Post on 10-Aug-2020
Office of Graduate StudiesUniversity of South Florida
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CERTIFICATE OF APPROVAL__________________
Ph.D. Dissertation__________________
This is to certify that the Ph.D. Dissertation of
YA-LI YAO
with a major in Medical Sciences has been approvedfor the dissertation requirement on April 9, 2001
for the Doctor of Philosophy degree.
Examining Committee:
_________________________________________________Major Professor : Edward Seto, Ph.D.
_________________________________________________Member : Burt Anderson, Ph.D.
_________________________________________________Member: Peter Medveczky, M.D.
_________________________________________________Member: Richard Jove, Ph.D.
REGULATION OF YY1, A MULTIFUNCTIONAL TRANSCIPTION FACTOR
by
YA-LI YAO
A dissertation submitted in partial fulfillmentof the requirements for the degree of
Doctor of PhilosophyDepartment of Medical Microbiology and Immunology
College of MedicineUniversity of South Florida
May 2001
Major Professor: Edward Seto, Ph.D.
DEDICATION
To my parents, Ta-Chung (Righteous and Magnanimous) and Shiao-Hung (Little
Rainbow).
ACKNOWLEDGEMENTS
I am eternally grateful to my Lord, Who gave me the chance to fulfill my
dream of earning a Ph.D. degree. I would like to thank my mentor, Dr. Edward Seto,
for his endless support and guidance. Without him I would never have accomplished
my goal. My gratitude also extends to my committee members, who gave me
invaluable advice and helped me meet the requirements of the department. My
special acknowledgement goes to my husband, Dr. Wen-Ming Yang, whose help and
encouragement accompanied me when I needed them the most.
I thank Dr. Nancy Olashaw for critical reading and the core facilities at the
Moffitt Cancer Center as well as the Protein Chemistry Core at the University of
Florida for technical support. I would also like to express my gratitude to Kathy
(Mom), Linda, Susan, Cheryl, Sally, and B.J. They made my life as a graduate
student much easier and happier.
i
TABLE OF CONTENTS
LIST OF FIGURES iv
ABSTRACT viii
INTRODUCTION 1
Transcription is an important step in gene regulation. 1
Transcription in prokaryotes. 2
Transcription in eukaryotes. 3
RNA polymerase II and the eukaryotic promoter. 4
Activators and transcriptional activation. 6
Repressors and transcriptional repression. 8
Chromatin and transcription. 9
Chromatin remodeling. 10
Histone acetyltransferases as co-activators. 11
Histone deacetylases as co-repressors. 13
YY1 is a transcriptional regulator. 13
General characteristics of YY1. 14
YY1 and transcriptional initiation. 15
OBJECTIVES 17
MATERIALS AND METHODS 20
Isolation of YY1 genomic clones. 20
ii
Plasmids. 20
Sequencing of the human YY1 promoter. 22
Primer extension. 22
Cell line, transfection, luciferase assays, and CAT assays. 23
Recombinant proteins. 23
In vitro acetylation reactions. 24
Immunoprecipitation and western blot analysis. 24
In vitro protein-protein interaction assays. 25
Histone deacetylation assays. 25
Immunofluorescence analysis. 26
Electrophoretic mobility shift assays. 26
Gel filtration analysis of YY1. 27
Purification of a YY1 complex by anion exchange and immobilized-
metal affinity chromatography. 27
Coomassie staining and silver staining. 27
Purification of a YY1 complex by anion exchange and antibody-
affinity chromatography. 28
Purification of the F-YY1 complex. 28
Radioimmunoprecipitation analysis (RIPA) of F-YY1. 28
Accession number. 29
RESULTS 30
Determination of the transcriptional start site of the human YY1
gene. 30
iii
Transcriptional analysis of the human YY1 promoter. 31
Sequence analysis of the human YY1 promoter. 33
E1A-mediated repression of the human YY1 promoter. 35
YY1 does not autoregulate its own promoter. 37
YY1 is acetylated by p300 and PCAF. 38
Lysine-to-arginine mutations within YY1 residues 170-200
significantly reduce the transcriptional repression activity of YY1. 45
Acetylation of YY1 residues 170-200 increases YY1 binding to
HDACs. 46
HDAC1 and HDAC2 deacetylate YY1 170-200 but not the C-
terminal region of YY1. 46
HDACs bind YY1 at multiple regions. 52
YY1 contains associated histone deacetylase activity, which localizes
to C-terminal residues 261-333 of YY1.
52
Acetylation of YY1 at the C-terminal zinc finger domain decreases
the DNA-binding activity of YY1.
54
YY1 exists in a high molecular weight protein complex. 57
Purification of native YY1 complexes. 57
Purification of a Flag-tagged YY1 complex. 65
DISCUSSION 70
REFERENCES 80
ABOUT THE AUTHOR End Page
iv
LIST OF FIGURES
FIG. 1. Step-wise assembly of the transcriptional initiation complex. 5
FIG. 2. Regulation of transcription: a model. 7
FIG. 3. Determination of the 5’ end of the human YY1 transcript. 30
FIG. 4. Expression of luciferase enzymatic activity driven by the
human YY1 promoter in transiently transfected cells. 32
FIG. 5A. DNA sequences upstream of the translational start codon of
the human YY1 gene. 33
FIG. 5B. Comparison of the YY1 promoter DNA sequences between
mouse and human. 34
FIG. 6. Adenovirus E1A proteins repress the YY1 promoter. 36
FIG. 7. The YY1 protein does not autoregulate the YY1 promoter. 37
FIG. 8. Multiple functional domains of transcription factor YY1. 38
FIG. 9A. Acetylation of YY1 by PCAF. 39
FIG. 9B. Acetylation of YY1 by p300. 40
FIG. 9C. Acetylation of YY1 is dependent on p300 or PCAF. 41
FIG. 9D. YY1 is acetylated in vivo. 42
FIG. 9E. Identification of regions of YY1 acetylated in vivo. 43
FIG. 9F, G. Determination of the number of acetylated lysines by mass
spectrometry. 44
v
FIG. 10. The effect of acetylation of YY1 residues 170-200 on the
transcriptional repressor activity of YY1. 45
FIG. 11. Increased HDAC-binding to YY1 residues 170-200 by
acetylation. 47
FIG. 12A. YY1 peptide deacetylation by HDACs. 48
FIG. 12B. YY1 protein deacetylation by HDACs. 49
FIG. 12C. Deacetylation of YY1 serial deletion proteins by HDAC1. 50
FIG. 13. Mapping of the HDAC-interaction domains of YY1. 51
FIG. 14A. Histone deacetylase activity of endogenous YY1 in HeLa
cells. 52
FIG. 14B, C. Identification of amino acid 261-333 as the histone
deacetylase activity domain of YY1. 53
FIG. 14D. Expression of F-YY1 deletion mutants. 54
FIG. 14E. Sub-cellular localization of F-YY1 deletion constructs. 55
FIG. 15A, B. The effect of acetylation on YY1’s sequence-specific DNA-
binding activity. 56
FIG. 16. Gel filtration analysis of YY1 in HeLa cells. 57
FIG. 17A, B. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and IMAC. (A) A
Coomassie-stained SDS-polyacrylamide gel of the Q
Sepharose fractions. (B) Western blot analysis of the Q
Sepharose fractions. 58
FIG. 17C, D. Purification of a native YY1 complex from HeLa cells using
vi
anion exchange chromatography and IMAC. (C). EMSA of
the Q Sepharose fractions using the AAV P5+1 YY1 binding
sequence as a probe. (D). Histone deacetylation assay of the
Q Sepharose fractions. 59
FIG. 17E, F. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and IMAC. (E) Western
blot analysis of the Ni2+ column fractions. (F) EMSA of the
300 mM imidazole-eluted fractions using the AAV P5+1
YY1 binding sequence as a probe. 60
FIG. 17G. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and IMAC. (G) Silver-
staining analysis of the second fraction from the 300 mM
imidazole-eluted fractions. 61
FIG. 18A, B. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and antibody-affinity
chromatography. (A) A Coomassie-stained SDS-
polyacrylamide gel of the Q Sepharose fractions. (B)
Western blot analysis of the Q Sepharose fractions. 62
FIG. 18C, D. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and antibody-affinity
chromatography. (C) EMSA of the Q Sepharose fractions
using the AAV P5+1 YY1 binding sequence as a probe. (D)
Histone deacetylation assay of the Q Sepharose fractions. 63
vii
FIG. 18E. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and antibody-affinity
chromatography: (E) Silver-staining analysis of fractions
from the anti-YY1 H-10 column (lane 2) and the protein G
control column (lane 1). 64
FIG. 18F. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and antibody-affinity
chromatography. (F) Histone deacetylation assay from the
protein G control column (protein G) and the anti-YY1 H-10
column (H-10) against the H4 peptide. 65
FIG. 19A. Silver-staining analysis of the second fractions from the
Flag-alone (lane 1) column and the F-YY1 column (lane 2). 66
FIG. 19B. Histone deacetylation analysis of the second fractions from
the Flag alone column and the F-YY1 column against the H4
peptide. 67
FIG. 20A. A representative autoradiograph showing proteins bound to
Flag alone or F-YY1. 68
FIG. 20B. A representative autoradiograph showing proteins bound to
different F-YY1. 69
FIG. 21 Summary of regulation of YY1 by acetylation and
deacetylation. 72
viii
REGULATION OF YY1, A MULTIFUNCTIONAL TRANSCRIPTIONAL
FACTOR
by
YA-LI YAO
An Abstract
of a dissertation submitted in partial fulfillmentof the requirements for the degree of
Doctor of PhilosophyDepartment of Medical Microbiology and Immunology
College of MedicineUniversity of South Florida
May 2001
Major Professor: Edward Seto, Ph.D.
ix
Yin Yang 1 (YY1) is a sequence-specific DNA binding transcription factor that plays
an important role in development and differentiation. It activates or represses many
genes during cell growth and differentiation and is also required for the normal
development of the mammalian embryo. Moreover, it has been shown that YY1 may
function as a transcriptional initiator. In this dissertation, regulation of human YY1 is
analyzed systematically at three levels: At the genomic level, one major
transcriptional initiation site of the YY1 gene was mapped to 478 bp upstream of the
ATG translational start site. The YY1 promoter was localized to within 277 bp
upstream of the major transcriptional initiation site and was shown to contain multiple
binding sites for transcriptional factor Sp1 but lack a consensus TATA box. Over-
expression of the adenovirus E1A protein represses expression of the YY1 promoter.
At the polypeptide level, the activity of YY1 is regulated through acetylation by p300
and PCAF and deacetylation by HDACs. YY1 was acetylated in two regions: both
p300 and PCAF acetylated the central glycine/lysine-rich domain of residues 170-
200, and PCAF also acetylated YY1 at the C-terminal DNA-binding domain.
Acetylation of the central region was required for the full transcriptional repression
activity of YY1 and targeted YY1 for active deacetylation by HDACs. However, the
C-terminal region of YY1 could not be deacetylated. Rather, the acetylated C-
terminal region interacted with HDACs, which resulted in stable histone deacetylase
activity associated with the YY1 protein. Furthermore, acetylation of the C-terminal
domain decreased the DNA binding activity of YY1. At the protein complex level,
YY1 was shown to form a complex with up to four different proteins consistently
throughout different purification methods. These proteins are likely to have important
x
regulatory roles in the transcriptional activity of YY1. Taken together, these findings
will provide valuable information to our understanding of the regulatory mechanisms
of transcription in general.
Abstract Approved: ______________________________________________Major Professor: Edward Seto, Ph.D.Associate Professor, Interdisciplinary Oncology Program
Date Approved: _______________________________
1
INTRODUCTION
Transcription is an important step in gene regulation. The Big Bang of
our knowledge on gene regulation happened in 1961, with the debut of the concept of
the prokaryotic operon (77, 78). Monod and Jacob pictured the perfect regulatory
circuitry of making proteins out of genes using an amplifiable intermediate, which
they called the messenger RNA (mRNA). In this model, genes of the same
biochemical pathway are grouped into an operon, which is immediately preceded by a
regulatory DNA element called the operator. A repressor protein binds the operator
and keeps the operon in the “off” position. Upon proper environmental cues, an
inducer molecule binds the repressor protein, inducing an allosteric modification. In
this configuration, the repressor no longer binds the operator, thus causing the operon
to switch to the “on” position. When the operon is “on,” mRNA is being made. This
process is referred called transcription. mRNA, the product of transcription, is the
template recognized by the ribosomes for making proteins (17, 50). This linear
relationship among DNA, mRNA, and proteins is so important that it is called the
Central Dogma of Biology, and transcription has been recognized as an important step
in gene regulation.
Much of the original operon model proposed by Monod and Jacob still holds
true today. However, some modifications are needed. For example, we now know
that all operons are not regulated by repression/de-repression. Some operons are
regulated by activation, and some operons are under control of multiple regulatory
2
mechanisms. Moreover, transcription regulation can occur at multiple stages,
including initiation, which was essentially what Monod and Jacob initially described,
as well as elongation and termination, two steps subsequent to the initiation stage.
Nevertheless, the concept that mRNA is made according to precisely regulated
interplay among DNA regulatory elements, repressors and activators remains as the
true essence of transcriptional regulation, and the importance of understanding how
genes are regulated will likely to continue rising with the complete sequencing of the
human genome.
Transcription in prokaryotes. The initial operon model soon expanded to
full understanding of transcriptional control in prokaryotes (for a historical review,
see reference 107). In prokaryotes, such as bacteria, transcription is catalyzed by an
enzyme called RNA polymerase. The RNA polymerase is a four-unit protein
consisted of two α subunits and two β subunits. During transcriptional initiation,
RNA polymerase scans the length of the bacterial DNA. When it encounters a
specific sequence about 35 bp upstream from the transcriptional initiation site, it
becomes associated with a specific protein called σ factor and anchors on the DNA.
Then it spreads along the DNA until it finds another sequence, which usually lies
about 10 bp upstream from the initiation site. This -10 sequence tells the polymerase
where to lay down the first nucleotide, and the polymerase faithfully unwinds the
DNA and starts incorporating nucleotides to make RNA. The σ factor then falls off,
and the polymerase becomes dedicated to the next phase of transcription, elongation.
Both the protein players, including the polymerase and the σ factor, and the DNA
sequences, including the -35 and -10 sequences, have tremendous influence on where,
3
when, and how often transcription starts. Regulation of transcriptional initiation
largely relies on manipulation of this system. For example, a repressor usually blocks
the binding of the RNA polymerase to either the -35 or the -10 sequence, and no
transcription can occur in this configuration. An activator may augment the
association between DNA and the RNA polymerase, causing more frequent firing of
the polymerase and higher levels of transcription. These findings significantly
enriched our understanding of transcriptional control. In the mean time, with the
continuous efforts from numerous researchers, it became apparent that transcription in
eukaryotes is regulated in very similar fashions. The differences between prokaryotic
transcription and eukaryotic transcription, in fact, are the complex layers and flavors
in regulations that are unique to eukaryotes.
Transcription in eukaryotes. The most significant difference between
prokaryotes and eukaryotes is the nucleus. The nucleus by itself provides excellent
regulation because mRNA export in eukaryotes is a highly regulated process (49, 79).
However, the consequences of having a nucleus are far beyond mere physical
barriers: In eukaryotic nuclei, DNA is packaged into chromatin (151). Even though
the basic layouts of transcription in eukaryotes are the same as those in prokaryotes,
chromatin inevitably inhibits access of the RNA polymerase to the DNA. Therefore,
as opposed to gene-specific activators/repressors observed in prokaryotes, genes in
eukaryotes are repressed in general due to the way DNA is organized. Furthermore,
prokaryotes are unicellular organisms, but many eukaryotes are multi-cellular
organisms with complex body plans. Prokaryotic gene regulators activate or repress
transcription in a swift fashion because prokaryotes need to respond to the
4
environmental changes quickly. Cells in complex eukaryotes, however, are
committed to specific functions after differentiation, and only a certain fraction of the
genome is active in a certain place at a certain time. As a result, transcriptional
control in eukaryotes is characterized by developmentally timed up- and down-
regulations of specific subsets of genes whose accessibility is determined by the
chromatin structure. The beauty of eukaryotic transcriptional control, therefore, lies
in the intricate connections among the RNA polymerase, activators, repressors, and
the DNA promoter elements organized within the chromatin along the temporal and
spatial axes.
RNA polymerase II and the eukaryotic promoter. The RNA polymerase
responsible for making mRNA in eukaryotes is the RNA polymerase (Pol) II. Pol II
enzyme in yeast is a multi-subunit protein complex comprising 12 polypeptides (174).
