-control. Taken together, these data suggest that sequence from +1 to +77

77

 

Table 9. Effect of downstream sequence on promoter activity
Constructs in Position of promoter Activity'
pGL2-basic fragment (% pGL3-control)
ST -74Zto-1 10.5:1: 1.6
SN -742 to +77 16.1 :t 4.1
ST2 -l71to-1 2.0:t0.7
SN2 -171 to +77 2.2 i 0.3

 

'Luciferase activities are normalized to B—galactosidase activities and expressed as mean 1
SD of six independent transfection experiments.

78

increase the activity of either the upstream promoter (Pl box and -570 Spl site) or the
downstream promoter (-364 to -1) or both, but it does not possess promoter activity of its own

to drive transcription from -100 start sites.

CHAPTER IV

DISCUSSION

79

80

Two regions of promoter activity have been identified in the 5' flanking region of the
gene for rat Type I hexokinase. The upstream promoter is associated with fragment -742 and -
516, the downstream one is with fiagment -364 to -1, based on the results of experiments with
promoter-reporter constructs. The major focus of the present study has been on the upstream
promoter. Footprinting analysis identified two protected regions, the P1 and P2 boxes.
Functional analysis revealed that PI box and -570 Spl site are important cis-elements for
hexokinase I promoter activity. It is evident that members of the Sp family play a prominent

role in regulation of the upstream promoter.

1. The Weam promoter: P1 box and -5 70 Sp] site

Promoter activity was markedly dependent on maintenance of sequence at both the
Spl binding site located within the P1 box (approximate position -540) as well as an additional
Spl site just upstream (approximate position -570) fiom the P1 box. These sites are located
more than the minimal 10 bp required to pennit simultaneous binding of Spl at both sites (1 10)
and the results with the reporter constructs clearly suggest that both sites must be occupied for
expression of full promoter activity. The inability to detect protection of the upstream Spl site
by DNase I footprinting presumably reflects a lower affinity for binding to this site, at least
under the conditions of the footprinting experiments. Multiple Spl sites are often associated
with regulation of TATA-less promoters in GC-rich regions, characteristic of housekeeping

enzymes (155, 156) such as Type I hexokinase.

81

It is interesting to note that, although intactness of both Spl sites (-540 and -5 70) is
required for full activity of the promoter in this region (see results in Fig. 5), the presence of a
single Spl site at the position (-540) normally occupied by the Spl site intrinsic to the P1 box
is suficient for maintaining promoter activity at approximately 50% of its full activity. Thus,
either deletion of the upstream (-570) Spl site or deletion of the Pl box in construct
pGL2SSdelP1(24), which brings the upstream site into about the same position as that
normally occupied by the -540 Spl site, results in approximately 50% of the 11111 promoter

activity.

2. Mtltiple members Qflpfamily of transcription/actors regulate the activity af upstrem

2r 017101 er

Gel shift and supershift experiments suggested that several members of the Spl family
of factors (122, 123) can bind to P1 box region and are likely to participate in regulating
expression of the Type I isozyme of hexokinase. Both Spl and Sp4 serve as activators of
transcription (124, 125). In contrast, Sp3 generally seems to play a repressive role in
transcriptional regulation (124, 125), although this may depend on both promoter type as well
as presence or absence of other transcription factors (127). The activity of the upstream
promoter region can be expected to depend on the relative levels Sp factors present in various
tissues.

Based on the fact that the sequence between -742 and -516 accounts for 85% of the

promoter activity in both PC12 cells and H9c2 cells (Fig 2), we think that the activities of Spl

82

family of transcription factors in a given tissue determine the activity of hexokinase I upstream
promoter in that tissue. The ubiquitous expression of Spl and Sp3 (123) as well as the frequent
involvement of the Sp family in regulating expression of housekeeping genes (155, 156)
suggests that the upstream promoter may be involved in basal expression of the ubiquitous
Type I isozyme. The transcriptional activator, Sp4, is expressed at particularly high levels in
brain (124), and this may well be related to the very high levels of Type I hexokinase in brain
when compared to other tissues (5 2).

Although the supershift experiment showed that with Sp 1 , Sp3 and Sp4 antibodies
added to the reaction, almost all the complexes were supershifted, it is still possible that the
remaining radioactivity is the result of some other transcription factor(s) bound to the Spl site.
Thus, there might be other factor(s) regulate hexokinase I promoter activity through binding to

P1 box sequence.

3. The P2 box and transcription initiation

The P2 box includes the most upstream (approximately -460) transcriptional start sites
(75). We think it is likely that protection of the P2 box results from binding of RNA
polymerase H or associated factors. Thus, the lack of specific sequence requirement (indicated
by the lack of effect of mutations on expression from pGL2SSsubP2 and pGL2BSsubP2) is
consistent with the existence of multiple transcriptional start sites throughout this region (75),

implying lack of sequence specificity in binding of the polymerase.

83

There are two Inr (initiator) elements flanking 5’- and 3’- end of the P2 box sequence
(Appendix A). Inrs are involved in transcriptional start site selection in many TATA-less
promoters (157). They usually overlap transcriptional start sites, with consensus sequence of
T/CT/CANT/AT/CT/C (158). The two Inrs around P2 box might specify the transcriptional
start sites at -460.

Recently, another element called MED-1 (Multiple start site Element Downstream) is
identified as an element regulating a window of multiple start sites in TATA-less promoter
(159). The MED-1 sequence is usually positioned at 20-45 bp downstream from the 3’- end of
multiple start site window, or up to 110 bp downstream from the 5’- end of the window. The
consensus sequence is GCTCCC/G. Thus, the MED-1 element located at -170 in hexokinase I
promoter region (see Appendix A) might be the determinant for transcriptional initiation at the

multiple start sites around -3 00 to -200.

4. The downstream promoter

The upstream promoter activity is clearly functional with the upstream transcriptional
start sites (75), located in the -460 and -3 00 regions, since the downstream transcriptional start
sites in the -100 region were not included in the shorter reporter constnicts (results in Fig. 2).
In view of the distance between the P1 box and the downstream start sites (-100), it seems
unlikely that this upstream promoter can drive transcription from the downstream start sites,
although this remains to be directly determined. It is more probable that the promoter activity

located in the -364 to -1 region is associated with transcription from the downstream start sites.

84

More extensive analysis of the downstream promoter is currently underway. However,
the results presented here (compare Figs. 2 and 11) already make it clear that distinct cis-
elements will be involved.

Recent report showed that myogenesis and MyoD down-regulated Spl, and that it is
the mechanism for the repression of GLUTI during muscle differentiation (160). In
contrast, level of hexokinase I did not Change under the same condition (personal
communication with Dr. A. Zorzano from Departament de Bioquimica i Biologia Molecular,
Universitat de Barcelona, Spain). This might be due to the second (downstream) promoter of

hexokinase I, which is not regulated by Spl factor.