Pol II needs additional protein factors to recognize promoters and initiate
transcription; these protein factors are termed general transcription factors (reviewed
in references 32, 175). It has been suggested that the general transcription factors for
Pol II, designated TFIID, -B, -F, -E, and -H, cooperate with Pol II in a highly
coordinated fashion to form the transcriptional initiation complex (20, 175) (Figure
1): First, TFIID recognizes the promoter by virtue of its TBP subunit binding to the
TATA box in the promoter. The TATA box is analogous to the -10 sequence in
prokaryotes, and TBP is analogous to the σ factor. Upon recognizing the TATA box,
TBP bends DNA and creates a binding surface for TFIIB, which associates with Pol II
and recruits Pol II to the promoter. TFIIF associates with both Pol II and TFIIB.
Through multiple interactions with TFIID, TFIIB, and TFIIF, Pol II is accurately
5
positioned on the promoter. Under some circumstances, this minimal complex can
initiate transcription without TFIIE and TFIIH.
The association of TFIIE with the polymerase is required for the subsequent
recruitment of TFIIH. TFIIH possesses an ATP-dependent helicase activity, which
unwinds DNA around the transcriptional initiation site and triggers transcriptional
initiation. It is thought that TFIIE and TFIIH are key players in converting the
process of transcription from the initiation phase to the elongation phase, an event
sometimes called promoter clearance.
Pol II
TFIIB
TBP
TFIID
TATA+1
TFIIE
TFIHTFIIF
TFIIA
TFIIE
TFIH
Pol II
TFIIB
TBP
TFIID
TATA+1
TFIIF
TFIIA
Pol II
TFIIB
TBP
TFIID
TATA+1
TFIIA
TFIIF
FIG. 1. Step-wise assembly of the
transcriptional initiation complex. A
thick line represents DNA. A bent
arrow depicts the direction of
transcription. The transcriptional
initiation site is indicated by “+1.”
TATA represents the TATA box
(adapted from reference 20).
6
Activators and transcriptional activation. Activators are usually DNA-
binding proteins with modular structures of activation domains that can be defined by
fusing to a heterologous DNA-binding motif. Typical activation domains have been
characterized as acidic (reviewed in reference 56), glutamine rich, or proline rich
(reviewed in reference 113) . Detailed structural and mutagenesis studies suggest that
the mechanisms of activation domains might involve ionic and hydrophobic
interactions between the repeated amino acids within the activation domains and their
target general transcription factors (37, 44, 157). However, in vitro, transcriptional
activation cannot occur without participation of another protein complex called the
Mediator (reviewed in reference 14). In yeast, the Mediator complex contains 20
subunits, many of which have been shown to be important in transcriptional control in
vivo (118). For example, inactivation of SRB4 (suppressor of RNA polymerase B 4)
immediately causes cessation of transcription by Pol II (156). Interestingly, a human
Mediator complex was also isolated as a co-activator complex specifically associated
with ligand-bound nuclear hormone receptors (53). Subsequent efforts of purification
demonstrate significant similarities between the human and the yeast Mediator
complexes (reviewed in reference 59). The consensus at this moment is that
transcriptional activators directly interact with components of the Mediator complex,
which in turn cause the polymerase to initiate transcription more often than the basal
level (Figure 2).
The Mediator complex is thought to tether directly to the C-terminal domain
of Pol II (85, 155). Affinity purification involving components of the Mediator
complex suggests that in vivo, the Mediator complex exists in a form of a RNA
7
polymerase holoenzyme containing Pol II, general transcription factors, the Mediator,
and proteins involved in chromatin remodeling (reviewed in references 87, 125, 169).
It has been further suggested that interactions between activators and any components
of the holoenzyme can lead to transcriptional activation (27). This view implies that
the holoenzyme represents a single target of transcriptional activation in vivo, and
perhaps repression as well, since an SRB complex purified as part of the holoenzyme
has been shown to act as a co-repressor (reviewed in reference 118). This view also
AcAc
TFs
Mediator
GeneralTranscription
Factors
co-activator complex
co-repressor complex
Pol II
FIG. 2. Regulation of transcription: a model. The basic unit of chromatin, the
nucleosome, is depicted as a flat cylinder of histone octamers wrapped around by
two turns of DNA. DNA-binding transcription factors (TFs) that activate
transcription have been shown to interact with the Mediator proteins, which in turn
associate with the general transcription machinery, causing increased levels of
transcription. TFs also interact with co-activators or co-repressors. Some co-
activators have HAT activity, and some co-repressors have HDAC activity
(adapted from reference 89).
8
directly contradicts the ordered-assembly view of the transcriptional initiation
complex formation. Recent evidence supports the idea of a stable holoenzyme
complex; however, it has not been proven that the ordered-assembly view is wrong.
Therefore, the detailed kinetics of transcriptional activation in vivo remains unclear.
Repressors and transcriptional repression. Transcription of eukaryotic genes
is also regulated by repressors, which turn off transcription. The mechanism of
repression is even less well understood than that of activation. Traditionally, it is
thought that repressors employ the following strategies to repress transcription: (1) by
competing with an activator for its binding site in DNA, such as competition of
transcription factor YY1 with serum-response protein for binding to the serum
response element in α-actin gene (28); (2) by physical interactions with the activator,
which result in either masking of the activation domain or dimerization leading to
inactivation of the activator. Examples include interactions between MDM2 and p53
(115) as well as MyoD dimerization with Id proteins (12); (3) by post-translational
modification of the activator, such as acetylation of HMG-1, which renders it unable
to activate IFN-β (reviewed in reference 150); (4) by interfering with the formation of
a functional initiation complex. Examples include the yeast SRB10 protein, which
phosphorylates the C-terminal domain of Pol II prior to initiation and marks the
polymerase incapable of transcriptional initiation (reviewed in reference 118). Recent
understanding of transcriptional repression adds another class of transcriptional
repressors: those involved in modification of chromatin by deacetylation, which
establishes a transcriptionally repressive chromatin environment.
9
Chromatin and transcription. Although it had long been suspected that
chromatin played a role in transcription, it was the proposal more than 25 years ago
that the eukaryotic chromatin is based on repeating units of nucleosomes first laid
down the ground stone for subsequent elucidation of the relationship between
chromatin and transcription (88). Nucleosomes, the basic units of chromatin, are
organized by DNA wrapping around a histone octamer, which composes of two
copies of histone proteins, namely H2A, H2B, H3 and H4 (83, 90, 132). Micrococcal
nuclease digestion reveals that these four histones are closely associated with 146
base pairs of DNA, forming the core particle of nucleosomes (123). A short stretch of
DNA, called linker DNA, connects two nucleosomes. Histone proteins have two
general structural domains: the globular histone fold domain and the flexible N-
terminal tails. The X-ray crystal structure of the core particle suggests that while the
DNA winds around the surface of the histone core, it winds two turns with grooves
aligned, creating a gap where the N-terminal tails of H2B and H3 pass through to the
outside of the core particle. The tails of H2A and H4 also extend from the flat
surfaces of the core particle to the outside (108). These exposed tails are thought to
contact neighboring core particles and can be critical in mediating higher-order
structures of chromatin beyond the nucleosomal level. In vivo and in vitro studies
show that these histone tails also contact DNA (39, 120, 166). However, the high
ionic crystal environment and the lack of linker DNA preclude direct observation of
histone tail-DNA contact (108, 166). Interestingly, nucleosomes have intrinsic
plasticity suggested by the observation that the twist of DNA around histone core is
slightly altered, and the lost of about 1 base per turn is readily accommodated at
10
various locations within the core particle (108). This structural plasticity also
suggests that nucleosomes can be subjected to remodeling.
Nucleosomes have a general inhibitory effect on transcription. In vitro studies
show that transcriptional initiation is stalled by packaging of promoters into
nucleosomes (86, 106). Early genetic studies in yeast also demonstrated that histone
proteins are important in transcriptional repression: When synthesis of normal core
histone H2B or H4 is repressed, promoters of inducible genes become accessible to
the general transcription machinery (52). Deletions of histone H2A/H2B genes (170)
and point mutations in H3 (67) have been identified as suppressor mutations in
switch-independent (SIN) genes, which allow inducible gene transcription in yeast
strains deficient for switch (SWI) genes or sucrose nonfermenting (SNF) genes, two
general activators in yeast (68, 122, 129). Most researchers agree that gene regulation
by modifying chromatin structures occurs in two flavors, chromatin remodeling and
histone acetylation/deacetylation, which are often found in the same protein
complexes.
Chromatin remodeling. Genetic studies suggest that two classes of activator
proteins, SWI and SNF, act to antagonize the negative effects of chromatin on
transcription (reviewed in references 13, 170). Subsequently, a multi-protein complex
containing the SWI/SNF proteins was purified and shown to remodel chromatin (25,
35, 74). Since then, the yeast SWI/SNF complex has become the paradigm of the
ATP-dependent chromatin remodeling complexes, which utilize energy from ATP
hydrolysis to change the chromatin structure and are often required for the full
functioning of a variety of transcription factors (22). To date, two families of ATP-
11
dependent chromatin remodeling complexes have been purified: the SWI/SNF family
and the ISWI family. The SWI/SNF family of chromatin remodeling machines
contains large protein complexes. They destabilize nucleosomes by disrupting the
contacts between DNA and histones, resulting in increased accessibility of
nucleosomal DNA to DNase I, restriction enzymes, or DNA-binding transcription
factors (35, 74, 105). In contrast, the ISWI family of chromatin remodeling machines
contains smaller protein complexes, which uses ISWI (imitation SWI) protein as the
ATPase (76, 159, 162). ISWI chromatin remodeling complexes enable sliding of
histone octamers to adjacent positions on the same strand of DNA (33, 58, 94).
Purification of these ATP-dependent chromatin remodeling complexes suggest that
the nucleosome is a dynamic structure subject to intricate modulations, and that
transcription and chromatin structure are tightly linked in vivo.
Histone acetyltransferases as co-activators. Several lysine residues in the
core histones can be acetylated at the ε-amino positions. This kind of histone
modification has been associated with various biological processes, one of them
transcriptional control (4, 65, 140, 163). It has been postulated that charge
neutralization caused by lysine acetylation reduces the affinity of histone tails to DNA
and increases the accessibility of regulatory elements to transcription factors (69).
Some researchers believe that lysine acetylation does not influence the strength of
interaction between histone tails and DNA but rather opens up the higher-order
structure of the chromatin by disrupting the interaction between histone tails
protruding from nucleosomes within the chromatin fiber (89). Nevertheless, there is a
positive correlation between the extent of histone acetylation and gene activity. For
12
example, the transcriptional inactive heterochromatin is associated with
hypoacetylated histones (65, 163).
The enzymes catalyzing the addition of acetyl groups to histones are histone
acetyltransferases (HATs). The first definitive link between transcriptional activation
and histone acetylation came with the identification that the yeast Gcn5 protein,
which is a transcriptional co-activator of many genes, is a HAT (19). Most
significantly, the HAT activity of Gcn5 is required for its transcriptional activation
activity (91), and there is a general increase in histone H3 acetylation in the promoter
of a Gcn5-regulated gene upon induction (21). Since then, many transcription factors
have been identified as HATs, including the TAFII250 subunit of TFIID (114),
p300/CBP (8, 124), PCAF (173), and SRC-1 (148). p300/CBP and SRC-1 family
members were first identified as transcriptional co-activators. They do not bind
specific DNA elements but potentiate transcriptional activation when associated with
an activator. An attractive model has emerged in which sequence-specific
transcriptional activators activate transcription by associating with HATs (Figure 2).
However, there is no direct evidence demonstrating that histone acetylation per se
activates transcription. Recently many transcription factors have been shown to be
acetylated, including p53 (54) as well as the basal transcription factors TFIIE and
TFIIF (75). It is possible that acetylation of transcription factors contributes to
transcriptional activation (or repression), in addition to acetylation of the core
histones. Recent studies, both biochemical and genetic, also suggest that core histone
acetylation and chromatin remodeling act in concert to activate transcription (34,
13
101). However, the temporal sequence of these two biological activities is still under
intense debate.
Histone deacetylases as co-repressors. Similar to the discovery that many
transcriptional co-activators are HATs, many transcriptional co-repressors are found
to possess histone deacetylase (HDAC) activity (171) (Figure 2). HDACs identified
to date are classified into families. The Class I Rpd3 family of HDACs, including
HDAC1, HDAC2, and HDAC3, has been shown to be important in transcriptional
repression (153, 171, 172). These HDACs are found in multi-protein complexes,
including many co-repressor complexes (reviewed in reference 36). Moreover, they
also associate with DNA-binding repressor proteins such as YY1 (171), Mad (93),
Ume6 (81), and nuclear hormone receptor co-repressors (3, 66, 121). Although
association with HDAC complexes may not account for all cellular repression
activities, more and more repressors appear to utilize this mode of transcriptional
repression. It will be extremely informative to test whether HDACs as co-repressors
only act in a gene-specific manner, or they also participate in epigenetic control of
gene repression.
YY1 is a transcriptional regulator. Transcription factor YY1 (Yin Yang 1)
represents a valuable system to study various aspects of eukaryotic transcriptional
control. YY1 was first discovered in a biochemical assay analyzing the activity of
the P5 promoter of the adeno-associated virus (AAV) (144). AAV is a defective
parvovirus; it cannot replicate without co-infection of adenovirus. The P5 promoter
of AAV is silent in the absence of adenovirus co-infection. In the presence of the
adenoviral E1A protein, which is normally introduced as a result of adenovirus
14
infection, the P5 promoter is activated and the lytic cycle of AAV begins. Using this
P5 promoter element, a cellular protein was discovered as the target of E1A-directed
promoter activation. This protein was named Yin Yang 1 for its dual transcriptional
activity: in the absence of E1A, it represses transcription from P5. In the presence of
E1A, YY1 activates transcription from P5. Yin and Yang represent the repression
and activation properties of YY1.
Independent of the analysis of the AAV P5 promoter, other experimental
systems also discovered YY1 as a transcriptional regulator. δ, the mouse homolog of
YY1, was identified as an activator of certain ribosomal protein genes (61). YY1,
also identified as NF-E1, represses transcription by binding to the immunoglobulin κ
3’enhanber (126). YY1 was also known as UCRBP, which binds to the upstream
conserved regions of the Moloney murine leukemia virus (MuLV) and represses
MuLV promoter activity (41). Since then, YY1 has been shown to be instrumental in
the regulation of many cellular and viral gene promoters, many of which have
important functions in cell growth and differentiation (reviewed in reference 143).
General characteristics of YY1. YY1 binds a specific DNA sequence
(CGCCATNTT) in the promoters by 4 C2H2 zinc fingers, which are located at the C-
terminus of the protein (reviewed in references 143, 154). Deletional and domain-
swapping experiments (5, 23, 24, 97, 99, 100, 144, 171) show that the C-terminal zinc
finger region of YY1 accounts for one of the two repression domains of YY1. The
other repression domain resides in the middle glycine/alanine-rich segment of YY1.
The N-terminal region of YY1 is identified as the activation domain of YY1,
suggesting that YY1 possesses transcriptional activation activity independent of its
15
repression activity. The human YY1 protein contains 414 amino acid residues with a
predicted molecular weight of 44 kDa. However, YY1 has an apparent size of about
65 kDa on SDS-polyacrylamide gels, suggesting that YY1 might have a unusual
structure. The amino acid sequence of YY1 is highly conserved among human,
mouse, and Xenopus, and a Drosophila homologue of YY1 has also been discovered
(144, 18, 41, 61, 126, 130). Knockout studies show that deletion of YY1 results in
peri-implantation lethality in mice (38), further demonstrating the importance of YY1
in fundamental biological processes such as development.
YY1 and transcriptional initiation. In addition to being a transcriptional
regulator, YY1 has also been suggested to be a transcriptional initiator. In vitro
studies show that YY1 binds directly to the initiator (Inr) element of the AAV P5
promoter (144), and YY1 binding to this Inr element is necessary for transcriptional
initiation (142). In a reconstituted transcription assay, purified YY1, TBP, and Pol II
are sufficient to initiate transcription from a supercoiled template (160). Crystal
structure studies of the zinc finger domain of YY1 bound to the Inr element of P5
suggest intrinsic directionality of YY1-mediated transcriptional initiation: YY1 binds
both the template strand and the non-template strand of DNA upstream of the Inr
element but only the template strand downstream of Inr, providing structural basis for
the correct direction of transcription (70).
In addition to the AAV P5 promoter, YY1 binds to the Inr elements of many
gene promoters, including the cytochrome oxidase Vb subunit promoter (10), DNA
polymerase β promoter (64), PCNA promoter (92), and the mouse Surf-1 promoter
(42, 43). However, the mechanistic basis for the initiator activity of YY1 is still
16
unclear. It has been shown that YY1 physically interact with TFIIB and Pol II both in
vitro and in vivo (30, 161). YY1 also interacts with TBP and TAFII55 (5).