5. Iifi'ect of + 1 to + 77 on the promoter activity

Transfection data showed that sequence between +1 and +77 increased promoter
activity by about 60%. But it is not an independent promoter, since it did not drive the
transcription from -100 start sites. It remains to be tested whether this region is acting at

the upstream or downstream promoter.

CHAPTER V

FUTURE WORK

85

86

1. To investigate roles of Spfactors in regulation of the upstream promoter

- T rapiection into Drosophila SL2 cells

The overexpression of Sp factors in PC12 cells did not increase hexokinase level in
the cells. It is perhaps due to the saturating amount of endogenous Sp factors. Drosophila
cell SL2 lacks endogenous Sp factors; thus it is a cell line frequently used to study the role
of Sp family of transcription factors. Co-transfection of SL2 cells with Sp expression
plasmids and hexokinase I promoter/reporter constructs should reveal how Sp factors
regulate activity of the upstream promoter, e. g, which factor is up-regulating or down-
regulating the promoter activity, and which factor has stimulating or suppressing effect on
other factors. These experiments will show the function of Sp factors on activity of the

upstream pfOIHOtCI'.

2. To test downstream promoter activity in H 9c2 cells

Downstream promoter activity associated with -364 to -1 region has been shown
in PC12 cells (Fig. 11). The same set of promoter-reporter constructs can be used to
transfect H9c2 cells to see if the same region is also active in H9c2 cell. If the profile of
promoter activity is different in H902 cells from that in PC12 cells (Fig. l 1), it might
suggest a mechanism for the different expression level of hexokinase I in different tissue

and cell lines.

87

3. To identify cis-elements in the downstream promoter

More extensive analysis of the downstream promoter is currently underway. The
fragment associated with downstream promoter, MT (-3 64 to -1) is being used in DNaseI
footprinting experiment with PC12 nuclear extract. This experiment should reveal the Cis-
elements in this promoter region. The protected fragment in the footprinting experiment
will be used in gel-shift and supershift experiments to identify the trans-factors bound to
the region. Finally, the functional significance of the cis-elements will be tested by
mutating the elements in promoter-reporter constructs which will be transfected into PC 12

cells.

4. T 0 may traascriptional regulation gfhexokinase I expression

By CAMP

It has been shown that hexokinase I mRNA is increased in cultured rat thyroid
FRTLS cells by thyroid stimulating hormone (T SH) through CAMP pathway (64). With the
availability of various promoter-reporter constructs, CAMP response element can be
identified by stimulating FRTLS cells which are transfected with nested deletion of
promoter-reporter constructs. When the element is deleted, the CAMP response will be

lost.

88

By energy demand

The levels of type I hexokinase in various rat brain regions have been correlated with
basal rates of glucose utilization. We want to study the regulation of hexokinase I in
response to changes in energy demand in PC12 cells. The energy demand and glucose
utilization of PC12 cells will be stimulated by increasing the extracellular KCl
concentration which constantly depolarizes PC12 cells. Once the condition is set up for
hexokinase level in response to changes in energy demand, it will be asked whether the
changes are due to transcriptional regulation. If that is the case, various promoter-reporter
constructs can be transfected to PC 12 cell under stimulating condition, and see which
fiagment keeps HKI promoter responsive to the change of energy demand, and what Cis-

element(s) and trans-factor(s) are involved in the regulation.

5. T 0 define transcriptional start site and promoter usage in different tissues and cell

lines

Levels of hexokinase I mRNA vary in different tissues and cell lines. It is of great
interest to study whether this variation is determined by different usage of transcriptional
start sites and /or promoters. RNase protection assay with mRNA from different sources
should reveal the preferential usage of transcriptional start sites. With the availability of
I-H(I promoter-reporter constructs, the activities of the upstream promoter and

downstream promoter can be tested in different cell lines. In addition, similar promoter

89

constructs linked to different reporter system in an in-vitro assay allow us to test promoter
activity driven by nuclear extract from different tissues. Comparison of start site and
promoter usage should give us valuable information about how HKI levels are regulated in

different tissues at the transcription level.

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APPENDICES

APPENDIX A

-742

-150

-100

+1

+51

APPENDIX A

DNA SEQUENCE AROUND HKI PROMOTER REGION

WWACATGGTGTCACGGGGGCAGGTAGTITGGGTITA
Sac I
GCAATGTGAACTCTGAC AA'I'ITGGGATGTAGAGCTGGTGGGCCATCGTGG
GACGCCAAGCATCATCC'I'I‘AGAG'I'I'I‘GGATCC’ITI‘AGGGCAGGCAGGCAC
AGGGACCCAGTGCGAGATCAGTGAAGCCGCCCAG’I’I‘TCGGC'ITCCGCTC_'_T
Sp 1
TI'I'I‘CCACGCCCACTTGCGTGCITCTWATGGGAGGGGTG

P1 box Bst XI 0 e
GGGGACGAGCCCTAATCTC CGAGGAAGGGGTGTGGCCCCGTI‘CGTGTTCI‘

 

 

“mu“ '4’ nu _‘-.'.

 

 

 

o Inr P2 box
CCAG’ITI‘GTGGCGTCCTGGATCTGTCCTCTGGTCCCCTCCAGATCGTGTC

Inr
CCACACCCACCCGT'I‘CAGGCATGGCAC'I‘GTGCCGCCACLKLGIGACCGTGC

e e 0 MI“ I
GCTCCTTACGTGGGGGACGTGCAGGGTQCTQCCTCCE'ITCCGGTGCGGGA

GGGAGCGGCCQTC I ' I '1 CTCCTGCTCTGGCTGGGAAGCCCCAGCCé'ITGCG

MAGGAGACITGCAGCCAATGGGGACTGAGGAAGTGGGCCGGCI‘GG
PSI I e
CGGTFGTCACCCTCCCGGGQACCGWCGAGGTCTGGAGAGCGCAGG

. Sac I
CAgACGCCCGCCCCGCCCGGGGACI‘GAGGGGGAGGAGCGAAGGGAGGAGG

 

 

e e e e e Spl 0 SP 1 o e
AGGTQGQGTCTCCQATCTGCCGCI‘ GGAGGACCQCT GgTCACCAGGGCI‘ AC

TGAGGAGCCACTGGCCCCACACCTGCITITCCGCATCCCCCACCGTCAGC
ATGATCGCCGCGCAACTACTGGCCTATI‘ACITCACCGAGCTGAAGGA'I‘GA
MIAAQLLAYYFTELKDD
CCAAGTCAAAAAGGTGAGCCCCGCCQQQGQQGCC

Q V K K NarI

Dots above DNA sequence represent transcriptional start sites.
Underlined sequences represent restriction sites.
Double underlined sequences represent transcriptional start sites, Inrs, important promoter elements

and potential Spl binding sites.

A in start codon ATG is designated as +1.

100

APPENDIX B

1

APPENDIX B

DNA SEQUENCE OF HKI UPSTREAM REGION

pBS39.5 (1 TO 3450)
(-3366 to +84, relative to start codon)

GAATI'CCAAG ACAGCCAGGA GTACACAGAG GGACCGTGTC TTGAAAAAAC
:5

51 AACCAACCAA CCAATCAACA TAACCCATGC ATGAAGCCT G AAGAGATGAC

101

151

201

251

301

351

401

451

501

551

601

651

701

751

801

851

901

951

TCAGTGGTAA AGAGT'I'I'I'T G ACT GCT CT AG CAGAGGACCT AAGTTCAATT
CCCAGCACCC ACT GGT GTCT CACAAACATC T'I'I'AACTCCA G'I'TCCAGGGG
ACCT GATG'IT CT CIT CT GAC CT CCTC AAGC ATCAGGAACA TGTATGGTAC
GT ACACACAC ACACACACAC ACACACACAC ACACACACAC ACACACACT A
TACATACATA TATACATGTA AGCAAAATCT TCATATAAAA AAACCTAAAA
AAATTAAAAA TAAATCCAGG CATAGTAGCG ATCACC'ITGG GCC'I'I‘GCGCT
TCCCAGGCAA GCGCT CT ACC ACT GAGCT AA ATCCCCAACC CCATAGTAGC
GATAACCTTF AAGCCCAGCA CTTGGGAGGC AGAGGTAGGC AGATCT CT GT
GAGTTCAAGG CCAGCCTGGT CT ATATAG’I'I‘ CCAGGACTGC C AAAGCT ATA
CAGTAAGACC CT G'I'I'I'CAAA AACTAATAAA AAACCAAC AA GAAACA'ITCA
TGAGGTAGCC TAGGGC'I'I' AA CCAAGAGAAG ATACAACACG GCCATAGTGC
TGCCTCCAGG GAATGGCCT G ATGGAAGT GA TAAGGCCTGC TCCGGCAGAC
CGT CT CCCCT ATCAGGGAGG CAGTCTTGAC TCAG'I'I'GCTI' ATCTGGCCCA
GT CT GCCT GA GGTGGTGGGG TGGGATGGAA AGAGTGCTGG CT AAGCCT CA
GAGTCCCGGC TCGAAATTCT ACGGCAACCT GCCGCTGGGC AAGATGGCCC
ACCAGCTGCG CACCCTACAC CT'TG'ITTGTA GAGAAGAGCC CT GGTCT C CA
:9

'I'TCGCATGGA CT CT GAGCAC TGTCTGAGTG TGCCCTGTGG AGGTGCTGGG

GCCTGGGAGG CCAGGGTGAG TGGCTGAGGT CAGGGGAGGC CAGCTCGGGA

101

3317

-3 267

-3217

-3167

-3117

-3067

-3017

-2967

-2917

-2867

-2817

-2767

-2717

-2667

-2617

-2567

-2517

-2467

-2417

-2367

«am »i-ifii “I!
r’

1001

1051

1101

1151

1201

1251

1301

1351

1401

1451

1501

1551

1601

1651

1701

1751

1801

1851

1901

1951

2001

2051

2101

2151

2201

2251

102

AGGAGGTCAG ATGTGCC’I'T C CT GTGGTGAG GTCGGAACCT GGACAGGTGT

AGGAGAATCG GGACGGAAGG GTGGTATCGA GGGGAGCCAC ACAGAGTGGA

TGTGTGGGGT TAAGGACAGT GGGACACACT AGTGCI‘GTCG GTCAGGGGCT
GGGTAAGAGA 'I'I‘CCGCGCTC AGAGTCCGGT AACAGGGCCA GTGTG'I'I'I’AA
ATI‘GGC'I'I‘CC ACCAGGATGC AGGGCCCAGA AGGGACTTTG AGCAGGGGCA
TAACGTAA’I'I‘ AAGG‘I'I‘GCTI‘ TTCAGTGAGA CTGTCGCGGC CTGCAGCACA
GAATGGCTI‘G GATGGCGGGT AGCAACCAAC CAGAAAGCCA GCTCAAATCG
TrCTrGTCAT A'I'I’I‘CTAGCT AGACAAGAGA AAAACTGGGG TGGAGG'I’GGT
GTTGGGAGTA GCAGTGAGTC TGACI‘TAGAG AGATATGCAT TAGGAAACAA
CACTAAGCCT CGGTGCCTGG GTGTATGTGT GTG'I’GTCTGT GTCC’I‘GAGTC
ACC'ITGGTAC AGGGGTTGAG GGAAGGGACA GCAGAGGTAC CTACCTGCCT
ACAAGCTGGT CCAGC'ITITC CCCAGCTCTG G'I'I'I‘GTI‘GGC TAAGGGTTGT
ATCCCCATAT AATCCCACAG A'I'I‘ATAGTGA TGGCTGAAGG TTGGGGGAGG
GGTCCATGAG CCCGACACTG GGGTTGGAGA ATGGGGTGGC CCATTCAACC
TACAGACCTC TGAGAAAACA AAACAACCCT CCATCCCCAA AGACITGCCA
GCCTTGAGAG CTGGCCGAAG TACA'ITI‘GCA GATTCAAA’I‘G CAGC’I‘CAGAA
GAACCAT'I'IT TGTCTI‘GACG CTGTCAGGCA ATATTGGTAG CGTGTGTGTG
TGTGTGTGTG TI‘AGTTTGAG A'I‘TTACGTGG GACTCAATGC TAATC'ITI'I'I‘
AAGGGAAGGG CAACTATAAC AGGCCCTCCC TGGGCTGGAA ATGGTGACAT
TCAGGTGACA GGTCCCCCAA ACTI'I‘AACCT GTAGGTTCCC GAGTCCG'ITC
TATGAGAGTA CCTCCCATCT AATCTA‘I'I‘AG ATACCAGTGT TTATGCTCTI‘
TCAGAAAAAT TACAGGGTGT AGGCATCAGC TCCATC‘ITAT GGGTAAAGAA
AGAAAGCCTG AGAGGTGACT CAGGACACAT CTGCACCAGC CA'I'I‘AGGCAG
GGTGGTGGCT GGAAGGGCCC TGGGATACTI‘ GAAGGGCACC CTGGGGTATC
TI'I‘GCCCCGA TGGATGCTGG GCCTAGCCAG GGGCTCCCAG TCCCCAGGCG

TGGGGTAGAA GTTGGACT CT ATAGTCACCT AAGGGCCT AT GTTGCAGTCC

-2317

-2267

~2217

-2l67

-2117

-2067

-2017

-l967

-l917

-l867

-1817

-I767

-l717

-l667

-1617

-1567

-1517

-1467

-1417

-1367

-l317

-1267

-1217

-ll67

-1117

-1067

ll .2. n.'.A‘“-/q

 

103

2301 TGGTCTCAGG CACACGGCC’I‘ GCAGGAGAGG CTTAAAAGAA GAGAAAC’I‘GC -1017
a
2351 ACACAATGGC TAGGTCACCG GCG'ITAAAGC TAAGCAAACC CAGCGCTACT -967