Sedimentation studies further confirm that YY1 co-purifies with components of the
general transcription machinery (109, 139). These lines of evidence suggest that
YY1, when bound to the Inr element, recruits Pol II to the transcriptional initiation
site, and this may account for the initiator activity of YY1.
17
OBJECTIVES
As we began to appreciate the unique properties of YY1, it became apparent
that we knew very little about how the activity of YY1 is regulated. For example,
when does YY1 function as a transcriptional activator and when as a repressor? How
does the role of YY1 as a transcriptional initiator reconcile with that as a
transcriptional regulator? Understanding how the activity of YY1 is regulated will
help us answer those questions and tremendously enhance our knowledge about the
basic mechanisms of transcription. Therefore, it is important that we understand how
the activity of YY1 is regulated.
It has been shown that YY1 is a stable phosphorylated protein expressed
ubiquitously regardless of cell cycle position or the differentiation status of the cell
(5). However, several lines of evidence also suggest that YY1 is regulated. For
example, expression of YY1 mRNA in NIH3T3 cells is affected by cell density and
growth factors such as insulin-like growth factor (IGF)-1 (40). The activity of YY1
also changes during myoblast differentiation and during aging (1, 98). Furthermore,
the DNA-binding activity of YY1 decreases during differentiation in human
teratocarcinoma cells (104).
A wide variety of transcription factors have been shown to associate with
YY1, suggesting that the activity of YY1 could be controlled by protein-protein
interactions. These YY1-interacting proteins include proteins of the basal
transcription machinery, such as TBP (5), TFIIB (160); sequence-specific DNA-
18
binding transcriptional activators, such as Sp1 (96, 141), c-Myc (145), ATF/CREB
(178), C/EBP (11); and various transcriptional co-regulators, such as E1A (100),
TAFII55 (30), p300/CBP (5, 95), and HDAC1, HDAC2, and HDAC3 (171, 172).
The YY1-p300/CBP and YY1-HDACs interactions are of particular interest. In
certain circumstances, the transcriptional activation activity of YY1 directly depends
on its association with p300/CBP (95). In contrast, it has been shown that the
transcriptional repression activity of YY1 is mediated by association of HDAC2 with
residues 170-200 of YY1, which corresponds to one of the repression domains of
YY1 (171). It is conceivable that by selectively associating with either HATs or
HDACs, YY1 becomes an activator or a repressor. However, it has not been shown
definitively that such an active selection system exists for YY1. Recently p300, CBP,
and another HAT, PCAF (p300/CBP associated factor), have been shown to acetylate
transcription factors in addition to their histone substrates (reviewed in reference 150).
Importantly, acetylation was a key regulatory mechanism for the regulation of those
transcription factors. Because YY1 interacts with both p300/CBP and HDACs, it is
possible that the activity of YY1 is regulated by acetylation and deacetylation.
Association between YY1 and p300/CBP or HDACs also suggests that YY1
might interact with cellular proteins to form protein complexes. It has been
discovered that many chromatin-modifying activities, including chromatin-
remodeling and histone acetylation/deacetylation, exist in forms of multi-subunit
protein complexes. For example, the yeast Gcn5 co-activator, which possesses HAT
activity, exists in two protein complexes: SAGA (Spt-Ada-Gcn5 acetyltransferase)
and ADA (48). HDAC1 and HDAC2 also exist in two major histone deacetylase
19
complexes: NuRD and SIN3 (reviewed in reference 2). The formation and
recruitment of these protein complexes have important influence on the biological
activities of the constituent proteins. It is possible that YY1 is also regulated in the
context of a protein complex in vivo.
Taken these lines of evidence together, this dissertation study was initiated to
test the hypothesis that the activity of YY1 is regulated by multiple mechanisms. Three
aims have been proposed:
Aim 1 is focused on regulation of YY1 at the genomic level, which includes
cloning and analysis of the human YY1 promoter.
Aim 2 is devoted to regulation of YY1 at the polypeptide level, which is
regulation of YY1 by acetylation and deacetylation.
Aim 3 is intended to study YY1 in a context of protein complexes, which
involves purification of a YY1 protein complex.
20
MATERIALS AND METHODS
Isolation of YY1 genomic clones. A human liver genomic library (ATCC
37333) in Charon 4A was screened with a 32P-labeled human YY1 cDNA fragment
(nt 1-315) using a standard protocol (136). λ phage DNA was purified from two
positive clones, digested with restriction enzymes, and analyzed by Southern blotting
(136). A 4.5 kb EcoRI fragment was subcloned into a pGEM7zf(+) vector (Promega)
for subsequent analyses.
Plasmids. An EcoRI/NcoI fragment was isolated from the 4.5 kb human YY1
λ clone described above, treated with Klenow to blunt ends, and ligated into pGL2-
Basic vector to create p-3600Luc. pGL2-Basic (Promega) contains a firefly luciferase
gene as a reporter and lacks eukaryotic promoter and enhancer sequences. p-3600Luc
was subsequently digested with NsiI/StuI and subjected to exonuclease III digestion to
create 5’ progressive deletions of the YY1 promoter linked to the luciferase reporter
gene. pRL-TK (Promega) was used as a control for normalization in co-transfection
experiments. pRL-TK contains a Renilla luciferase genes under control of the herpes
simplex virus thymidine kinase promoter.
Glutathione S-transferase was expressed from pGSTag (133). GST-YY1 (1-
414) was expressed from pGST-YY1 (171), and deletion constructs of GST-YY1
were generated by restriction enzyme digestion and re-ligations of pGST-YY1. GST-
p53 expression plasmid has been described (72). GST-p300 and GST-PCAF were
21
expressed from plasmids pGEX2T-p300 (aa 1195 to 1810) and pGEX5X-PCAF (aa
352 to 832), respectively (26).
Gal4-YY1 was expressed from pM1-YY1, which was constructed by inserting
the full-length YY1 cDNA in-frame with the Gal4 DNA-binding domain in pM1
(134). pM1-YY1 (K170-200R) was prepared using adapter oligonucleotides
containing AAG (lysine) to AGG (arginine) mutations within YY1 aa 170 to 200 of
pM1-YY1. pG5CAT-Control was constructed by inserting five Gal4 DNA-binding
sites into the BglII site of pCAT-Control (Promega), which contains the
chloramphenicol acetyltransferase (CAT) gene downstream of the SV40 promoter and
enhancer sequences. Flag-YY1 was expressed from pCEP4F-YY1, which was
generated by inserting the full-length YY1 cDNA into the pCEP4F vector (179).
Serial Flag-YY1 deletion constructs were made by restriction enzyme digestion and
re-ligations of pCEP4F-YY1. pET15b-YY1, which expressed non-tagged full-length
YY1 in E. coli in an inducible system, was constructed by cloning the full-length YY1
cDNA into pET15b (Novagen). pGEM7Zf3X-HD1, which was used to generate in
vitro translated HDAC1, was made by subcloning full length HDAC1 into the
pGEM7Zf3X vector.
Plasmids pCMV12S, pCMV13S, and pCMV-YY1 have been described
previously (7, 171). pCMV12S directs expression of the E1A 243R protein, and
pCMV13S expresses the E1A 289R protein. The following plasmids have also been
previously described: pBJ5-HD1F (153), which expresses HDAC1 C-terminally
tagged with a Flag epitope; pBJ5.1-HD1F (H199F) HDAC1 point mutant (63);
pME18S-FLAG-HDAC2, which expresses Flag-tagged HDAC2 (93).
22
Sequencing of the human YY1 promoter. The dideoxy chain-termination
sequencing method was employed to obtain the complete sequence of one strand of
the human YY1 promoter using p-3600Luc and its deletion derivatives as templates
and GLprimer 1 (Promega) as primer. Custom-designed oligodeoxynuclotide primers
and GLprimer 2 (Promega) were used to obtain the sequence of the complementary
strand.
Primer extension. Primer extension was performed essentially as described
previously with minor modifications (136). An antisense oligonucleotide
corresponding to the human YY1 promoter sequence from position +76 to +100 was
synthesized and end-labeled with [γ-32P]ATP and T4 polynucleotide kinase. HeLa
total RNA was isolated using the acid phenol-guanidinium thiocyanate method (31).
10 µg of the HeLa total RNA or yeast total RNA was mixed with 105 cpm of the
labeled oligonucleotide primer and the mixture was ethanol-precipitated. The DNA-
RNA mixture was then re-dissolved in 30 µl of hybridization buffer [40 mM PIPES
(pH 6.4), 1 mM EDTA (pH 8.0), 0.4 M NaCl and 80% formamide], denatured at 85oC
for 10 min, and annealed at 30oC overnight. The annealed hybridization mixture was
then ethanol-precipitated, washed, and re-dissolved in 20 µl of reverse transcriptase
buffer [50 mM Tris-HCl (pH 7.6), 60 mM KCl, 10 mM MgCl2, 1 mM each of dATP,
dCTP, dGTP and dTTP, 1 mM dithiothreitol (DTT), 1 U/µl RNase inhibitor and 50
µg/ml actinomycin D]. 50 U of avian myeloblastosis (AMV) reverse transcriptase
was then added, and the reactions were incubated at 37oC for 2 h. At the end of
incubation, the reactions were stopped with EDTA, treated with DNase-free RNase,
and phenol-chloroform extracted. Single-stranded DNA was recovered by ethanol
23
precipitation, washed, and dissolved in 4 µl of TE (pH 7.4). Samples were added with
6 µl of formamide loading buffer [80% formamide, 10 mM EDTA (pH 8.0), 1 mg/ml
xylene cyanol and 1 mg/ml bromophenol blue], heated at 95oC for 5 min, and
resolved on a 6% polyacrylamide/7 M urea gel. The gel was then dried and images
were obtained by autoradiography.
Cell line, transfection, luciferase assays, and CAT assays. HeLa cells were
maintained in Dulbecco’s modified Eagle’s media supplemented with 10% fetal
bovine serum and 100 mg/ml penicillin and streptomycin. For each transfection
reaction, 3x105 cells were seeded into a 60-mm tissue culture dish. Sixteen h later,
10 µg plasmids were transfected using the calcium phosphate co-precipitation method
(47). Forty-eight h after transfection, cells were harvested. For luciferase assays,
luciferase activity was determined using the Dual Luciferase Reporter Assay system
(Promega). For CAT assays, cells were harvested by scraping and lysed by repeated
freezing/thawing, and extracts were assayed for CAT activity by thin-layer
chromatography (46).
Recombinant proteins. The GST fusion constructs of p300, PCAF, p53, and
YY1 deletion mutants were expressed in E. coli DH5α, bound to glutathione-agarose
beads (Sigma), washed extensively in phosphate-buffered saline (PBS), and eluted
with 25 mM reduced glutathione. The eluate was then dialyzed against a buffer
containing 20 mM Tris-HCl (pH 8), 0.5 mM EDTA, 100 mM KCl, 20% glycerol, 0.5
mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF).
Non-tagged YY1 was expressed in E. coli BL21 (DE3), induced with 0.2 mM
IPTG, and captured by Ni2+ resin (ProBond, Invitrogen). Unbound bacterial proteins
24
were removed with 50 mM imidazole, and bound YY1 was eluted with 500 mM
imidazole. Flag-tagged YY1 used in electrophoretic mobility shift assays (EMSA)
was purified on an anti-Flag column (Sigma) under stringent conditions following the
manufacturer’s suggestions.
In vitro acetylation reactions. Purified GST-YY1 and serial deletion proteins
were incubated at 30oC for 30 min with 0.25 µCi [3H]acetyl coenzyme A (CoA)
(Amersham) and purified GST-p300 (0.2 µg) or GST-PCAF (1 µg) in 30 µl of
acetylation buffer containing 50 mM Tris-HCl (pH 8.0), 5% glycerol, 0.1 mM EDTA,
50 mM KCl, 1 mM DTT, 1 mM PMSF, and 10 mM sodium butyrate. Proteins were
resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-
polyacrylamide gels were fixed by Coomassie blue staining and subjected to signal
amplification (Amplify, Amersham) prior to exposing to X-ray film.
For subsequent mass spec analyses, 0.5 pmol of the YY1 peptide
(GRVKKGGGKKSGKKSYLSGGAGAAGGRGADP) was acetylated in acetylation
buffer for 2 hr with CAT-assay grade acetyl CoA (Amersham).
Immunoprecipitation and western blot analysis. Immunoprecipitation of
endogenous YY1 from HeLa cell extract was performed with H-10 anti-YY1 mAb
(Santa Cruz) and GammaBind protein G Sepharose (Amersham). Endogenous Max
and 14-3-3 were immunoprecipitated using anti-Max mAb (Pharmingen) and anti-14-
3-3 H-8 mAb (Santa Cruz), respectively. Immunoprecipitation of Flag-YY1 deletion
proteins was done using anti-Flag M2 affinity gel (Sigma) following the
manufacturer’s suggestions.
25
Western blot analyses were performed using standard protocols (62).
Immunoprecipitated proteins were detected with diluted primary antibodies [1:1000
of anti-acetyl lysine (Upstate), 1:5000 of H-10, 1:500 of anti-Max, 1:1000 of H-8,
1:5000 of anti-Flag M2 (Sigma), 1:1000 of anti-HDAC1, HDAC2, or HDAC3 rabbit
anti-serum (93, 158, 168)] followed by 1:7500 diluted alkaline phosphatase-
conjugated secondary antibodies (Promega). The blots were subsequently developed
with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Promega).
In vitro protein-protein interaction assays. 35S-labeled HDAC1 was
generated from pGEM7Zf3X-HD1 using T7 RNA polymerase and the TNT
Reticulocyte Lysate System (Promega). GST-YY1 (170-200) was either acetylated
with cold acetyl CoA or mock acetylated and was then captured onto glutathione-
agarose beads. 5 µl of in vitro translated HDAC1 was mixed with the beads in the
presence of PBS plus 0.2% NP-40 at room temperature for 1 h. Beads were washed
extensively in PBS plus 0.2% NP-40. Bound proteins were eluted by boiling in
Laemmli sample buffer, separated by SDS-PAGE, and detected by Coomassie blue
staining and autoradiography.
Histone deacetylation assays. HeLa cells were transfected with 10 µg of
pCEP4F-YY1 deletion plasmids by the calcium phosphate co-precipitation method
(47). Forty-eight h after transfection, cells were harvested, lysed in PBS plus 0.1%
NP-40, and immunoprecipitated with anti-FLAG M2 affinity gel (Sigma). Histone
deacetylase activities of the immunoprecipitated Flag-YY1 were determined using a
peptide corresponding to residues 2 to 24 of histone H4 as described (153) except that
incubation was performed at room temperature overnight. 400 nM (final
26
concentration) of TSA (Sigma) or a 5 column-volume (cv) of Flag peptide (Sigma)
was added to the immunoprecipitate 30 min prior to addition of the H4 substrate
peptide, if appropriate. Histone deacetylation assays on chromatographic fractions
were performed using the same H4 peptide as substrate.
Immunofluorescence analysis. HeLa cells were grown on chamber slides
(Nalge Nunc International) for about twenty-four h and transfected with 10 µg of F-
YY1 deletion constructs. Two days later, cells were washed with ice-cold PBS, fixed
with 4% paraformaldehyde for 10 min, rinsed again with PBS, covered with 400 µl of
1% bovine serum albumin (BSA) in PBS for 1 h at room temperature, washed again
in PBS, and then treated with 1:200 dilution of anti-Flag FITC conjugate antibody
(Upstate) for 1 h at room temperature. Subsequently, cells were subjected to
extensive washing with PBS and cover slips were applied with one drop of anti-fade
mounting medium with DAPI (Vector) before analysis under a fluorescence
microscope.
Electrophoretic mobility shift assays. Single-stranded
oligodeoxynucleotides corresponding to a consensus YY1-binding site (142) or a p53
cognate sequence (54) were labeled individually with [γ32P]ATP and T4
polynucleotide kinase, heated together at 65oC, and allowed to anneal by slow cooling
to room temperature. Binding reactions were performed in a 12 µl reaction volume
containing 12 mM Hepes (pH 7.9), 10% glycerol, 5 mM MgCl2, 60 mM KCl, 1 mM
DTT, 0.5 mM EDTA, 50 mg/ml BSA, 0.05% NP-40, 0.1 mg poly(dI-dC),
approximately 1 ng of purified proteins or 5 µl of chromatographic fractions, and 5
fmol radiolabeled DNA. Reactions were incubated for 10 min at room temperature
27
and separated on 4% non-denaturing polyacrylamide gels. The gels were then dried
and exposed to film.