2401 CCTGGGCAGC AACTGCAAAG CG'ITI'CTT CA GGTCCCTCAC CT GTAGAATC -9 l 7
2451 AGAGCGGT AG TCGCCTCTGC ATGTCTGAGT TCTTACATGT CGTAATGTAC -867
2501 AAACACGATI‘ TCCCCTCAAT CACCGCCCGG AACAGTACCT CCAAC'ITCCC -817
2551 AGACCCGGAT GCCCCAAGAG CCAGAGTAGG GTGGGAAAAT CGGGACAGGC -767

2601 CCCCAAA'ITC CACTCGGGGG CC'I'I' GAGCT C TTACATGGTG TCACGGGGGC -717
:>
2651 AGGTAGTI'I‘G GGT'I'I'AGCAA TGTGAACT CT GACAA’ITI‘GG GATGTAGAGC -667

2701 TGGTGGGCCA TCGTGGGACG CCAAGCATCA TCC'ITAGAGT TTGGATCCTI‘ -617
2751 TAGGGCAGGC AGGCACAGGG ACCCAGTGCG AGATCAGTGA AGCCGCCCAG -567
2801 TTTCGGC'I'I' C CGCTC'ITITT CCACGCCCAC TTGCGTGC’I'I‘ CT CCAACAGT -517

2851 GTGGATGGGA GGGGTGGGGG ACGAGCCCT A ATCTCCGAGG AAGGGGTGTG -467
2)
2901 GCCCCGTTCG TGTTCTCCAG TI'I‘GTGGCGT CCTGGATCT G TCCT CT GGTC -417

2951 CCCTCCAGAT CGTGTCCCAC ACCCACCCGT TCAGGCATGG CACTGTGCCG -367

3001 CCACGCGTGA CCGTGCGCT C C’I'I‘ACGTGGG GGACGTGCAG GGTGCTGCCT -317
:>
3051 CCI'TTCCGGT GCGGGAGGGA GCGGCCGTCT TI‘CTCCTGCT CT GGCT GGGA -267

3101 AGCCCCAGCC ATTGCGCTGC AGAGGAGACT TGCAGCCAAT GGGGACT GAG -217
Z)
3151 GAAGTGGGCC GGCTGGCGGT TGTCACCCT C CCGGGGACCG GAGCTCCGAG -167
:9

3201 GTCTGGAGAG CGCAGGCAGA CGCCCGCCCC GCCCGGGGAC TGAGGGGGAG -117
3251 GAGCGAAGGG AGGAGGAGGT GGAGTCTCCG ATCTGCCGCT GGAGGACCAC -67

3301 TGCTCACCAG GGCI‘ACI‘GAG GAGCCACTGG CCCCACACCT GC’I‘I'ITCCGC 17
+1
3351 ATCCCCCACC GTCAGCATGA TCGCCGCGCA ACTACTGGCC TA'I'I‘ACTTCA +34
MI AAQ LLAYYFT
3401 CCGAGCTGAA GGATGACCAA GTCAAAAAGG TGAGCCCCGC CGGCGCCGCC +84

ELK DDQVKK

:9 Represent 5’ end points of HKI upstream sequence in promoter-reporter constructs.

APPENDIX C

APPENDIX C

SCHEMATIC REPRESENTATION OF RESTRICTION SITES

IN HKI UPSTREAM REGION

pBS39.5 (1 TO 3366)

104

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APPENDIX D

APPENDIX D

LIST OF RESTRICTION SITES IN HKI UPSTREAM REGION

pBS39.5 (1 TO 3366)

AceIII CAGCTCnnnnnnn'nnnn_

Cutsat: 0 1003 1351 1585 1746 1803 2089 2685 3366
Size: 1003 348 234 161 57 286 596 681

A011 c'cc_c

Cutsat: 0 832 1163 1286 1315 2454 2523 2793 2810
Size: 832 331 123 29 1139 69 270 17

Cutsat: 2810 2998 3061 3071 3166 3224 3229 3286 3347
Size: 188 63 10 95 58 5 57 61

Cuts at: 3347 3366

Size: 19
MIN A'CryG__T
Cuts at: 0 238 314 2485 3003 3366
Size: 238 76 2171 518 363
AluI AG'CT

Cutsat: 0 426 545 855 993 1341 1368 1555 1564
Size: 426 119 310 138 348 27 187 9

Cuts at: 1564 1575 1760 1793 2079 2379 2627 2699 3193

Size: 11 185 33 286 300 248 72 494
Cuts at: 3193 3366
Size: 173

A1wIGGATCnnnn'n_

Cutsat: 0 2738 2751 2942 3366
Size: 2738 13 191 424

A1wNICAG_nnn'CTG
Cuts at: 0 1148 2412 3366
Size: 1148 1264 954

109

110

Apal G_GGCC'C

Cutsat: 0 1226 2169 3366
Size: 1226 943 1197

ApoI f'AATTJ
Cutsat: 0 l 814 2605 3366
Size: 1 813 1791 761
AvaI C'yCGr__G

Cutsat: 0 994 1988 2613 3180 3232 3366
Size: 994 994 625 567 52 134

AvaII G'GwC_C
Cutsat: 0 31 135 199 1558 1651 1961 2431 2769
Size: 31 104 64 1359 93 310 470 338
Cuts at: 2769 2947 3185 32.94 3366
Size: 178 238 109 72

AvrII C'CI‘AG_G

Cuts at: 0 609 3366
Size: 609 2757

Bad GrTACnnnnGTnnnnnnnnnn_nnnnn‘

Cutsat: 0 1498 1531 2022 2055 3366
Size: 1498 33 491 33 1311

BamHI G'GATC_C

Cuts at: 0 2743 3366
Size: 2743 623

Banl G'Ger_C

Cutsat: 0 1462 1536 2185 3366
Size: 1462 74 649 1181

BanII G_rGCy'C

Cutsat: 0 890 1226 1662 2169 2235 2629 2877 3195
Size: 890 336 436 507 66 394 248 318

Cutsat: 3195 3366
Size: 171

T‘-
I‘

 

111

Bbvl GCAGCnnnnnnnn'nnnn_

Cutsat: 0 636 842 1305 1802 2418 3031 3103 3144
Size: 636 206 463 497 616 613 72 41

Cuts at: 3144 3366
Size: 222

Bed CCATC

Cutsat: 0 672 776 845 1313 1631 1732 2016 2083
Size: 672 104 69 468 318 101 284 67

Cuts at: 2083 2211 2709 2856 3366
Size: 128 498 147 510

Bee83I CIT GAGnnnnnnnnnnnnnn_nn'

Cuts at: 0 209 1774 2643 3366
Size: 209 1565 869 723

Bcefl ACGGCnnnnnnnnnnnn‘n_

Cutsat: 0 654 837 2330 3060 3366
Size: 654 183 1493 730 306

BciVI GGATACnnnnn_n'