Gel filtration analysis of YY1. HeLa nuclear extract was prepared according
to the Dignam method and dialyzed into TM buffer [50 mM Tris-HCl (pH 7.9), 122.5
mM MgCl2, 1 mM EDTA, 10% glycerol, 1 mM DTT, 0.1 mM PMSF] plus 0.1 M
KCl. 2.5 mg of dialyzed HeLa nuclear extract was applied to a calibrated Superdex
200 HR 10/30 column (Pharmacia) and fractions were collected.
Purification of a YY1 complex by anion exchange and immobilized-metal
affinity chromatography (IMAC). Nuclear extract derived from 6 L of HeLa cells
was loaded on a Q Sepharose column (Pharmacia). Bound proteins were eluted in
buffer A [20 mM Hepes (pH 7.8), 5 mM MgCl2, 20% glycerol, 0.5 mM DTT, 0.2 mM
EDTA, 0.1 mM PMSF] with a linear gradient (5 cv) of KCl from 150 mM to 1 M.
The presence of YY1 was monitored with western blotting and EMSA. Fractions
containing YY1 were pooled, dialyzed into PBS (pH 7.4) containing 10% glycerol, 5
mM β-mercaptoethanol, and 1 mM PMSF, and loaded on a column with 1 mL bed
volume of Ni2+ resin (ProBond, Invitrogen). The column was washed extensively
with wash buffer [PBS (pH 7.4), 50 mM imidazole, 10% glycerol, 5 mM β-
mercaptoethanol, and 1 mM PMSF]. Bound YY1 complex was eluted with elution
buffer [PBS (pH 7.4), 10% glycerol, 5 mM β-mercaptoethanol, and 1 mM PMSF]
containing 300 mM imidazole.
Coomassie staining and silver staining. Detection of YY1 protein
complexes with Coomassie staining and silver staining was performed using standard
protocols (6).
28
Purification of a YY1 complex by anion exchange and antibody-affinity
chromatography. Nuclear extract derived from 6 L of HeLa cells was loaded on a Q
Sepharose column (Pharmacia). Bound proteins were eluted in buffer A [20 mM
HEPES (pH 7.8), 5 mM MgCl2, 20% glycerol, 0.5 mM DTT, 0.2 mM EDTA, 0.1 mM
PMSF] with a linear gradient (5 cv) of KCl from 150 mM to 1 M. The presence of
YY1 was monitored with western blotting and EMSA. Fractions containing YY1
were pooled, dialyzed into PBS (pH 7.4) containing 10% glycerol and 1 mM PMSF,
and loaded on a protein G column (GammaBind, Amersham). The flowthrough
fraction was collected and loaded onto an antibody column made by coupling H-10
anti-YY1 mAb (Santa Cruz) to protein G beads. The antibody column was then
washed extensively with PBS (pH 7.4) containing 10% glycerol, 100 mM KCl, and 1
mM PMSF. Bound proteins were either used directly in histone deacetylation assays
or eluted with 100 mM glycine (pH 2.5).
Purification of a Flag-tagged YY1 complex. 1.5x109 HeLa cells were
transiently transfected with plasmid pCEP4F-YY1 using the calcium phosphate co-
precipitation method (19). Cells were harvested, pooled, and lysed by sonication in
PBS (pH 7.4) plus 1 mM PMSF. Cell lysate was applied to an anti-Flag M2 column
(Sigma) and the adsorbed protein complex was washed extensively with PBS (pH 7.4)
containing 10% glycerol and 1 mM PMSF. Bound proteins were eluted with excess
Flag peptide (100 µg/ml) in 5 cv of PBS (pH 7.4) containing 10% glycerol and 1 mM
PMSF.
Radioimmunoprecipitation analysis (RIPA) of F-YY1. HeLa cells were
transiently transfected with full-length F-YY1 (pCEP4F-YY1) and its serial deletion
29
plasmids using the calcium phosphate co-precipitation method (47). Sixteen h later,
cells were labeled with [35S]methionine and cysteine (11.0 mCi/ml, NEN) for four h.
Labeled cells were harvested and lysed by sonication in PBS (pH 7.4) containing
0.1% NP-40 and 1 mM PMSF. Anti-Flag M2 agarose beads (Sigma) were added to
the cell lysates and washed extensively with PBS (pH 7.4) containing 0.1% NP-40
and 1 mM PMSF. The beads were boiled in Laemmli sample buffer and proteins
were separated by SDS-PAGE. Gels were then dried and exposed X-ray film.
Accession number. The nucleotide sequence data of the human YY1
promoter reported in this dissertation appears in GenBank, EMBL, and DDBJ
Nucleotide Sequence Databases under the accession number AF047455.
30
RESULTS
Determination of the transcriptional start site of the human YY1 gene.
To determine the transcriptional initiation site of the YY1 gene, primer extension was
performed using primers spanning the putative transcriptional start site and total HeLa
RNA. Identical reactions using yeast tRNA were performed side by side as negative
controls. One primer consistently yielded a strong signal indicating one strong
transcriptional start site. Alignment with a dideoxynucleotide sequence ladder from
FIG. 3. Determination of the 5’ end
of the human YY1 transcript.
Purified total RNA from HeLa cells
(lane 5) or tRNA from yeast (lane 6)
were used as templates in primer
extension analysis using a 25 bp 32P-
labeled antisense oligonucleotide
probe and AMV reverse transcriptase.
A sequencing ladder of the YY1
promoter was created in parallel and
is shown on the left with radiolabeled
nucleotides (lanes 1-4). The arrow
indicates the most likely start site of
transcription.
.
GATC HeLa t
otal R
NA
yeas
t tRNA
1 2 3 4 5 6
GAATCGAGAGGGCGAACGGGC
5'
3'
+1
31
the same primer showed that the strong signal corresponds to a G within a GC-rich
region of the YY1 promoter (Fig. 3) However, due to the cap structure of the mRNA,
the true transcription initiation site might be the A immediately preceding this G.
Transcriptional analysis of the human YY1 promoter. A 3.6 kb
EcoRI/NcoI fragment of the genomic clone containing sequences upstream of the
ATG codon in the YY1 cDNA was subcloned into pGL2-Basic and the resulting
plasmid was named p-3600Luc. The pGL2-Basic vector contains a firefly luciferase
gene but no eukaryotic promoter or enhancer sequences.
When transiently transfected into HeLa cells, p-3600Luc resulted in a 180-fold
increase in luciferase activity compared to pGL2-Basic alone (Fig. 4). To determine
the 5’ boundary of the YY1 promoter, serial deletion constructs of p-3600Luc were
generated and assayed for luciferase activities in HeLa cells after transient
transfection. As shown in Figure 4, deletion constructs p-2100Luc, p-1729Luc, p-
1514Luc, p-1248Luc, p-1110Luc, p-917Luc, p-478Luc, and p-277Luc exhibited
similar levels of luciferase activities to p-3600Luc. Deletion of the promoter to +54
drastically reduced the luciferase activity, suggesting the presence of a positive cis-
acting element between positions -277 and +54. However, a significant increase in
luciferase activity was observed between p-1514Luc and p-1248Luc, indicating a
negative cis-acting element was present between nt -1514 and -1248 of the YY1
promoter. Further deletions of the YY1 promoter to position +379 completely
abolished the luciferase activity to the background level (compared to pGL2-Basic).
These results suggest that a minimal sequence of 54 bp located downstream of the
32
transcriptional initiation site of the YY1 gene contains significant levels of the
promoter activity, and this activity is enhanced by the sequence between positions
-277 and +54.
-3600 Luciferase
-2100
-1514
-1248
-1110
-917
-478
+54
+379
0 100 200 300
relative luciferase activity
-1729
+475
-277
≈
≈
p-3600Luc
p-2100Luc
p-1729Luc
p-1514Luc
p-1248Luc
p-1110Luc
p-917Luc
p-478Luc
p-277Luc
p+54Luc
p+379Luc
pGL2-Basic
FIG. 4. Expression of luciferase enzymatic activity driven by the
human YY1 promoter in transiently transfected cells. The left panel
shows schematic drawings of various fragments of the human YY1
gene 5’ sequence subclones upstream of a luciferase reporter gene. A
bent arrow indicates the direction of transcription. Reporter constructs
were transfected into HeLa cells by the calcium phosphate co-
precipitation method, harvested, and assayed for luciferase activity.
All relative luciferase activities, as shown in the right panel, are
normalized with control Renilla luciferase expressions. Data shown
represent the average of three independent experiments with standard
deviations.
33
Sequence analysis of the human YY1 promoter. p-3600Luc and its serial
deletion derivatives were utilized to determine the promoter sequence of the human
YY1 gene. The complete DNA sequence of the human YY1 gene is shown in Figure
5A, which exhibits remarkable similarity to the mouse YY1 promoter (Fig. 5B). The
FIG. 5A. DNA sequences upstream of the translational start codon of
the human YY1 gene. The major transcriptional initiation site (+1) is
indicated by a bent arrow. Transcription factor binding sites are
underlined and indicated below each binding site sequence.
-1717 CCTCATTTCTCTTGCTCTCACAATTGGTGTTTATGGG -1681
-1680 GAAGTATCAACTACTTGCAGTGCCATTTCCCCATCAATTACTAGAGAGTGGGGAAAGTGAAATTTAAAAAGCATTAAGAC -1601
-1600 ATGTAAAAGTTCTCCCACAACTGGTTTCCATTCATGAAATATTTATGCAGAGTTTATAAGCTATAAAGCCAGAGATGACT -1521
-1520 TAATTCAACAGATTTGACTTTTCCAAACTGAGTGGGTGCAGTACTCCAAGGGGAATTATTCAGGTTTCAAAGCTCAGATT -1441
-1440 CAAAGAGAAAACGATCTAATTTGTCACTCCTTACACTAATATTCTCAAAAGTCAAGTCCAATGCGATTTAGCAATAAGAA -1361
-1360 GGGACTAGGATTTATAAAGTTGCCTTTTAATGAGAATGTATAGTCTACTTGTTTTAACAAGTTGAGCCTAAGATTAATGT -1281
-1280 ATTGCGTACAGTTCAGAAAATCATGGGCCTCAACTGCATGAACACTATTACAAAACAGTAAATGTTGATAATATTTAAGT -1201
-1200 TGAACAAATTCACAGAGGCGTTAAATCGGCCAAACGAGTACAACAAAGACACACCTTTCCAACTCTTCAATTTGTCATAG -1121
-1120 TTTCCTTAAAAATCAGGAAATCTTTGTTTTGCTTTAAGGCAAATTGTCAGGTCGACCAAAAGGACAGATAAGAGCAGAAA -1041
-1040 CACTTCGCTGCAATGTAACTCATTCAGGAAGGTTTAACTTGCCGCTAATCCGTGCCACAAAAAAAAATCTAGGCTCTGTT -961
-960 GCAGGTACAATGGAGGACACGGCTGAAAAAATTTGGAATTTTAAATGAGACAAATGCAAAACC TGGTGGGCGTAAAAAGG -881
-880 AGCACCTATGAAAGTGACAAATAGGGGGAAAGGGTGGGCAAGGGAAACAATGGCTGACTGGAG AGCAAAGAAGGGGAAGC -801
-800 TCAGGAGAAAATTTTAGAAAGCCGCCAGGACCCTGTAGTTCTAAGATCTACGGGGAAACAGGC ACCCAACGGCTGCGTCT -721
-720 CAGGTTTCCGCGGGTCACTAAAGAATAACGGACATCCTCCCAACGGTGGCCCTGGGGCTCCGC GGGCGCTTCCGCCGAGC -641
-640 TCGCGCCGACCCCGCGCTCGGCCCCGCACCCCGCCGGGCGCTCGCGGCGAGATACCGGACGCT GCCCGCGTCGCCCGATT -561
-560 TTGTCCGTTCGGTCCTCCACACTCACCCCGCGGCCATCGCTCGCCCGAAGCCAGGCGACAAGA ACAAACACCTCCCGACG -481
-480 CGAAAAAGGAAGCACAGGCGATTCTCGTCAAAGCAGACTTTATTGGGGCGACAGGGCCGCCCC GCACGCGCCAGCCGCTC -401
-400 CCCGCGCCGCCGGGCCGCCCACCCGCCTCAACCCCGCTCCCGGCCGGCCCCTCCCTCCCTTCT CCTCAGCTCCCGCCCCC -321
-320 GTGGTGCCCGGGGCCGCGCGGACCGCTCACCGGCTCCCAAGGCAGCGGCTGTAGCGGCGACGC CCGTTCCCGAGTGCGGC -241
-240 CCCGGCCCGAGGCGGCGGGTTTTGTGGCTGTGGCACCGCGAAGGGCGGCACGGCGCGACACCG GGAAGCGGGAGGCGGTG -161
-160 GCGGCGGCGGCGGCGCGCTGACGTCACGCGCCGGGCCAGCCAGGGCGCGTGCGAGCCGCCCCGCCCCCGGTCCCATCGGC -81
-80 CCCAATCCGGGAGGAGCCCGGCGAGTGGGCGGGGCCGCGGAGGCCAGCGGACAGATCGATTGGCCGAGAGGAGAATCGAG -1
1 AGGGCGAACGGGCGAGTGGCAGCGAGGCGGGGCGGGCTGAGGCCAGCGCGGAAGTCTCGCGAGGCCGGGCCCGAGCAGAG 80
81 TGTGGCGGCGGCGGCGAGATCTGGGCTCGGGTTGAGGAGTTGGTATTTGTGTGGAAGGAGGCGGAGGCGCAGGAGGAGGA 160
161 AGGGGGAAGCGGAGCGCGGCCCGGACGGGAGGAGGCGCGGCCAGGGCGGGCGGTTGCGGCGAGGCGAGGCGAGGCGGGGA 240
241 GCCGAGACGAGCAGCGGCCGAGCGAGCGCGGGCGCGGGCGCACCGAGGCGAGGGAGGCGGGAAGCCCCGCGCCGCCGCGG 320
321 CGCCCGCCCCTTCCCCCGCCGCCCGCCCCCTCTCCCCCCGCCCGCTCGCCGCCTTCCTCCCTCTGCCTTCCTTCCCCACG 400
401 GCCGGCCGCCTCCTCGCCCGCCCGCCCGCAGCCGAGGAGCCGAGGCCGCCGCGGCCGTGGCGGCGGAGCCCTCAGCATG 476
MZF1
Nkx-2
v-myb/c-myb
v-myb
CREB/ATF
Sp1
Sp1
Sp1
CDP/Cut
34
human YY1 promoter possesses a region rich in GC nucleotides (80% GC from -427
to +96) and lacks a TATA box, which is typical of the 5’ untranslated regions of
many transcription factors. Multiple GC boxes are located at positions -57, +29 and
+231, which are binding sites for the Sp1 family transcription factors (80). In
addition to the GC boxes, the human YY1 promoter also contains putative binding
sites for several ubiquitous and lineage-specific transcription factors. A binding site
for CDP/CUT, which functions as a transcriptional repressor (60), is found just 20 bp
upstream from the transcriptional start site. Further upstream is a binding site for
10 20 30 40 50 60hYY1 - AGGCACCCAACGGCTGCGTCTCAGGTTTCCGCGGGTCACTAAAGAATAACGGACATCCTC ::::::::::: : : : ::: ::::::::::::::: ::::::::::::::: ::mYY1 - AGGCACCCAACCGGCGGGGCTCCGGTTTCCGCGGGTCATAAAAGAATAACGGACACACTT 10 20 30 40 50 60
70 80 90 100 110hYY1 - CCAACGGTGGCCCTGGGGCTCCGCGGGC-GCTTCCGCCGAGCTCGCGCCGACCCCGCGCT ::::::::: :: :::: :::: :: :::::::: : :: :::: :mYY1 - CCAACGGTGAACCCTAGGCTGCGCGCGCCGCTTCCGC-----TTGC--------CGCGGT 70 80 90 100
120 130 140 150 160 170hYY1 - CGGCCCCGCACCCCGCCGGGCGCTCGCGGCGAGATACCGGACGCTGCCCGCGTCGCCCGA :: : :: :::::: :: :::: : : : : ::::: : :: :mYY1 - CGCCTTCGGCGCCCGCCACCGGCACGCGACCAAAGA---------GCCCGTGGAGCACA- 110 120 130 140 150
180 190 200 210 220 230hYY1 - TTTTGTCCGTTCGGTCCTCCACACTCACCCCGCGGCCATCGCTCG---CCCGAAGCCAGG ::::: : :: ::: : : :: : ::: :: ::: :: : :mYY1 - -----------CGGTCGGCTACGCTC---CGTCCGCTACCGCACGAGACCCCGAGTAACG 160 170 180 190 200
240 250 260 270 280 290hYY1 - CGACAAGAACAAACACCTCCCGACGCGAAAAAGGAAGCACAGGCGATTCTCGTCAAAGCA : ::: :::: : :: : ::::: ::::::::::: ::::::::: ::::mYY1 - AGTAAAGG-CAAAACGAACGGGAAACCAAAAATCAAGCACAGGCGTTTCTCGTCAGAGCA 210 220 230 240 250 260