Cutsat: 0 1593 2184 3366
Size: 1593 591 1182

BfaI C‘TA_G

Cutsat: 0 127 610 1129 1365 1369 2223 2360 3366
Size: 127 483 519 236 4 854 137 1006

B111 ACTGGGnnnn'n_

Cutsat: 0 741 1393 1676 2230 2766 2790 3366
Size: 741 652 283 554 536 24 576

BglI GCCn_nnn'nGGC

Cutsat: 0 957 3366
Size: 957 2409

BglII A'GATC_T

Cutsat: 0 491 3366
Size: 491 2875

BmgI GkGCCC

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112

Cutsat: 0 932 1224 2167 2186 3366
Size: 932 292 943 19 1180

Bpll GAGnnnnnCT C

Cuts at: 0 990 2008 3366
Size: 990 1018 1358

Bme CT GGAGnnnnnnnnnnnnnn_nn'

Cutsat: 0 171 639 2900 2938 3224 3310 3366
Size: 171 468 2261 38 286 86 56

BpulOI CC'TnA_GC

Cutsat: 0 974 1590 3366
Size: 974 616 1776

Bpu11021 GC'TnA_GC

Cutsat: 0 791 2380 3366
Size: 791 1589 986

BsaI GGTCTCn'nnnn_

Cutsat: 0 899 2308 3366
Size: 899 1409 1058

BsaAI yAC'GTr

Cutsat: 0 250 1876 3025 3366
Size: 250 1626 1149 341

353111 Gr'CG_yC

Cutsat: 0 2718 2927 3220 3366
Size: 2718 209 293 146

85311 C'CnnG_G

Cutsat: 0 194 385 402 609 656 889 952 961
Size: 194 191 17 207 47 233 63 9

Cutsat: 961 1458 1466 1502 1928 1929 2168 2169 2189
Size: 497 8 36 426 1 239 1 20

Cutsat: 2189 2190 2227 2243 2401 2885 3180 3181 3196
Size: 1 37 16 158 484 295 1 15

Cutsat: 3196 3232 3233 3307 3366
Size: 36 1 74 59

113

BsaWI w'CCGG_w

Cutsat: 0 1175 3055 3187 3366
Size: 1175 1880 132 179

BsaXI ACnnnnnCl' CC

Cutsat: 0 1397 1410 1923 2536 3073 3191 3270 3366
Size: 1397 13 513 613 537 118 79 96

35131 CAACAC
Cutsat: 0 636 1401 1450 3366
Size: 636 765 49 1916
85061 CCCGT

Cuts at: 0 2645 2904 2977 3366
Size: 2645 259 73 389

BseRI GAGGAGnnnnnnnn_nn'

Cutsat: 0 212 3137 3263 3275 3278 3333 3366
Size: 212 2925 126 12 3 55 33

BsgI GTGCAGnnnnnnnnnnnnnn_nn'

Cutsat: 0 2116 2332 3056 3366
Size: 2116 216 724 310

851131 CG_ry'CG

Cutsat: 0 3075 3366
Size: 3075 291_

BsiHKAI G_wGCw'C

Cutsat: 0 919 2629 3195 3366
Size: 919 1710 566 171

leI CCnn_nnn'nnGG

Cutsat: 0 194 471 662 712 713 834 939 1022
Size: 194 277 191 50 1 121 105 83

Cutsat: 1022 1182 1577 1667 1668 1930 1934 2088 2228
Size: 160 395 90 1 262 4 154 140

Cutsat: 2228 2249 2250 2554 2577 2616 2802 2891 2923
Size: 21 1 304 23 39 186 89 32

 

114

Cutsat: 2923 3026 3165 3182 3187 3202 3233 3234 3366
Size: 103 139 17 5 15 31 1 132

BsmAI GTCTCn'nnnn_

Cutsat: 0 172 707 899 1271 2308 3119 3279 3366
Size: 172 535 192 372 1037 811 160 87

8511131 CGT CT Cn'nnnn_

Cuts at: 0 707 3366
Size: 707 2659

BsmFI GGGACnnnnnnnnnn'nnnn_

Cutsat: 0 44 212 788 1074 1135 1246 1539 1893
Size: 44 168 576 286 61 111 293 354

Cutsat: 1893 1947 2225 2417 2606 2729 2782 2882 2933
Size: 54 278 192 189 123 53 100 51

Cutsat: 2933 2949 3044 3156 3198 3250 3366
Size: 16 95 112 42 52 116

Bsp241 CCAnnnnnnGTCnnnnnnnn_nnnnn'

Cuts at: 0 2947 2979 3366
Size: 2947 32 387

Bsp12861 G_dGCh'C

Cutsat: 0 890 919 934 1226 1662 2169 2188 2235
Size: 890 29 15 292 436 507 19 47

Cuts at: 2235 2629 2877 3195 3366
Size: 394 248 318 171

BspGI CTGGAC

Cutsat: 0 1041 1561 3366
Size: 1041 520 1805

BspLUl 11 A'CATG_T

Cutsat: 0 238 314 2485 3366
Size: 238 76 2171 881

BspMI ACCT GCnnnn'nnnn_

Cutsat: 0 836 1552 2640 3346 3366
Size: 836 716 1088 706 20

115

8er ACT G_Gn'

Cutsat: 0 166 188 748 1188 1389 1672 2034 2237
Size: 166 22 560 440 201 283 362 203

Cutsat: 2237 2773 2797 2917 3331 3366
Size: 536 24 120 414 35

BsrBI CCG'CT C
Cutsat: 0 2454 2812 3071 3366
Size: 2454 358 259 295
13er1 GCAATG_nn'

Cutsat: 0 2674 3109 3366
Size: 2674 435 257

Ber1 r'CCGG_y

Cutsat: 0 2367 3158 3366
Size: 2367 791 208

BerI T'GTAC_A

Cuts at: 0 2496 3366
Size: 2496 870

BstAPI GCAn_nnn'nTGC

Cutsat: 0 2412 3366
Size: 2412 954

BstEII G'GTnAC_C

Cuts at: 0 2363 3366
Size: 2363 1003

B500 CCAn_nnnn'nTGG

Cuts at: 0 905 2850 3366
Size: 905 1945 516

BstYI r'GATC_y

Cutsat: 0 491 2743 2934 3366
Size: 491 2252 191 432

Bsu36I CC’TnA_GG

116
Cuts at: 0 757 2279 3366
Size: 757 1522 1087
Cac81 GCn'nGC

Cutsat: 0 409 512 687 788 991 1291 1339 1749
Size: 409 103 175 101 203 300 48 410

Cutsat: 1749 1762 2319 2757 2761 2835 3160 3164 3214
Size: 13 557 438 4 74 325 4 50

Cutsat: 3214 3224 3366
Size: 10 142

Cjel CCAnnnnnnGTnnnnnnnnn_nnnnnn'
Cuts at: 0 357 391 952 986 1078 1079 1112 1113
Size: 357 34 561 34 92 1 33 1

Cutsat: 1113 1563 1597 1657 1691 2946 2980 3366
Size: 450 34 60 34 1255 34 386

CjePI CCAnnnnnnnTCnnnnnnnn_nnnnnn'

Cutsat: 0 227 260 874 907 1330 1363 1368 1401
Size: 227 33 614 33 423 33 5 33

Cutsat: 1401 1487 1520 2153 2186 2200 2233 2382 2415

Size: 86 33 633 33 14 33 149 33

Cutsat: 2415 2713 2746 2881 2914 2947 2980 3366

Size: 298 33 135 33 33 33 386
CviJIrG'Cy

Cutsat: 0 14 86 391 426 463 510 514 545
Size: 14 72 305 35 37 47 4 31

Cuts at: 545 608 615 641 666 685 746 790 795
Size: 63 7 26 25 19 61 44 5

Cuts at: 795 809 847 855 888 951 960 973 989
Size: 14 38 8 33 63 9 13 16

Cutsat: 989 993 1086 1148 1187 1205 1224 1289 1306
Size: 4 93 62 39 18 19 65 17

Cutsat: 1306 1337 1341 1368 1457 1555 1564 1575 1589
Size: 31 4 27 89 98 9 11 14

Cutsat: 1589 1633 1660 1689 1751 1760 1764 1793 1923

 

Size:

Cuts at:

Size:

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Size:

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Size:

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CviRI TG'CA

Cuts at:

Size:

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Size:

DdeI C'TnA_G

Cuts at:

Size:

Cuts at:

Size:

Cuts at:

Size:

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Size:

DpnI GA'TC

Cuts at:

Size:

DraI 'I'I'I‘AAA

Cuts at:

Size:

1923

2901

2294 2321

913

44 27

11 145

7 52

21 7

173 20

119 11

0 79 1219

79 1140 74

27 28

0 100 139

100 39
924 974
1 l 50

DraIII CAC_nnn'GTG

Cuts at:

0 1095 3366

1934 2079 2106 2139 2158 2167 2221

283

194

117

29 62 9 4 29 130

2226
27 33 19 9 54 5

2226 2233 2285 2317 2330 2359 2379 2570 2599

32 13 29 20 191 29

2599 2620 2627 2699 2707 2792 2806 2875 2901

72 8 85 14 69 26

3074 3094 3102 3108 3135 3158 3162 3193

8 6 27 23 4 31

3193 3312 3323 3330 3366

7 36

1790 2133 2294
343 161

1437 1778
341 12

1293
144

2349 2415 2469 3037 3119 3132 3366

66 54 568 82 13 234

797 913
116

422 730 757 791

308 27 34 6
1590 1710
120

1168 1424 1453 1494
256 29 41 96

1710 1794 2108 2119 2279 2305 2380 2475 2733

84 314 11 160 26 75 95 258
2733 3146 3240 3316 3366
413 94 76 50
0 381 493 2745 2783 2936 2959 3281 3366
381 112 2252 38 153 23 322 85
0 1198 3366
1198 2168

 

118

Size: 1095 2271
DrdI GACnn_nn'nnGTC

Cutsat: 0 2271 3366
Size: 2271 1095

DrdII GAACCA

Cuts at: 0 1803 3366
Size: 1803 1563

Bad y'GGCC_r

Cutsat: 0 639 1762 3072 3366
Size: 639 1123 1310 294

EagI C'GGCC_G

Cutsat: 0 3072 3366
Size: 3072 294

Earl CT C'I‘T Cn'nnn_

Cutsat: 0 85 217 878 2333 3366
Size: 85 132 661 1455 1033

150047111 AGC'GCT

Cutsat: 0 412 2394 3366
Size: 412 1982 972

E00571 CT GAAGnnnnnnnnnnnnnn_nn'

Cutsat: 0 109 1655 2411 3366
Size: 109 1546 756 955

56601091 rG'GnC_Cy

Cutsat: 0 135 199 949 1222 1922 1961 2165 2166
Size: 135 64 750 273 700 39 204 1

Cutsat: 2166 2283 2431 2598 2618 2769 3366
Size: 117 148 167 20 151 597

15le G'AA'IT_C

Cutsat: 0 1 3366
Size: 1 3365

EcoRII 'CCwGG_

119

Cutsat: 0 14 193 365 402 514 530 655 889
Size: 14 179 172 37 112 16 125 234

Cutsat: 889 951 960 1037 1211 1465 1928 2168 2189

Size: 62 9 77 174 254 463 240 21
Cuts at: 2189 2226 ‘2243 2298 2400 2930 3306 3366
Size: 37 17 55 102 530 376 60
Paul CCCGCnnnn'nn_

Cutsat: 0 1308 3054 3231 3236 3366
Size: 1308 1746 177 5 130

Fnu4HI GC'n_GC

Cutsat: 0 650 832 856 1287 1294 1791 2407 2793
Size: 650 182 24 431 7 497 616 386

Cutsat: 2793 2998 3045 3072 3117 3133 3286 3366
Size: 205 47 27 45 16 153 80

FokI GGATGnnnnnnnnn'nnnn_

Cutsat: 0 786 1111 1228 1323 1718 2225 2570 2703
Size: 786 325 117 95 395 507 345 133

Cutsat: 2703 2715 2866 3336 3366
Size: 12 151 470 30

FspI TGC'GCA

Cuts at: 0 859 3366
Size: 859 2507

GdiII C'GGCC_1’

Cuts at: 0 639 1762 3072 3366
Size: 639 1123 1310 294

HaeI wGG'CCw

Cutsat: 0 510 666 685 960 989 3366
Size: 510 156 19 275 29 2377

HaeII r_GCGC‘y

Cutsat: 0 414 2396 3366
Size: 414 1982 970

HaeIII GG'CC

120

Cutsat: 0 391 510 641 666 685 746 847 951
Size: 391 119 131 25 19 61 101 104

Cutsat: 951 960 989 1187 1224 1289 1689 1764 1923

Size: 9 29 198 37 65 400 75 159
Cuts at: 1923 2167 2221 2285 2317 2599 2620 2707 2901
Size: 244 54 64 32 282 21 87 194
Cuts at: 2901 3074 3158 3330 3366
Size: 173 84 172 36
Hgal GACGCnnnnn'nnnnn_

Cutsat: 0 1826 2726 2916 3228 3366
Size: 1826 900 190 312 138

HgiEII ACCnnnnnnGG'I‘

Cuts at: 0 1042 3366
Size: 1042 2324

HhaIG__CG'C
Cutsat: o 398 413 860 1167 2395 3017 3116 3212
Size: 398 15 447 307 1228 622 99 96

Cutsat: 3212 3366
Size: 154

Hin4I GAbnnnnanC

Cutsat: 0 890 990 2008 2456 2511 2741 2877 2939
Size: 890 100 1018 448 55 230 136 62

Cutsat: 2939 3073 3190 3270 3299 3366
Size: 134 117 80 29 67

I-Iinfl G'AnT_C

Cutsat: 0 98 728 801 909 1055 1159 1172 1416
Size: 98 630 73 108 146 104 13 244

 