300 310 320 330 340 350hYY1 - GACTTTATTGGGGCGACAGGGCCGCCCCGCACGCGCCAGCCGCTCCCCGCGCCGCCGGGC :::::::::: ::: ::::::: :: : : ::: ::::::: :mYY1 - GACTTTATTGCGGC---------------CACGCGC--GCGGTCGCACGCCCCGCCGG-C 270 280 290 300
360 370 380 390 400 410hYY1 - CGCCCACCCGCCTCAACCCCGCTCCCGGCCGGCCCCTCCCTCCCTTCTCCTCAGCTCCCG :: : :: ::: :: : ::: ::: :::::: :: :::::: :mYY1 - AGCACGCCAGCCCCAGGCGCGCGCCC---------------CCCTTCACCCCAGCTC--G 310 320 330 340
420 430 440 450 460 470hYY1 - CCCCCGTGGTGCCCGGGGCCGCGCGGACCGCTCACCGGCTCCCAAGGCAGCGGCTGTAGC ::::: :: ::: :::::::: : :: ::::::::: :::: : : :mYY1 - TTACCGTGC--GCCTGGGTCGCGCGGAAAGTTCGCCGGCTCCCGAGGCGTAAGGCGCACG 350 360 370 380 390 400
480 490 500 510 520 530hYY1 - GGCGACGCCCGT-TCCCGAGTGCGGCCCCGGCCC--GAGGCGGCGGGTTTTGTGGCTGTG : :::::::: ::: : ::: : ::::: ::: : : :::::: ::::mYY1 - GCCGACGCCCCACTCCTGTCAGCGACTGGGGCCCCGGAGACCGGCACTTTTGTCACTGTT 410 420 430 440 450 460
540 550 560 570 580 590hYY1 - GCACCGCGAAGGGCGGCACG--GCGCGACACCGGGAAGCGGGAGGCGGTGGCGGCGGCGG :::::::: : : ::: ::::::: ::: ::: : :: ::: ::::mYY1 - GCACCGCGGACGCCGGGGATTCGCGCGACGCCG---AGC----GCCGTAAGCGACGGC-- 470 480 490 500 510
600 610 620 630 640hYY1 - CGGCGCGCTGACGTCACGCGCCGGG---CCAGCCAGGGCGCGTGCGAGCCGCCCCGCCCC :: ::::::::::::::::::: :::::::: ::::::::: :::::: ::::::mYY1 - --ACGAGCTGACGTCACGCGCCGGGGGGCCAGCCAGCGCGCGTGCG-GCCGCCACGCCCC 520 530 540 550 560 570
650 660 670 680 690 700hYY1 - CGGTCCCATCGG-----CCCCAATCCGGGAGGAGCCCGGC---GAGTGGGCGGGGCCGCG : : ::: ::: :::::::: ::::::: :: :: :::: ::::::::: ::mYY1 - C--TACCAGCGGGGGCCCCCCAATC-GGGAGGACCCTGGTTGGGAGTAGGCGGGGCCCCG 580 590 600 610 620 630
710 720 730 740 750hYY1 - GAGGCCAGCGGACAGAT-CGATTGGCCGAGAGG-AGAATCGAGAGGGCGAACGGGCGAGT : : : : :: : ::::::: : :::: :: : :::::: :::: : :::::mYY1 - GCGCCTCGAGGGGCCCTGCGATTGGTAGCGAGGGAGGAGCGAGAGCCGGAACAG-CGAGT 640 650 660 670 680
760 770 780 790 800 810hYY1 - GGC--AGCGAGG-CGGGGCGGGCTGAGGCCAGCGCGGAAGTCTCGCGAGGCCGGGCCCGA :: : :: :: ::::::::: ::: : ::::::::::::::::::::: ::: ::mYY1 - GGGGAATCGGGGACGGGGCGGGGCGAGAGCCCCGCGGAAGTCTCGCGAGGCCGAGCCGGA 690 700 710 720 730 740
820 830 840 850 860hYY1 - GCAGAGTGTGGCGGCGGCGGCG------AGATCTGGGCTCGGG-TTGAGGAGTTGGTATT :::::::::::::::::::::: ::::::::::::::: ::::::::::::::::mYY1 - GCAGAGTGTGGCGGCGGCGGCGGCGGCGAGATCTGGGCTCGGGGTTGAGGAGTTGGTATT 750 760 770 780 790 800
870 880 890 900 910 920hYY1 - TGTGTGGAAGGAGGCGGAGGCGCAGGAGGAGGAAGGGGGAAGCGGAGCGC-GGCCCGGA- ::::::::::::::::::::::::::::: :::::: :::: : ::: ::::::::mYY1 - TGTGTGGAAGGAGGCGGAGGCGCAGGAGGC---AGGGGGCAGCGTAACGCCGGCCCGGAG 810 820 830 840 850 860
930 940 950 960 970hYY1 - --CGGGAGGAGGCGCGGCCA------GGGCGGGCGGTTGCGGCGAGGCGAGGCGAGGCGG :::::::::::::::::: :::: ::: : : :::::::::: : ::::::mYY1 - GGCGGGAGGAGGCGCGGCCACAGCCAGGGCAGGCAGCCGAGGCGAGGCGAAGGCAGGCGG 870 880 890 900 910 920
980 990 1000 1010 1020 1030hYY1 - GGAGCCGAGACGAGCAGCGGCCGAGCGAGCGCGGG-CGC----GGGCGCACCGAGGCGAG ::: : ::: :::::::::::::::::: : : ::: ::::::::::: : :mYY1 - GGAAGCAGGACAGGCAGCGGCCGAGCGAGCGAGCGACGCAGCGGGGCGCACCGAT-CTCG 930 940 950 960 970 980
1040 1050 1060 1070 1080 1090hYY1 - GGAGGCGGG-AAGCCCCGCGCCGCCGCGGCGCCCGCCCCTTCCCCCGCCGCCCGCCCCCT ::::::::: :::::::: ::::: ::::::::::::::::::::::::::::::::mYY1 - GGAGGCGGGGAAGCCCCG----GCCGCCGCGCCCGCCCCTTCCCCCGCCGCCCGCCCCCT 990 1000 1010 1020 1030 1040
1100 1110 1120 1130 1140 1150hYY1 - CTCCCCCCGCCCGCTCGCCGCCTTCCTCCCTCTGCCTTCCTTCCCCACGGCCGGCCGCCT :::::::::::::: ::: :::::::::::::::::::::::::::::::::::::::::mYY1 - CTCCCCCCGCCCGCCCGCTGCCTTCCTCCCTCTGCCTTCCTTCCCCACGGCCGGCCGCCT 1050 1060 1070 1080 1090 1100
1160 1170 1180 1190hYY1 - CCTCGCCCGCCCGCCC--------GCAGCCGAGGAGCCGAGGCCGCC-------GCGGCC :::::::::::::::: :::::: ::::::::: :::::: ::::::mYY1 - CCTCGCCCGCCCGCCCTCCCTCCCGCAGCCCAGGAGCCGACGCCGCCTGCCGCGGCGGCC 1110 1120 1130 1140 1150 1160
1200 1210hYY1 - GTGGCGGCGGAGCCCTCAGC---- ::::::::::::::::::::mYY1 - GTGGCGGCGGAGCCCTCAGCCATG 1170 1180
FIG. 5B. Comparison of the YY1 promoter DNA sequences between mouse
and human. The human YY1 promoter sequence [hYY1 (-74 to +476)] is
aligned with the mouse YY1 sequence [mYY1 (-751 to +434)] (135).
Identical nucleotide positions are indicated by double dots.
35
CREB/ATF (position -142) (57) as well as three Myb binding sites (51) in tandem
(positions -736 to -673). Far upstream at position -1522, a consensus binding site for
the mouse tinman homeodomain factor Nkx-2 is found. Nkx-2 has been shown to
antagonize the transcriptional repression activity of YY1 in the cardiac α-actin
promoter through competitive binding during myogenesis (28). There is a binding
site for MZF-1, a myeloid zinc finger transcription factor (116), at position -1640
close to the Nkx-2 site. Interestingly, the three Sp1 binding sites (GC boxes), the
CREB/ATF binding site, and the three Myb binding sties are conserved between the
mouse and the human YY1 promoters, suggesting that transcription factors that bind
to those sites might play an important role in the regulation of the mRNA level of
YY1.
E1A-mediated repression of the human YY1 promoter. YY1 is an
important regulator of the cardiac α-actin promoter, and the adenovirus E1A protein
blocks myogenesis as well as cardiac-specific gene transcription (119). It has also
been shown that E1A relieves YY1-mediated transcriptional repression of the AAV
P5 promoter (144). It is possible that E1A directly affects transcription of the YY1
gene. To test this possibility, E1A expression plasmids (pCMV12S or pCMV13S)
and a YY1 promoter construct (p-3600Luc) were transiently transfected into HeLa
cells to examine the effect of E1A on YY1 transcription. Transcription initiated from
the human YY1 promoter was significantly down-regulated by over-expressed E1A
12S and 13S proteins as measured in luciferase assays (Fig. 6A).
To further examine whether E1A represses YY1 promoter expression through
upstream cis-acting sequences or through basal transcriptional machinery, a minimal
36
YY1 promoter, p-277Luc, was utilized in co-transfection/reporter assays. Figure 6B
shows that the luciferase activity of p-277Luc was repressed by both E1A 12S and
13S proteins, suggesting that the mechanism of YY1 repression by E1A does not
involve upstream sequence-specific DNA binding factors. This E1A-mediated
.
0
20
40
60
80
100
120
0
20
40
60
80
100
120
rela
tive
luci
fera
se a
ctiv
ity
rela
tive
luci
fera
se a
ctiv
ity
p-3600 luc p-3600 luc + E1A 12S
p-3600 luc + E1A 13S
p-277 luc p-277 luc + E1A 12S
p-277 luc + E1A 13S
A B
FIG. 6. Adenovirus E1A proteins repress the YY1 promoter.
Luciferase assays were performed after transient
transfections into HeLa cells with p-3600Luc or p-277Luc
and pRL-TK plasmids in the presence or absence of
plasmids encoding the adenovirus 12S or 13S proteins.
Results were presented as the mean of three independent
transfections with standard deviations and normalized with
control Renilla luciferase expressions.
37
repression of the YY1 promoter most likely results from E1A modulating the activity
of the basal transcription elements surrounding the YY1 transcriptional initiation site.
YY1 does not autoregulate its own promoter. It is known that many
transcription factor autoregulate their own promoters. Some transcription factors bind
directly to DNA elements located in their own promoters and regulate their own
expression, while others activate or repress expression of a separate set of
transcription factors, which in turn modulate transcription from their gene promoters.
Although the human YY1 promoter does not appear to contain a YY1-binding
.
120
100
80
60
40
20
0p-3600Luc p-3600Luc
+pCMV-YY1
rela
tive
luci
fera
se a
ctiv
ity
FIG. 7. The YY1 protein does
not autoregulate the YY1
promoter. Luciferase assays
were performed after transient
transfections into HeLa cells
with p-3600Luc and pRL-TK
plasmids in the presence or
absence of a plasmid encoding
the YY1 protein. Results are
presented as the mean of three
independent transfections with
standard deviations and
normalized with control
Renilla luciferase expressions.
38
consensus sequence, YY1 might alter expression levels of other transcription factors
which are capable of binding the YY1 promoter and regulate expression of YY1.
Therefore, a YY1 expression plasmid (pCMV-YY1) was transfected into HeLa cells
to test if over-expression of YY1 had any effect on its own promoter (p-3600Luc).
Figure 7 shows that YY1 had no significant effects on the activity of its own
promoter. It is possible that the concentration of endogenously-expressed YY1 is
sufficiently high to negate any effects from over-expressed YY1 introduced by
transfection. However, our data suggest that YY1 most likely does not autoregulate
its own promoter.
YY1 is acetylated by p300 and PCAF. Acetylation has been shown to
FIG. 8. Multiple functional domains of transcription factor
YY1. YY1 has one transcriptional activation domain at the
N-terminus and two repression domains, one encompassing
residues 170 to 200 and the other one residing at the C-
terminus. The amino acid sequence of the central repression
domain (residues 170 to 200) is given with lysine residues
underlined. His, histidine-rich domain; GA,
glycine/alanine-rich domain; GK, glycine/lysine-rich
domain; Zn fingers, zinc fingers.
54 80 154 414200 295
transcriptionalactivation
domain
transcriptionalrepression
domain
transcriptionalrepression
domain
170
DNA-binding domain
Zn FingersSpacerAcidic GKGAHisAcidic
GRVKKGGGKKSGKKSYLSGGAGAAGGRGADP
39
regulate the activity of many transcription factors including sequence-specific DNA-
binding factors p53 (54), GATA-1 (15, 73), E2F (110, 111), and MyoD (137).
Analysis of the YY1 amino acid sequence reveals that YY1 contains multiple lysine
residues that are potential substrates for acetylation. Interestingly, within YY1’s
histone deacetylase interaction domain (residues 170-200), there are 6 lysines
arranged in pairs (Fig. 8). As described previously, YY1 interacts with the HAT p300
(5, 95) which interacts with another HAT, PCAF (173). Remarkably, both p300 and
PCAF catalyze the acetylation of transcription factors (reviewed in reference 150).
To determine if p300 or PCAF acetylated YY1, we performed in vitro acetylation
reactions using GST-tagged p300 and PCAF purified from E. coli as enzymes. YY1
serial deletion constructs were similarly purified from E. coli as GST-fusion proteins
..
97
68
43
29
GST1-
414
170-
200
∆ 170-
200
261-
333
MW(kD)
8 9 10 11 12
GST1-
414
398-
414
333-
414
260-
414
13 14 15 16 17
29
97
68
43
MW(kD)1-
200
1-26
11-
333
1-41
4
1-12
21-
396
1-17
0
1 2 3 4 5 6 7
97
68
43
MW(kD)
FIG. 9A. Acetylation of YY1 by PCAF. Serial GST-YY1
deletion proteins were incubated with GST-PCAF and
separated by SDS-PAGE. The gels were then exposed to X-
ray film to detect acetylated proteins. Arrows indicate
acetylated YY1 proteins. Auto-acetylated forms of PCAF
were detected as three bands around 68 kD.
40
and used as substrates. GST-PCAF acetylated various deletion constructs of YY1
(Fig. 9A, lanes 1-5, 8-11, 13, 15, 16) but not GST alone (lanes 12, 17). Unlike PCAF,
GST-p300 efficiently acetylated GST-YY1 (170-200) as well as GST-YY1 (1-200)
(Fig. 9B, lanes 3, 12, 15). GST alone was not acetylated by p300, showing that the in
vitro acetylation was specific to YY1 (Fig. 9B, lane 1). Moreover, acetylation of YY1
was dependent on p300 or PCAF and was not due to YY1 auto-acetylation (Fig. 9C).
Taken together, we found that PCAF acetylated YY1 at residues 170-200 and at its C-
terminus. p300, in contrast, efficiently acetylated YY1 only at residues 170-200 and
only when YY1 was in a C-terminal truncated form, implying that p300-mediated
acetylation of YY1 is dependent on the conformation of YY1.
..
97
68
43
29
GST1-
414
170-
200
∆ 170-
200
261-
333
∆ 261-
333MW
(kD)
1 2 3 4 5 6
1-41
420
0-26
1
260-
414
398-
414
7 8 9 10 11 12
170-
200
333-
414
97
68
43
29
MW(kD)
1-12
2
1-17
01-
200
1-26
11-
333
1-39
61-
414
13 14 15 16 17 18 19
MW(kD)
97
68
43
29
FIG. 9B. Acetylation of YY1 by p300. Serial GST-YY1 deletion
proteins were incubated with GST-p300. Arrows indicate YY1 proteins
acetylated by p300.
41
To determine if YY1 was acetylated in vivo, we immunoprecipitated
endogenous YY1 from HeLa cells and then western blotted the precipitated material
with anti-acetyl lysine antibody (Fig. 9D, lanes 2 and 3). Neither YY1 nor acetylated
YY1 was seen in experiments in which YY1 antibody was omitted from the
immunoprecipitation reaction (mock IP, lane 1). Unlike YY1, the transcription
factors Max and 14-3-3 were not acetylated in vivo (lanes 4 and 5). This finding
indicates that YY1 is acetylated in vivo, and protein acetylation is restricted to specific
proteins such as YY1.