Cutsat: 1416 1496 1781 1881 1991 2117 2265 2446 3272

Size: 80 285 100 110 126 148 181 826

Cutsat: 3272 3366
Size: 94

thl GGTGAnnnnnnn_n'

121
Cuts at: 0 374 977 1037 1491 1955 1966 2126 2267
Size: 374 603 60 454 464 1 1 160 141

Cutsat: 2267 2357 2429 2512 3165 3296 3366
Size: 90 72 83 653 131 70

Kpnl G_GTAC'C

Cutsat: 0 1540 3366
Size: 1540 1826

MaeIII 'GTnAC_

Cutsat: 0 1178 1497 1943 1954 2114 2273 2363 2639
Size: 1178 319 446 11 160 159 90 276

Cutsat: 2639 3006 3171 3366
Size: 367 165 195

MboII GAAGAnnnnnnn_n'

Cutsat: 0 102 204 320 639 895 1810 2350 2417
Size: 102 102 116 319 256 915 540 67

Cuts at: 2417' 3366
Size: 949

Mlul A'CGCG_T

Cuts at: 0 3003 3366
Size: 3003 363

Mmel TCCrACnnnnnnnnnnnnnnnnnn_nn'

Cutsat: 0 1012 1654 2242 2565 2867 3366
Size: 1012 642 588 323 302 499

MnlI CCI‘Cnnnnnn_n'

Cutsat: 0 21 126 230 233 469 475 595 663
Size: 21 105 104 3 236 6 120 68

Cuts at: 663 710 752 806 933 950 969 979 996
Size: 47 42 54 127 17 19 10 17

Cutsat: 996 1021 1072 1386 1468 1511 1527 1640 1717
Size: 25 51 314 82 43 16 1 13 77

Cutsat: 1717 1738 1935 2021 2105 2320 2445 2474 2524
Size: 21 197 86 84 215 125 29 50

Cutsat: 2524 2548 2852 2880 2952 2962 3058 3115 3141

122

Size: 24 304 28 72 10 96 57 26

Cutsat: 3141 3187 3191 3235 3241 3253 3256 3259 3285
Size: 46 4 44 6 12 3 3 26

Cuts at: 3285 3311 3366
Size: 26 55

Msel T'TA_A

Cutsat: 0 182 354 459 617 1110 1197 1259 1899
Size: 182 172 105 158 493 87 62 640

Cutsat: 1899 1974 2332 2374 3366
Size: 75 358 42 992

MslI CAynn'nm’l‘G

Cutsat: 0 903 1129 1214 1952 2832 3366
Size: 903 226 85 738 880 534

MspI C'CG__G

Cutsat: 0 692 806 1176 2368 2527 2555 3056 3159
Size: 692 114 370 1192 159 28 501 103

Cutsat: 3159 3181 3188 3233 3366
Size: 22 7 45 133

MspAlI CmG'CkG

Cutsat: 0 834 855 3288 3366
Size: 834 21 2433 78

Mon GCnn_nnn'nnGC

Cutsat: 0 404 743 752 792 957 1312 1561 1757
Size: 404 339 9 40 165 355 249 196

Cutsat: 1757 2164 2291 2327 2390 2412 2460 2567 3228
Size: 407 127 36 63 22 48 107 661

Cuts at: 3228 3366
Size: 138

Neil CC's_GG

Cutsat: 0 806 2527 2555 3181 3182 3233 3234 3366
Size: 806 1721 28 626 1 51 1 132

NgoAIV G'CCGG_C

 

123

Cutsat: 0 3158 3366
Size: 3158 208

NlaIII _CATG'
Cutsat: o 79 83 242 318 602 908 1658 2473
Size: 79 4 159 76 284 306 750 815
Cuts at: 2473 2489 2637 2989 3366
Size: 16 148 352 377

NlaIV GGn'nCC

Cutsat: 0 32 200 950 1036 1085 1224 1464 1538
Size: 32 168 750 86 49 139 240 74

Cutsat: 1538 1652 1963 1986 2167 2187 2234 2433 2600

Size: 114 311 23 181 20 47 199 167
Cutsat: 2600 2619 2745 2770 2771 2902 2949 3186 3322
Size: 19 126 25 1 131 47 237 136
Cuts at: 3322 3331 3366
Size: 9 35

NsiI A_TGCA'T
Cutsat: 0 81 1439 3366
Size: 81 1358 1927

NSpI r__CATG'y

Cuts at: 0 242 318 2473 2489 3366
Size: 242 76 2155 16 877

PM CCAn_nnn'nTGG

Cutsat: 0 471 662 2088 2923 3366
Size: 471 191 1426 835 443

PleI GAGTCnnnn'n_

Cutsat: 0 92 722 809 903 1180 1424 1504 1875
Size: 92 630 87 94 277 244 80 371

Cutsat: 1875 1999 2111 2259 3280 3366
Size: 124 112 148 1021 86

PspSII rG'GwC_Cy

Cutsat: 0 135 199 1961 2431 2769 3366
Size: 135 64 1762 470 338 597

 

124

PstI C_TGCA'G

Cutsat: 0 1295 2323 3121 3366
Size: 1295 1028 798 245

PvuII CAG'CT G

Cuts at: 0 855 3366
Size: 855 2511

Real T‘CATG_A

Cuts at: 0 598 3366
Size: 598 2768

RleAI CCCACAnnnnnnnnn_nnn'

Cutsat: 0 1093 1631 2983 3349 3366
Size: 1093 538 1352 366 17

RsaIGT'AC
Cutsat: 0 22 248 252 1508 1538 1771 2009 2498
Size: 22 226 4 1256 30 233 238 489
Cutsat: 2498 2536 3366
Size: 38 830

SacI G_AGCT‘C

Cuts at: 0 2629 3195 3366
Size: 2629 566 171

SanDI GG'GwC_CC

Cuts at: 0 2769 3366
Size: 2769 597

Sap] GCTCI'I’Cn'nnn_

Cuts at: 0 878 3366
Size: 878 2488

Sau961 G'GnC_C

Cutsat: 0 31 135 199 389 745 846 949 1185
Size: 31 104 64 190 356 101 103 236

Cutsat: 1185 1222 1223 1558 1651 1688 1922 1961 2165
Size: 37 1 335 93 37 234 39 204

 

125

Cutsat: 2165 2166 2219 2283 2431 2598 2618 2705 2769
Size: 1 53 64 148 167 20 87 64
Cutsat: 2769 2900 2947 3156 3185 3294 3329 3366
Size: 131 47 209 29 109 35 37
Sau3AI‘GATC__
Cutsat: 0 379 491 2743 2781 2934 2957 3279 3366
Size: 379 112 2252 38 153 23 322 87
SchI CC'n_GG
Cutsat: 0 16 195 367 404 516 532 657 806
Size: 16 179 172 37 112 16 125 149
Cutsat: 806 891 953 962 1039 1213 1467 1930 2170
Size: 85 62 9 77 174 254 463 240
Cutsat: 2170 2191 2228 2245 2300 2402 2527 2555 2932
Size: 21 37 17 55 102 125 28 377
Cutsat: 2932 3181 3182 3233 3234 3308 3366
Size: 249 1 51 l 74 58
SfaNI GCATCnnnnn'nnnn_
Cutsat: 0 238 1206 2082 2203 2548 2734 3358 3366
Size: 238 968 876 121 345 186 624 8
SfcI C'TryA_G
Cutsat: 0 1291 1700 1979 2269 2319 2441 3117 3366
Size: 1291 409 279 290 50 122 676 249
S111 GGCCn_nnn'nGGCC
Cutsat: 0 957 3366
Size: 957 2409
SgrAI Cr'CCGG_yG
Cutsat: O 2367 3366
Size: 2367 999
SimI GG'GTC_
Cutsat: 0 556 1651 2551 2769 3366
Size: 556 1095 900 218 597

Smal CCC'GGG

Cuts at:
Size:

Smll C‘TyrA__G

Cuts at:
Size:

SnaBI TAC'GTA

Cuts at:
Size:

SpeI A'C’I‘AG_T

Cuts at:
Size:

126

0 3182 3234 3366
3182 52 132

0 224 1753 2622 3366
224 1529 869 744

0 250 3366
250 3116

0 1128 3366
1128 2238

Sse83871 CC_TGCA'GG

Cuts at:
Size:

0 2323 3366
2323 1043

Sse8647I AG'GwC_CT

Cuts at:
Size:

SspI AAT'ATI‘

Cuts at:
Size:

StuI AGG'CCT

Cuts at:
Size:

StyI C'waG_G

Cuts at:
Size:

Tail _ACGT'

Cuts at:
Size:

Tan T'CG_A

Cuts at:
Size:

0 135 3366
135 3231

0 1832 3366
1832 1534

0 685 3366
685 2681

0 385 609
385 224 893

1502 3366
1864

0 252 1256
252 1004 622

1878 3027 3036 3366
1149 9 330

0 811 1077 3366
811 266 2289

127

TanI GACCGAnnnnnnnnn_nn'

Cutsat: 0 172 1128 1458 2986 3366
Size: 172 956 330 1528 380

TatI wGTACw

Cuts at: 0 22 1771 2498 3366
Size: 22 1749 727 868

TauI GCSGC

Cuts at: 0 832 1287 2793 2998 3072 3286 3366
Size: 832 455 1506 205 74 214 80

TfiI G'AwT_C

Cutsat: 0 1055 1159 1781 2446 3366
Size: 1055 104 622 665 920

ThaI CG'CG

Cuts at: 0 1165 1286 3005 3366
Size: 1165 121 1719 361

TseI G'CwG_C

Cutsat: 0 649 855 1293 1790 2406 3044 3116 3132
Size: 649 206 438 497 616 638 72 16

Cutsat: 3132 3366
Size: 234

Tsp451 'GTSAC_

Cuts at: 0 1497 1943 1954 2114 2273 2363 2639 3006
Size: 1497 446 11 160 159 90 276 367

Cutsat: 3006 3171 3366
Size: 165 195

Tsp4CI AC_n'GT

Cutsat: 0 35 552 701 921 1118 1282 2534 2848
Size: 35 517 149 220 197 164 1252 314

Cutsat: 2848 2994 3012 3360 3366
Size: 146 18 348 6

Tsp5091 'AA'I'I‘_

 

128

Cutsat: 0 1 146 351 814 1199 1256 2057 2605

Size: 1 145 205 463 385 57 801 548
Cuts at: 2605 2683 3366
Size: 78 683

TSpRI CAsTGnn'

Cutsat: 0 108 166 426 924 1123 1195 1279 1418
Size: 108 58 260 498 199 72 84 139

Cutsat: 1418 1672 2041 2780 2791 2853 2997 3304 3331
Size: 254 369 739 11 62 144 307 27

Cutsat: 3331 3366
Size: 35

Tthl 1 ll GACn'n_nGTC

Cutsat: 0 35 1820 3366
Size: 35 1785 1546

Tthl 1 111 CAArCAnnnnnnnnn_nn'

Cutsat: 0 189 242 863 2516 2738 3366
Size: 189 53 621 1653 222 628

UbaDI GAACnnnnnnTCC

Cutsat: 0 205 2535 3366
Size: 205 2330 831

UbaEI CACCTGC

Cutsat: 0 3338 3366
Size: 3338 28

 

129

Enzymes that do cut:

AceIII AciI AflIII AluI AlwI AleI Apal ApoI
Aval AvaII AvrII BaeI Baml-II Banl BanIl Bbvl
Bch Bce83l Bcefl BciVl BfaI Bfil BglI BglII
Bmgl BplI Bme Bu 101 Bpul 1021 Bsal BsaAI BsaI-Il
BsaJI BsaWI BsaXI BSbI BscGI BseRI BsgI BSiEI
BsiHKAI BSlI BsmAl BsmBI BsmF I Bsp24 Bsp1286l BspGI
BspLUl 11 BSpMI Ber BsrBI Ber1 Ber1 Ber1 BstAPI
BstEII BstXI BstYl Bsu36l Cac8I Cjel CjePI CviJI
CviRl DdeI DpnI Dral DraIII Drdl DrdII Eael
Eagl Earl Eco47III E00571 EcoOlO9I EcoRI EcoRII FauI
Fnu4HI FokI FspI GdilI Hael Haell Haelll Hgal
HgiEII HhaI Hin4I Hinfl thl KpnI MaeIII MboII
MluI Mmel Mnll Msel Msll Mspl MspAlI Mwol
NciI NgoAIV NlaIII NlaIV NsiI NspI PflMl PleI
PspSII Pstl PvuII Rcal RleAI RsaI SacI SanDI
SapI Sau96I Sau3AI SchI SfaNI SfcI Sfil SgrAI
SimI SmaI Smll SnaBI SpeI Sse8387l Sse8647I Sspl
Stul StyI Tail Taql TanI Tatl TauI TfiI
Thal TseI Tsp4SI Tsp4Cl Tsp509l TspRI Tthllll TthlllII
UbaDI UbaEI

Enzymes that do not cut:

AatII Ach AflII Ahdl ApaLI AscI Bbsl Bcgl
BclI BsaBI BsmI BspEI Bssl-III BSSSI BstDSI BstZl7I
ClaI Ecil EcoNI EcoRV F sel Hincll HindIII Hpal
Mscl MunI NarI Ncol NdeI Nhel NotI Nrul
NspV PacI PH] 1081 PinAI Pmel Pmll PshAI Pspl4061
PvuI RerI Sacll SalI Scal SexAI Sgfl SmiI

SphI Srfl Sunl Vspl Xbal Xcml Xhol anI

      

HICHIG

IIIIIIIIIIIIIIIIII“