To determine which region of YY1 was acetylated in vivo, we performed
similar IP-western experiments, this time using YY1 serial deletion constructs fused
to a Flag epitope. Flag-tagged YY1 (F-YY1) deletion constructs were transiently
transfected into HeLa cells and immunoprecipitated with anti-Flag antibody.
200
97
68
43
29
18
MW(kD)
YY1 (170-200)
YY1 (1-414)
p300
PCAF
Acetyl-CoA
+-
+
++++
+--
-
-
+
+
+-
-++
-
-
---
-
1 2 3 4 5
FIG. 9C. Acetylation of YY1 is
dependent on p300 or PCAF. In
vitro acetylation reactions were
performed in the presence or
absence of p300 or PCAF.
Arrows indicate that YY1 was
acetylated only in the presence
of p300 or PCAF.
42
Acetylated forms of F-YY1 were detected by western blotting with anti-acetyl lysine
antibody (Fig. 9E, top panel). To ensure that all F-YY1 deletions were expressed,
blots were also probed with anti-Flag antibody (Fig. 9E, bottom panel). The results of
these experiments show that YY1 is acetylated in vivo at residues 170-200 as well as
in the C-terminal residues 261-414. Taken together, our in vitro and in vivo
acetylation results suggest that there are two acetylation domains on YY1: residues
170-200, which are acetylated by both p300 and PCAF, and the C-terminus, which is
acetylated by PCAF only.
Because both p300 and PCAF acetylate YY1 at residues 170-200, we were
interested in determining the specificity of acetylation by these two enzymes. Using
GST-p300 or GST-PCAF, we in vitro acetylated a synthetic peptide corresponding to
FIG. 9D. YY1 is acetylated in vivo.
Endogenous YY1 (lanes 2 and 3), Max
(lane 4), and 14-3-3 (lane 5) were
immunoprecipitated from HeLa cells
and analyzed for acetylation by western
blotting using anti-acetyl lysine
antibody (bottom panels) or for
expression using their perspective
antibodies (top panels). "Mock IP"
indicates that no primary antibody was
used in immunoprecipitation reactions.
Arrows indicate highlighted protein
bands.
200
97
68
43
29
MW(kD) IP
α-YY1
Westernα-YY1α-Max
α-14-3-3
IP α-M
ax
IP α-1
4-3-
3
3 4 5
Westernα-acetyl lysine
200
97
68
43
29
mock
IP
IP α-Y
Y1MW(kD)
1 2
43
YY1 residues 170-200. We then compared the mass spectra produced by mass
spectrometry. Figure 9F shows that mock-acetylated YY1 peptide had a Mr of 2861
(panel A, 2861 m/z). When this peptide was acetylated with PCAF, another peak
emerged with a Mr of 2903 (panel B, 2903 m/z). Compared to the mock-acetylated
peptide, this additional peak had a mass corresponding to one additional acetyl group
(2903-2861=42), suggesting that the YY1 peptide was acetylated once by PCAF.
Interestingly, when we compared the spectrum from the p300-acetylated peptide with
FIG. 9E. Identification of regions of YY1 acetylated in
vivo. Flag-tagged YY1 serial deletion proteins were
transiently expressed in HeLa cells, immunoprecipitated
with anti-Flag antibody, and analyzed by western blotting
using anti-acetyl lysine antibody and anti-Flag antibody.
Arrows indicate immunoprecipitated YY1 proteins that
were acetylated.
MW 1-12
21-
170
1-20
0
1-26
11-
333
1-39
6
1-41
4
55-4
14
∆170-
200
∆261-
300
∆261-
333
333-
414
261-
414
170-
414
kD 1-17
026
1-33
3
∆261-
333
1-41
4
14
18
29
68
43
97200
14
18
29
68
43
97200
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
α-acetyl-lysine
α-Flag
44
that of the mock-acetylated peptide, we found 3 additional peaks at 2903, 2945, and
2987 m/z, which contained one, two, and three additional acetyl groups, respectively
(Fig. 9G). This result strongly suggests that YY1 can be acetylated by p300 at three
different lysines between residues 170 and 200.
.
Mass (m/z)
2900 3000 31002900 3000 3100
cou
nts
2800 2800
unmodifiedunmodified
monoacetylated
YY1 (170-200) PCAF acetylatedYY1 (170-200)
Mass (m/z)
2900 3000 3100 32002900 3000 31002800
unmodified unmodified
monoacetylated
diacetylated
triacetylated
YY1 (170-200) p300 acetylatedYY1 (170-200)
cou
nts
G
F
FIG. 9F, G. Determination of the number of acetylated lysines by mass
spectrometry. A YY1 peptide containing residues 170 to 200 was in vitro
acetylated by PCAF or p300 and subjected to mass spec analysis. Left panels
are spectra from mock-acetylated peptides. Right panels contain spectra from
acetylated as well as unacetylated peptides.
45
Lysine-to-arginine mutations within YY1 residues 170-200 significantly
reduce the transcriptional repression activity of YY1. To understand the effect of
acetylation on the transcriptional activity of YY1, we mutated the six lysines within
YY1 residues 170-200 to arginines. Arginine substitutions preserve the charges of the
affected amino acid residues but prevent acetylation in vivo by HATs. Both wild-type
and mutant YY1 were fused to a Gal4 DNA-binding domain and transfected into
HeLa cells in combination with a CAT reporter driven by the SV40 promoter
Gal4Gal4-YY1
Gal4-YY1 (K170-200R)
+--
+-
- +-- +
--
+-
- +--
Gal4-YY1
YY1
Western α-YY1
1 2 3 4 5 6
1 2 3 4 5 6
Gal4-YY1 (K170-200R)
FIG. 10. The effect of acetylation of YY1 residues 170-200
on the transcriptional repressor activity of YY1. HeLa cells
were transfected with Gal4 DNA-binding domain alone
(Gal4), Gal4-YY1 fusion construct (Gal4-YY1), or Gal4-
YY1 mutant with the 6 lysines mutated to arginines (Gal4-
YY1 (K170-200R)). Transcriptional activities were analyzed
by CAT assays using a CAT reporter containing five Gal4
binding sequences in tandem, and a representative
autoradiogram is shown. Western blot analyses were
performed to verify the expression levels of the effector
proteins.
46
containing five Gal4 binding sites. Consistent with previous findings (144, 171),
wild-type Gal4-YY1 was a potent transcriptional repressor (Fig. 10, top panel, lanes 2
and 5). However, when the six lysines were mutated and no longer able to be
acetylated, YY1 lost much of its repression activity (Fig. 10, top panel, lanes 3 and 6).
This finding suggests that acetylation of YY1 is necessary for the maximum
transcriptional repression activity of YY1. Western blot analysis showed that the loss
of repression was not due to lack of expression of the mutant YY1 proteins (Fig. 10,
bottom panel, compare lane 3 to lane 2, lane 6 to lane 5).
Acetylation of YY1 residues 170-200 increases YY1 binding to HDACs.
Using GST pull-down assays, we previously demonstrated that HDACs interact with
YY1 residues 170-200 (79). Therefore, we asked if acetylation of YY1 in this region
would affect YY1’s interaction with HDACs. Figure 11 shows that [35S] methionine-
labeled HDAC1 bound more efficiently to PCAF-acetylated and p300-acetylated
GST-YY1 (170-200) than to unacetylated GST-YY1 (170-200) (compare lane 1 to
lane 2, lane 3 to lane 4). The same amount of GST-YY1 (170-200) was used in all
reactions as shown by Coomassie staining. Similar results were obtained using in
vitro transcribed and translated HDAC2 (data not shown). In short, our data suggest
that acetylation of YY1 at residues 170-200 significantly increases the binding of
YY1 to HDACs.
HDAC1 and HDAC2 deacetylate YY1 170-200 but not the C-terminal
region of YY1. It is possible that the binding of HDACs to acetylated YY1 (170-
200) results in the subsequent deacetylation of YY1 (170-200). To test this
hypothesis, a synthetic peptide corresponding to YY1 residues 170-200 was
47
chemically labeled with [3H]sodium acetate and used as a substrate in deacetylation
assays. Figure 13A shows that immunopurified Flag-tagged HDAC1 and HDAC2 (F-
HDAC1 and F-HDAC2) deacetylated the YY1 (170-200) peptide. Deacetylation of
YY1 (170-200) by F-HDAC1 and F-HDAC2 was abolished by treatment with
tricostatin A (TSA), a potent inhibitor of HDAC1 and HDAC2. Furthermore, if F-
HDAC1 and F-HDAC2 were immunoprecipitated in the presence of an excess
competitor, Flag peptide, deacetylase activity was not observed on the YY1 peptide.
Similarly, endogenous HDAC1 and HDAC2 immunopurified from HeLa cells
FIG. 11. Increased HDAC-binding
to YY1 residues 170-200 by
acetylation. A representative
autoradiogram of in vitro translated
HDAC1 captured by acetylated or
mock-acetylated GST-YY1 (170-
200) is shown here. "Input"
represents one-tenth the amount of
HDAC1 used in each binding
reaction. Reaction mixtures were
separated by SDS-PAGE, and the
gels were stained with Coomassie
blue prior to exposure to film to
confirm that equal amounts of
GST-YY1 (170-200) were used in
the binding reactions.
.
HDAC1
1 2 3 4
input
97
68
43
29
MW(kD)
18
200
PCAF acet
ylate
d
mock
p30
0 ace
tylat
ed
p300 a
cety
lated
mock
PCAF ac
etyla
ted
GST-YY1(170-200)
GST-YY1(170-200)
Coomassie
48
(HDAC1 and HDAC2) also deacetylated the YY1 (170-200) peptide, and
deacetylation by endogenous HDAC1 and HDAC2 was also TSA-sensitive (Fig.
12A)..
F-HDAC1F-HDAC2
HDAC1HDAC2
200
400
600
800
1000
YY
1 (1
70-2
00)
dea
cety
lase
act
ivit
y (C
PM
)
Flag +----
+-
---
+
--
--
+
---
- +
----
-
----
competitorTSA
1200
1400
1600
--
-
-
---
-+
+
---
+-
---
+-
+-
---+-
--
+
--
--+-
+
--
--
+-
--
--
FIG. 12A. YY1 peptide deacetylation by HDACs.
Endogenous HDAC1 and HDAC2 and over-expressed
Flag-tagged HDAC1 and HDAC2 (F-HDAC1, F-
HDAC2) were immunoprecipitated from HeLa cells.
A YY1 (residues 170-200) peptide was labeled with
[3H]acetate and 20,000 cpm of the labeled peptide was
used in deacetylation reactions.
49
To provide further evidence that HDACs deacetylated YY1, we used an F-
HDAC1 immunoprecipitate to deacetylate full-length GST-YY1 and GST-YY1 (170-
200), both of which were in vitro acetylated with PCAF. As expected, YY1 (170-
.
Flag F-HDAC1 F-HDAC1H199F
1000
2000
3000
his
ton
e d
eace
tyla
se a
ctiv
ity
(cp
m)
2 3 4 5 6 7 81
9768
43
29
MW(kD)
PCAF acetylated YY1 (170-200)
HDAC1HDAC1(H199F)
p300 acetylated YY1 (170-200)
97
68
43
29
MW(kD)
Coomassie1 2 3 4 5 6 7 8
FIG. 12B. YY1 protein deacetylation by HDACs. Left panels: GST-
YY1 (170-200) was in vitro acetylated by p300 or PCAF. Half of the
acetylated GST-YY1 (170-200) was mixed with protein G beads alone
and the other half was mixed with immunoprecipitated wild type or
mutant (H199F) HDAC1. Samples were then separated by SDS-PAGE,
and the gels were subsequently stained with Coomassie blue and
exposed to film. Arrows indicate the position of GST-YY1 (170-200).
Right panel: Transiently expressed wild type and H199F mutant
HDAC1 were immunoprecipitated from HeLa cells and tested for their
histone deacetylase activity against a H4 peptide.
50
200) was efficiently deacetylated by HDAC1 (Fig. 12B, top panel, compare lane 1 to
lane 2). As a control, YY1 (170-200) was not deacetylated by an HDAC1 mutant that
was devoid of deacetylation activity ((24), Fig. 12B, right panel and top panel, lane
3). A Coomassie-stained gel (bottom panel) showed that the same amount of YY1
(170-200) was present in each reaction. Similarly, YY1 (170-200) acetylated by p300
was also deacetylated by HDAC1 (Fig. 12B, compare lane 5 to lane 6). To our
surprise, and in contrast to YY1 (170-200), full-length YY1 was not appreciably
deacetylated by HDAC1 (Fig. 12C, lane 1). This observation suggests that
200
97
68
43
29
MW(kD)
1 2 3 4 5 6 7 8Coomassie
200
97
68
43
29
MW(kD)
YY1 (1-414)YY1 (170-200)YY1 (261-333)YY1 (261-414)
HDAC1
+
-
-
+ -+
-+-+
-+
--
-- -
-
+
-
--
+
+
--
-+
--
--
+
--
+
-
---
1 2 3 4 5 6 7 8
FIG. 12C. Deacetylation of
YY1 serial deletion proteins by
HDAC1. Serial deletion
proteins of GST-YY1 were in
vitro acetylated by PCAF. Half
of the GST-YY1 proteins were
mixed with protein G beads and
the other half mixed with
HDAC1 immunoprecipitate.
Samples were then separated by
SDS-PAGE, and the gels were
subsequently stained with
Coomassie blue and exposed to
film. Arrows indicate the
positions of GST-YY1 deletion
proteins.
51
deacetylases only target specific regions of YY1. In support, we also found that
HDAC1 did not deacetylate two additional PCAF-acetylated GST-YY1 proteins, YY1
(261-333) and YY1 (261-414) (Fig. 12C, lanes 5 and 7). We conclude that only
residues 170-200 of YY1, and not the C-terminal zinc finger region of YY1, can be
deacetylated by HDAC1.
..
1-200
1-261
++
1-333 +1-396 +
+
261-333 ++261-414
333-414 -1-414 +
∆170-200
1-170 -
HDAC-binding domains170 200 261 333
56-414 +170-414 +
Flag -
1-17
01-
200
1-26
11-
333
1-39
6
1-41
455
-414
∆ 170-
200
HDAC1HDAC2HDAC3 IgH
1 2 3 4 5 6 7 8
MW(kD)
68
43
97
Flag
55-4
14
1-41
4
∆ 170-
200
333-
414
261-
414
170-
414
261-
333
9 12 131110 1514 16
α-HDAC2α-HDAC3
FIG. 13. Mapping of the HDAC-interaction domains of YY1. Serial
Flag-tagged YY1 deletion constructs were transfected into HeLa cells,
immunoprecipitated with anti-Flag antibody, and analyzed by western
blotting for the presence of HDAC1, HDAC2, and HDAC3 (lanes 1-8)
or HDAC2 and HDAC3 only (lanes 9-16). The bottom panel
summarizes the HDAC-binding domains of YY1. "+" indicates
positive interactions between YY1 and HDACs; "-" denotes the
absence of YY1-HDAC interactions.
52
HDACs bind YY1 at multiple regions. Based on our previous result that
YY1 interacts with HDACs at residues 170-200 in vitro (171), the inability of
HDAC1 to deacetylate the C-terminal zinc finger region of YY1 may arise from a
general failure of HDACs to interact with YY1 in the zinc finger region. To test this
possibility, we performed co-immunoprecipitation analyses to map the interaction
domain of YY1 with HDACs in vivo (Fig. 13). Surprisingly, our results showed that
in addition to residues 170-200, YY1 also interacted with HDACs at residues 261-333
in vivo. These results, together with those of our previous in vitro acetylation-
deacetylation experiment, suggest that YY1 interacts with HDACs at two domains:
residues 170-200, where bound HDACs deacetylate YY1, and the C-terminal residues
261-333, where bound HDACs do not result in deacetylation of YY1.
YY1 contains associated histone deacetylase activity, which localizes to C-
terminal residues 261-333 of YY1. After we precisely mapped the HDAC-binding
FIG. 14A. Histone deacetylase
activity of endogenous YY1 in HeLa
cells. Endogenous YY1 was
immunoprecipitated from HeLa cells
and assayed for deacetylase activity
against the H4 peptide. "+ TSA"
indicates addition of TSA to 400 nM
prior to addition of the peptide
substrate.
.
200
400
600
800
TSA +-
his
ton
e d
eace
tyla
seac
tivi
ty (
cpm
)
α-YY1 +- +-
53
domains in YY1, we sought to determine the functional effect of this interaction. We
found that immunoprecipitated endogenous YY1 from HeLa cells also contained
histone deacetylase activity, which was inhibited by TSA (Fig. 14A). Using serial F-
YY1 deletions, we determined the histone deacetylase activity domain of YY1, which
localized to residues 261-333 (Figs. 14B and 14C). This region of YY1 was
necessary and sufficient for the histone deacetylase activity associated with YY1
(Figs. 14B and 14C). Most strikingly, the YY1 histone deacetylase activity
domain completely overlapped with one of the HDAC-interacting domains of YY1.
Furthermore, the histone deacetylase activity associated with YY1 residues 261-333
.
vector
1-122
1-170
1-200
1-261
1-333
1-396
56-414
∆ 170-200
YY1(1-414)
200 400 600 800 1000
histone deacetylase activity (cpm)
∆ 261-333
261-333
333-414
261-414
1200 1400
170-414
histone deacetylase activity domain261 333
HDAC-binding domains170 200 261 333
FlagFlag-YY1 (1-414)
competitor
Flag-YY1 (261-333)
1000
2000
TSA
+ -+
+
+ ++ + +
+- - - --
- ---
-
- -
- ----- +
+ ++
-- -
-
-
--
-
his
ton
e d
eace
tyla
se
acti
vity
(cp
m)
B C
FIG. 14B, C. Identification of amino acid 261-333 as the histone deacetylase
activity domain of YY1. Flag-tagged YY1 deletion constructs were transfected
into HeLa cells, immunoprecipitated with anti-Flag antibody, and assayed for
deacetylase activity against the H4 peptide. "+ TSA" indicates addition of TSA
to 400 nM prior to addition of the peptide substrate. "+ competitor" indicates
addition of excess Flag peptides prior to addition of the peptide substrate.
54
was highly specific, because the activity was sensitive to TSA (Fig. 14B) and
competed by excess Flag peptide (Fig. 14B). A representative western blot shows
that different F-YY1 deletion mutants expressed equally well (Fig. 14D). These
results strongly suggest that stable interaction between HDACs and YY1 contributes
to YY1’s histone deacetylase activity. Immunofluorescence analysis confirmed that
F-YY1 deletion proteins that did not exhibit histone deacetylase activity localized to
either the nucleus or both the nucleus and cytoplasm, ruling out the possibility that the
lack of histone deacetylase activity was due to abnormal localization of the mutants
(Fig 14E).
Acetylation of YY1 at the C-terminal zinc finger domain decreases the
DNA-binding activity of YY1 . Because the C-terminal acetylation domain (residues
261-333) of YY1 overlaps with the zinc finger DNA-binding domain (residues 261-
414) of YY1, we tested whether acetylation of YY1 at the zinc finger domain would
affect the DNA binding activity of YY1 . Indeed, when in vitro acetylated by PCAF,
GST-YY1 bound less avidly than did unacetylated GST-YY1 to the Inr element of the
68
43
29
MW(kD) 1-
122
1-17
01-
200
1-26
1
1-33
3Flag 1-
396
FIG. 14D. Expression of F-YY1 deletion
mutants. Overexpressed F-YY1 deletion
constructs and Flag alone from the
parental vector were immunoprecipitated
from HeLa cells using anti-Flag
antibody, separated by SDS-PAGE, and
analyzed by western blotting using anti-
Flag antibody. Arrows indicate the
positions of F-YY1 deletion proteins.
55
AAV P5 promoter, which contains a consensus YY1 binding site (Fig. 15A, compare
lane 7 to lane 8). Similar results were obtained when a GST-YY1 deletion construct
that contained only the zinc finger domain of YY1 was tested for its DNA binding
properties (Fig. 15A, compare lane 5 to lane 6). In contrast, and consistent with
earlier reports, acetylated GST-p53 bound to its recognition sequence better than
unacetylated GST-p53 (Fig. 15A, compare lane 3 to lane 2). Thus, the decrease in
DNA binding activity caused by acetylation is specific to YY1. To rule out the
(1-414)
(1-261)
(1-200)
(1-170)
DAPIFITC merge
(1-122)
F-YY1
FIG. 14E. Sub-cellular
localization of F-YY1 deletion
constructs. Various F-YY1
deletion constructs were
transiently transfected into
HeLa cells. Transfected HeLa
cells were then fixed and
probed with anti-Flag FITC
conjugate antibody. The nuclei
were stained with DAPI.
Images were obtained from a
fluorescence microscope.
Merged images from FITC and
DAPI stains indicate sub-
cellular localization of F-YY1
deletion constructs.
56
possibility that this decrease in DNA binding activity was associated with the GST
fusion constructs, we tested non-tagged YY1 purified from E. coli, as well as Flag-
tagged YY1 purified from HeLa cells. Both forms of YY1 exhibited decreased DNA
binding activity upon acetylation (Fig. 15B), proving that the DNA-binding activity of
YY1 decreases when the zinc finger domain of YY1 is acetylated.
.
A
acet
ylate
d p53
mock
acet
ylate
d p53
acet
ylate
d YY1 (
1-41
4)
acet
ylate
d YY1 (
261-
414)
free probe1 2 3 4 5 6 7 8
mock
acet
ylate
d YY1 (
1-41
4)
mock
acet
ylate
d YY1 (
261-
414)
p53 p
robe a
lone
YY1 pro
be alo
ne+ -+ -acetylated
E. coliYY1
HeLaFlag-YY1
free probe1 2 3 4
B
FIG. 15A, B. The effect of acetylation on YY1’s sequence-specific DNA-
binding activity. Representative EMSAs of GST-tagged YY1, bacterially
expressed non-tagged YY1 and HeLa cell-expressed Flag-tagged YY1..
Purified proteins were in vitro acetylated or mock acetylated with PCAF
and mixed with 32P-labeled probes containing either a YY1 or p53 binding
sequence. Protein-DNA complexes were resolved on non-denaturing
polyacrylamide gels. Black arrows indicate the positions of full-length
YY1- and p53-DNA complexes. The empty arrow indicates the position of
the complex between DNA and the zinc finger domain of YY1.
57
YY1 exists in a high molecular weight protein complex. To determine if
YY1 exists in a protein complex in the cell, gel filtration chromatography was
employed. Nuclear extract from HeLa cells was applied to a Superdex 200 column.
Proteins were fractionated and differentially eluted according to their molecular
weights, and the presence of YY1 was demonstrated by western blot analysis. Figure
16 shows that YY1, which normally runs at about 68 kD on SDS-polyacrylamide
gels, eluted at about 150 kD, suggesting that YY1 associated with other proteins in
HeLa nuclear extract. YY1 could also be detected in earlier fractions (Fig. 16,
fractions 1 and 3); however, because those fractions were outside the fractionation
range of this column, the precise molecular weight of the higher molecular weight
YY1 complex could not be determined.
Purification of native YY1 complexes. To purify a YY1 complex active in
transcription from HeLa nuclear extract, anion exchange chromatography was chosen
M97
68
43
kD 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33Input
200kD 150kD 66kD 29kD 12.4kD
fractions
YY1
FIG. 16. Gel filtration analysis of YY1 in HeLa cells. HeLa nuclear extract
was fractionated on a Superdex 200 column. Eluted fractions were analyzed
for the presence of YY1 by western blot analysis. Calibrated molecular
weights are indicated above the fraction numbers.
58
as the initial purification step. The DNA-binding activity of the YY1 complex was
monitored using EMSA, and the histone deacetylase activity associated with YY1 was
determined using histone deacetylation assay against a labeled H4 peptide. Figures
17A and 17B show that YY1 was eluted from a Q Sepharose column at 200 mM to
.
M W 11 12 13 14 17 18 19 20 21 23Inp
ut
9768
43
kD200
29
18
fractions
A
.
M W 11 12 13 14 17 18 19 20 21 23 25 27
YY1
Inp
ut
97
68
43
kD200
fractions
B
FIG. 17. Purification of a native YY1 complex from
HeLa cells using anion exchange chromatography and
IMAC. (A) A Coomassie-stained SDS-polyacrylamide
gel of the Q Sepharose fractions. (B) Western blot
analysis of the Q Sepharose fractions. An arrow
indicates the position of YY1.
59
500 mM of salt concentrations. Fractions containing YY1 also exhibited strong DNA
binding activity (Fig. 17C) and histone deacetylase activity (Fig. 17D). Q Sepharose
Input 11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 330
500
1000
1500
his
ton
e d
eace
tyal
se a
ctiv
ity
(cp
m)
fractions
D
11 12 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33Input
free probe
fractions
C
FIG. 17. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and IMAC. (C) EMSA of the Q
Sepharose fractions using the AAV P5+1 YY1 binding sequence as a
probe. (D) Histone deacetylation assay of the Q Sepharose fractions.
Fractions from the Q Sepharose column were assayed for histone
deacetylase activity against the H4 peptide.
60
1 2 3 4 5 6 7 8 9
300mM
10
free probe
fractions
F
M input 1 2 3 1 2 1 2 3 4 5 6 7 8 9 10
FT Wash 300mM
YY168
43
kD fractions
E
FIG. 17. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and IMAC. (E) Western blot analysis
of the Ni2+ column fractions. An arrow indicates the position of YY1.
FT, flowthrough fractions; Wash, wash fractions; 300 mM, 300 mM
imidazole-eluted fractions. (F) EMSA of the 300 mM imidazole-eluted
fractions using the AAV P5+1 YY1 binding sequence as a probe.
61
fractions 13 to 17 were pooled and further fractionated using a Ni2+ column. YY1
contains a stretch of histidines at the N-terminus, which potentially serve as binding
moieties to the Ni2+ column. Bound YY1 and the YY1-associated proteins were
eluted with 300 mM imidazole. Figure 17E and 17F show that the eluted fractions
containing YY1 were still active in sequence-specific DNA-binding; however, they
did not contain significant histone deacetylase activity (data not shown). Several
kD
200
97
68
43
29
18
M 300 mM
*
*
**
YY1
G
FIG. 17. Purification of a native YY1
complex from HeLa cells using anion
exchange chromatography and IMAC.
(G) Silver-staining analysis of the
second fraction from the 300 mM
imidazole-eluted fractions. A black
arrow indicates the YY1 protein band.
Empty arrows with stars indicate the
positions of protein bands that are
present in YY1 complexes purified by
other methods presented in this
dissertation.
62
protein bands from fraction 2 of the 300 mM imidazole fractions were stained with
silver, suggesting that those proteins associate with YY1 after both anion exchange
and immobilized metal affinity chromatography steps.
Because the final fractions from the Ni2+ column lost their histone deacetylase
activity, a different purification scheme was employed to purify a YY1 complex
97
68
43
kD
200
29
18
M Inp
ut
2 4 6 8 10 1214 16 1820 22 24 2826 30 fractions
A
M W 2 4 6 8 10 12 14 16 18 20 22 24
YY1
Inp
ut
97
68
43
kD200
26 fractions
B
FIG. 18. Purification of a native YY1 complex from HeLa cells using
anion exchange chromatography and antibody-affinity chromatography. (A)
A Coomassie-stained SDS-polyacrylamide gel of the Q Sepharose fractions.
(B) Western blot analysis of the Q Sepharose fractions. An arrow indicates
the position of YY1.
63
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Input
free probe
fractions
C
FIG. 18. Purification of a native YY1 complex from HeLa cells
using anion exchange chromatography and antibody-affinity
chromatography. (C) EMSA of the Q Sepharose fractions using
the AAV P5+1 YY1 binding sequence as a probe. (D) Histone
deacetylation assay of the Q Sepharose fractions. Fractions from
the Q Sepharose column were assayed for histone deacetylase
activity against the H4 peptide.
Input 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 fractions0
1000
2000
3000
his
ton
e d
eace
tyla
se a
ctiv
ity
(cp
m)
D
64
retaining histone deacetylase activity. HeLa nuclear extract was first fractionated on
the Q Sepharose column as described above, and fractions containing YY1 (Fig. 18A
and 18B, fractions 8 to 18) were pooled. These fractions still possessed sequence-
specific DNA-binding activity and histone deacetylase activity (Fig. 18C and 18D).
Pooled Q Sepharose fractions were passed through a protein G column and the
flowthrough fraction re-loaded on an antibody affinity column containing protein G
resin coupled to the H-10 mAb against YY1. Bound protein complexes were eluted
200
68
97
43
29
kD
YY1
1 2
protein G
H-10
*
**
*
18
E FIG. 18. Purification of a native YY1
complex from HeLa cells using anion
exchange chromatography and antibody-
affinity chromatography. (E) Silver-staining
analysis of fractions from the anti-YY1 H-
10 column (lane 2) and the protein G control
column (lane 1). A black arrow indicates
the position of the YY1 protein band.
Empty arrows show the presence of multiple
protein bands that exist in the fractions from
the H-10 column but not in those from the
protein G column. Stars indicate the
positions of protein bands that are present in
YY1 complexes purified by other methods
presented in this dissertation.
65
from both columns with acidic glycine. The protein components of the eluted YY1
complex were compared to those from the protein G column (Fig. 18E). The YY1
complex from the H-10 column still retained histone deacetylase activity (Fig. 18F).
Purification of a Flag-tagged YY1 complex. To purify a YY1 complex
using a different method, Flag-tagged YY1 (F-YY1) was over-expressed in HeLa
FIG. 18. Purification of a native YY1 complex from HeLa cells using anion
exchange chromatography and antibody-affinity chromatography. (F)
Histone deacetylation assay from the protein G control column (protein G)
and the anti-YY1 H-10 column (H-10) against the H4 peptide.
Protein G H-10
1000
2000
3000
4000
5000
6000
his
ton
e d
eace
tyal
se a
ctiv
ity
(cp
m)
F
66
cells, and the proteins associated with F-YY1 were purified from whole cell lysates on
an antibody column against the Flag epitope. As a control, empty Flag vector was
transiently transfected into HeLa cells and the whole cell lysate purified on an anti-
Flag column. The protein components of the F-YY1 complex and the proteins eluted
from the Flag alone control were compared on silver-stained SDS-polyacrylamide
gels (Fig. 19A). Protein bands that are present in the F-YY1 lane but not in the Flag
lane represent proteins specifically associate with YY1, including the over-expressed
F-YY1 as well as a degradation product of F-YY1, F-YY1’. Fractions from the F-
200
68
97
43
29
kD Flag F-YY1
*
F-YY1
F-YY1'
1 2
*
*
*
FIG. 19A. Silver-staining analysis of the
second fractions from the Flag-alone (lane
1) column and the F-YY1 column (lane 2).
Black arrows indicate the positions of the
full-length F-YY1 (F-YY1) and a
degradation product of F-YY1 (F-YY1’).
Empty arrows show the presence of
multiple protein bands that exist in the
fractions from the F-YY1 column but not
in those from the Flag alone column. Stars
indicate the positions of protein bands that
are present in YY1 complexes purified by
other methods presented in this
dissertation.
67
YY1 column contained significant histone deacetylase activity after concentration
through ultrafiltration (Fig. 19B).
Proteins specifically associate with F-YY1 were also visualized by
radioimmunoprecipitation analysis (RIPA) (Fig. 20A). HeLa cells were transiently
transfected with F-YY1 or Flag alone and the proteins within the cells metabolically
labeled with 35S-containing amino acids. Proteins were subsequently
immunoprecipitated from cell lysates with anti-Flag beads. Several protein bands
specific to F-YY1 were visible upon comparison to the Flag control. RIPA was also
used as a tool to analyze patterns of protein association specific to different regions of
YY1. Amino acid residues 261-333 were shown to be involved in YY1-associated
histone deacetylase activity. Therefore, F-YY1 (261-333) and F-YY1 (∆261-333)
were transiently transfected into HeLa cells and subjected to RIPA. However, despite
numerous attempts, comparisons among immunoprecipitated proteins from Flag, F-
.
Flag F-YY1
100
200
300
400
500
his
ton
e d
eace
tyal
se a
ctiv
ity
(cp
m)
FIG. 19B. Histone deacetylation
analysis of the second fractions
from the Flag alone column and the
F-YY1 column against the H4
peptide.
68
YY1, F-YY1 (261-333), and F-YY1 (∆261-333) failed to suggest proteins that were
specifically associated with constructs that had been shown to contain associated
histone deacetylase activity [F-YY1 and F-YY1 (261-333)] (Fig. 20B).
kD
200
97
68
43
29
F-YY1
Flag F-YY1
F-YY1'
1 2
*
*
*
*
Fig. 20A. A representative autoradiograph
showing proteins bound to Flag alone or F-
YY1. Black arrows indicate the positions of
the full-length F-YY1 (F-YY1) and a
degradation product of F-YY1 (F-YY1’).
Empty arrows show the presence of multiple
protein bands that were present in the F-
YY1 immunoprecipitate (lane 2) but not in
the Flag alone immunoprecipitate (lane 1).
Stars indicate the positions of protein bands
that are present in YY1 complexes purified
by other methods presented in this
dissertation.
69
..
kD
200
97
68
43
29
F-YY1
Flag F-YY1
F-YY1'
F-YY1 (∆ 261-333)
F-YY1 (∆ 261-333)
F-YY1 (261-333)
FIG. 20B. A representative autoradiograph showing
proteins bound to different F-YY1 deletion mutants.
Black arrows indicate the positions of the full-length F-
YY1 (F-YY1), a degradation product of F-YY1 (F-
YY1’), and one of the F-YY1 deletion mutants [F-YY1
(∆261-333)].
70
DISCUSSION
In this dissertation, the isolation and characterization of a genomic clone of
human YY1 is presented. Deletional analysis reveal that the human YY1 promoter
possesses many characteristics typical of housekeeping genes. For example, the YY1
gene does not contain a TATA box, and the elements critical for basal transcription
reside within a small region around the transcriptional start site. One unusual
observation is that plasmid p+54Luc, which contains sequences downstream of the
transcriptional initiation site, still possesses significant activity compared to the wild-
type YY1 promoter. Only one transcriptional start site of the human YY1 gene has
been detected by primer extension, and no other start sites were found around or
downstream of position +54. It is possible that minor transcription initiation sites
exist downstream of the mapped +1 site, but they are too weak to be detected by
primer extension analysis. It is also possible that the YY1 gene contains alternative
start sites that are utilized only when the natural start site is not present.
A notable feature of the human YY1 promoter is its similarity to the mouse
YY1 promoter (135). The proximal promoter region between positions -741 and
+476 in the human gene and that between positions -751 and +434 in the mouse gene
share a remarkable 70% identity. The human YY1 promoter is quite active with a
small sequence of position -277 to position +475. Similarly, the mouse YY1
promoter only requires a region as short as from position -58 to position +32 for
activity. Moreover, many transcription factor binding sites that are present in the
71
human YY1 promoter are also present in the mouse promoter. An Sp1 binding site
plays an important role in the expression of the mouse YY1 gene (135), and three Sp1
sites are found within the region critical for the activity of the human YY1 promoter.
These observations suggest that the human and the mouse YY1 promoter are
controlled by a similar regulatory mechanism.
The adenovirus E1A protein can convert YY1 from a repressor to an activator.
Many studies have been devoted to the mechanistic details of this interesting
phenomenon. It was elegantly demonstrated that physical interactions among E1A,
YY1, and p300 are critical in this conversion from repression to activation (95). Our
study shows here that E1A down-regulates the YY1 promoter and that a minimal
YY1 promoter is sufficient for E1A-mediated repression. These findings suggest an
alternate, albeit non-mutually exclusive, model of how the transcriptional activity of
YY1 is affected by E1A. Under physiological concentrations of YY1, YY1 represses
transcription in certain cell types. However, the presence of the E1A protein
decreases the expression level of YY1, which results in activation of genes normally
repressed by YY1. In this model, the activity of YY1 is similar to that of the
Drosophila Krüpple transcription factor, which activates or represses target genes
depending on its cellular concentrations (138). It will be interesting to study if the
expression of YY1 could be regulated by other viral and cellular transcription factors.
At the polypeptide level, the activity of the multifunctional transcription factor
YY1 is shown to be regulated by acetylation and deacetylation. Acetylation and
deacetylation of YY1 represents a novel and complex means of regulating the activity
of a DNA-binding transcription factor (Fig. 21). YY1 is acetylated in two regions:
72
one at the previously identified HDAC-interacting domain of residues 170-200, and
the other at the C-terminal DNA-binding zinc finger domain. Residues 170-200 of
YY1 are acetylated by p300 and PCAF, while the C-terminal zinc finger domain is
acetylated only by PCAF (Fig. 21). Acetylation of these two regions results in
54 80 154 414200 295170
transcriptionalactivationdomain
transcriptionalrepression
domain
transcriptionalrepression
domain
DNA-binding domain
Zn FingersSpacerAcidic GKGAHisAcidic
HDAC?
histones
HDAC
in vivo acetylated domains
p300 acetylated domain
PCAF acetylated domains
in vivo HDAC-binding domains
histone deacetylase activity domain
170 200
170 200 261 398
170 200 261
261 333
170 200 414261
333
in vitro HDAC-binding domain170 200
deacetylated domain170 200
FIG. 21. Summary of regulation of YY1 by acetylation and deacetylation.
YY1 is acetylated in two regions: residues 170-200 and the C-terminal
DNA-binding domain. Acetylation of residues 170-200 targets YY1 for
deacetylation. Acetylation of the C-terminus of YY1 results in stable
association of histone deacetylase activity with YY1 as well as decreased
DNA-binding activity. The in vivo association between YY1 and HDACs
at the C-terminal region is probably mediated through an unidentified
protein (depicted as a question mark in an oblong circle).
73
dramatically different outcomes: First, when residues 170-200 of YY1 are acetylated,
YY1 becomes a more effective transcriptional repressor and binds HDACs more
efficiently. However, upon binding to acetylated YY1 residues 170-200, HDACs also
actively deacetylate this region, possibly resulting in a negative feedback loop.
Second, YY1 possesses histone deacetylase activity toward histone H4 by associating
with HDACs using the C-terminal zinc finger region. This is most likely a result of
active targeting of acetylated YY1 zinc finger domains by HDACs. However, our
data do not indicate that association of the YY1 C-terminus with HDACs brings about
deacetylation of YY1 in this region. In contrast, the interaction between YY1 and
HDACs at residue 170-200 is likely to be a dynamic and highly regulated process and
does not result in associated histone deacetylase activity stable enough to be detected
in our experimental system. Finally, acetylation of YY1 zinc fingers decreases YY1’s
DNA-binding activity, which will have further impact on YY1’s activity as a
transcription factor.
This differential regulation of YY1 by acetylation and deacetylation suggests a
complex regulatory system unlike that of any other transcription factors known to
date. In cells where YY1 functions primarily as a transcriptional repressor,
acetylation of YY1 at the central HDAC-binding region and the C-terminal DNA-
binding region most likely will result in an intricate network of negative-feedback
regulation: Acetylation of YY1 at residues 170-200 augments YY1’s repressor
activity, but acetylation of this region also targets YY1 for deacetylation. In the mean
time, acetylation of the C-terminal DNA-binding region of YY1 most likely stabilizes
interaction between YY1 and HDACs, but acetylation of this region in turn decreases
74
YY1’s DNA binding activity. To date, we have not been able to determine the
relative levels of acetylation between the central region and the C-terminus of YY1
under physiological conditions; therefore, the relative contribution of acetylating these
two regions is unknown. Interestingly, we also found that TSA had little effect on the
acetylation status of YY1 (data not shown), suggesting that regulation of YY1 by
acetylation is more prominent than by deacetylation. This finding is also in
agreement with our discovery that deacetylation of YY1 only occurs at residues 170-
200, while acetylation of YY1 can happen at the C-terminal DNA-binding domain as
well.
In many experimental systems, YY1 can also activate transcription, and it is
still uncertain how this is accomplished. Different models, including bending of
DNA, the relative distance between the YY1 binding site and the transcriptional
initiation site, as well as protein-protein interactions have been proposed (reviewed in
references 143, 154). Increasingly more evidence shows that interactions with other
proteins are probably the most important factors in YY1-mediated transcriptional
activation. It has been suggested that interactions of YY1 with other cellular proteins
or viral proteins can either disrupt the quenching activity of YY1 on other
transcriptional activators or stimulate transcription with associated enzymatic
activities such as HATs (95, 144, 154). Our findings here provide an additional
speculation that on promoters activated by YY1, YY1-associated p300 and PCAF can
activate transcription by both acetylating core histones and acetylating YY1 at the C-
terminal zinc finger domain (PCAF only), which in turn decreases the overall histone
deacetylation activity at the promoter.
75
In addition to histones and several non-histone chromatin proteins, many
transcription factors have been shown to be regulated by acetylation. These
transcription factors include p53 (54), HIV Tat (84), E1A (176), GATA-1 (15, 73),
EKLF (177), MyoD ((131, 137), E2F (110), TFIIE, TFIIF (75), CIITA (149), TCF
(167), HNF-4 (147), UBF (128), TAL1/SCL (71), and nuclear receptor co-activators
ACTR, SRC-1, and TIF2 (29). Many of these factors are acetylated by both
p300/CBP and PCAF; therefore, it is not surprising that YY1 is also acetylated by
both p300 and PCAF. The p300-interaction domain of YY1 has been mapped to the
C-terminal 17 residues (95, 97), which were not acetylated by p300 in our study. This
finding is reminiscent of acetylation of p53 by p300/CBP, where the N-terminus of
p53 interacts with CBP (55, 102) while the region acetylated by p300 is at the C-
terminus of p53 (54). Moreover, the arrangement of the 6 lysines within the p300-
acetylation domain of YY1 closely resembles the sequence of the p300-acetylated
region of histone H2B (150), indicating that YY1 contains an authentic p300-
acetylation domain. Interestingly, the PCAF-interacting domain of p300 overlaps
with its YY1-interacting domain (5, 173), which suggests that acetylation of YY1 by
PCAF versus by p300 might be either cooperative or mutually exclusive in vivo,
depending on whether or not YY1 binding to p300 can be competed by PCAF.
Furthermore, in our in vitro acetylation studies, full-length GST-YY1 was acetylated
by PCAF and not by p300, which further suggests that the conformation of YY1,
perhaps affected by selective interaction with p300 and PCAF, is important to YY1
acetylation. We also found that in HeLa cells, overexpression of PCAF, but not p300,
partially alleviated the transcriptional repressor activity of a Gal4 DNA-binding
76
domain-YY1 fusion protein. However, in NIH3T3 cells, overexpression of p300, but
not PCAF, relieved repression from Gal4-YY1 (data not shown). In this regard, it
will be important to identify the in vivo triggers directing PCAF versus p300
acetylation. p53 is particularly interesting among acetylated transcription factors
because it has been elegantly demonstrated that DNA damage (UV and ionizing
radiation) causes p53 acetylation at two distinct lysines, one by p300 and the other by
PCAF (103). To date, this is the only report linking specific environmental cues to
acetylation of transcription factors by HATs, and yet it is still uncertain why
acetylation would require two HATs. It is interesting to note that while an increase in
DNA-binding activity has been observed for most acetylated DNA-binding
transcription factors, HMG I(Y) binds DNA less when it is acetylated (117). The
most striking observation is that only when HMG I(Y) is acetylated by CBP, not by
PCAF, is there a decrease in its DNA-binding activity (117). This phenomenon is
reminiscent of YY1 acetylation in its zinc finger domain by PCAF but not by p300,
and the consequent reduction in the DNA-binding activity of YY1.
We were also interested in finding out if acetylation of YY1 also contributes
to cellular events other than transcriptional control. So far, we have no evidence to
suggest that acetylation changes the sub-cellular localization of YY1 (data not
shown). YY1 has been shown to be a rather stable protein expressed at comparable
levels in both growing and differentiating cells (5). Interestingly, acetylation affects
the conformation of HNF-4 (147), the half-live of E2F (110), and promotes protein-
protein interactions between Rch1 and importin-β (9). Therefore, it will be
informative to test whether acetylation of YY1 may have similar consequences.
77
Recently it was demonstrated that YY1 null mutations resulted in peri-
implantation lethality in mice (38). In Drosophila, lack of a maternally derived YY1
homolog results in severe defects in pattern formation (16, 45). This Drosophila
homolog of YY1 was identified as PHO, which is encoded by pleiohomeotic, a
member of the Polycomb group (PcG) genes (18). PcG genes encode proteins
necessary for maintaining the repression state of homeotic genes in Drosophila, which
is absolutely essential for patterning (127, 146, 152). It has been proposed that the
Drosophila YY1 homolog, PHO, binds to PcG response elements and interacts with
other proteins to form a repressor complex (18). A Drosophila protein, dMi-2, has
been found to participate in PcG-mediated repression (82). dMi-2 shares high
sequence homology to the human autoantigen Mi-2. Xenopus Mi-2 homolog was
recently shown to be a subunit of a histone deacetylase complex with nucleosome
remodeling activity (164, 165). Incidentally, repression mediated by PcG proteins
resembles that of mating-type silencing in yeast and position-effect variegation in
Drosophila, both of which contain transcriptionally silent chromatin structures (112).
Therefore, it is possible that dMi-2 also forms a complex containing histone
deacetylase activity and nucleosome remodeling activity, and the interaction between
dMi-2 and PcG proteins like PHO is important in proper expression of homeotic
genes. Our study showing stable interactions between HDACs and YY1 also suggests
that association with chromatin-modifying activities is central to the functions of
YY1. YY1, PHO, and the Xenopus YY1 homolog FIII are almost completely
identical in the zinc finger region in amino acid sequence, reinforcing the role of YY1
as a DNA-targeting factor in nucleating a repressor complex capable of modulating
78
chromatin structures. Our data demonstrating that YY1 interacts with HDACs at two
different regions open the possibility that these two HDAC-interacting regions might
have distinct effects on YY1's role in transcriptional control. Perhaps the stable
association of YY1 with HDACs at the C-terminal zinc finger region represents an
ancient mode of the functions of YY1, which is to form a repressor complex
associated with the promoter. However, regulation of the affinity of the central region
of YY1 for HDACs by acetylation might have evolved as a more sophisticated means
of control, which defines a novel functional consequence of non-histone factor
acetylation.
In this dissertation, four different methods of purifying the YY1 complex are
presented: anion exchange chromatography followed by IMAC, anion exchange
chromatography followed by antibody-affinity chromatography, antibody-affinity
chromatography directly against a Flag-epitope, and RIPA. Upon comparison of the
YY1 complexes purified with different methods, it became apparent that four protein
bands exist in YY1 complexes purified by all four methods. These proteins most
likely represent genuine YY1-associated proteins.
Among the four candidate YY1 complex constituents, only one appears to
have a molecular weight higher than 200 kD. Gel filtration analysis of HeLa nuclear
extract suggests that YY1 exists in potentially two protein complexes, one with a very
high molecular weight, and the other one with a molecular weight of 150 kD. This
high molecular weight YY1-associated protein very likely represents a component of
the higher molecular weight YY1 complex. Other YY1-associated proteins with
molecular weights lower than 97 kD may or may not exist exclusively in the 150 kD
79
YY1 complex. Because the apparent molecular weight of YY1 on SDS-
polyacrylamide gels is different from the predicted molecular weight of YY1 based on
amino acid composition, it is difficult to estimate the stoichiometric composition of
the YY1 complex(s).
IMAC using Ni2+ resin following anion exchange chromatography may not be
an ideal method to purify a YY1 complex. First, this method intrinsically lacks a
proper control. As a result, without comparison to the YY1 complexes purified by
other methods, it would have been extremely difficult, if not impossible, to identify
true YY1-associated protein. Second, the amounts of proteins bound to the Ni2+
column, except for YY1 itself, appear to be quite low. This might account for the
lack of histone deacetylase activity from the eluate of this column. However, it is not
to exclude the possible application of Ni2+ resin in purifying other protein complexes.
Because the Flag epitope of the F-YY1 construct provides a strong epitope for
the anti-Flag beads, and the YY1 part of the F-YY1 construct is free to interact with
other proteins, purification of the F-YY1 complex might be the best method to purify
a YY1 complex based on its ease and true representation of the protein-protein
interactions. This method can be easily scaled up to facilitate large quantity
purification and subsequent micro-sequencing using a recombinant adenovirus
expressing F-YY1. Similarly, a recombinant adenovirus expressing F-YY1 (261-333)
can be made to infect HeLa cells and to purify a protein complex specifically
associated with residue 261-333 of YY1. This will provide valuable information
about the critical protein components involved in the histone deacetylase activity
associated with YY1.
80
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ABOUT THE AUTHOR
Ya-Li Yao received a Bachelor's degree in Biological Sciences from University of
California, Davis in 1995. She enrolled in the Ph.D program of Molecular Medicine at
the University of Texas, Health Sciences Center at San Antonio in 1996. Subsequently,
she transferred to the Ph.D. program in the Department of Medical Microbiology and
Immunology at the University of South Florida. She was awarded a Master's degree in
Medical Sciences in 1999 and a Ph.D. degree in 2001.
While in the Ph.D. program at the University of South Florida, Ya-Li Yao
received a graduate student fellowship from the American Heart Association as well as a
travel award from the University. She co-authored several publications in peer-reviewed
journals and received the Time Warner Road Runner ETD (Electronic Thesis and
Dissertation) Award in 2001.