From the Wistar Institute, Philadelphia, Pennsylvania
19104 and
Harvard Microchemistry and Proteomics Analysis
Facility, Harvard University, Cambridge, Massachusetts 02138
Received for publication, September 3, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Eukaryotic genes are under the control of
regulatory complexes acting through chromatin structure to control gene
expression. Here we report the identification of a family of
multiprotein corepressor complexes that function through modifying
chromatin structure to keep genes silent. The polypeptide composition
of these complexes has in common a core of two subunits, HDAC1,2 and
BHC110, an FAD-binding protein. A candidate X-linked mental retardation
gene and the transcription initiation factor II-I (TFII-I) are
components of a novel member of this family of complexes. Other
subunits of these complexes include polypeptides associated with cancer
causing chromosomal translocations. These findings not only delineate a
novel class of multiprotein complexes involved in transcriptional
repression but also reveal an unanticipated role for TFII-I in
transcriptional repression.
The genome of eukaryotes is packaged into chromatin, the
fundamental unit of which is the nucleosome. The higher order chromatin structure is formed by arrangement of nucleosomes into an array. Such a
higher order chromatin structure presents a barrier to cellular
processes such as transcription, DNA replication, and DNA repair.
Therefore, controlling accessibility to the nucleosomal DNA provides an
important regulatory point in these processes (1). One way to modulate
nucleosomal structure is through enzymatic modification of histones by
acetylation, phosphorylation, or methylation.
A number of transcriptional regulatory complexes have been identified
that contain histone acetylation or deacetylation activities. It was
previously shown that the hyperacetylated chromatin correlates with
active genes whereas the repressed genes exhibit a pattern of
hypoacetylation (2, 3). This contention was strengthened by the
discovery of the association of a number of transcriptional corepressors with histone deacetylation activity. The
HDACs1 identified in
mammalian cells can be divided into three classes. Homologs of the
yeast protein Rpd3 are members of the Class I HDACs (4, 5). Included in
this class are HDAC1, HDAC2, HDAC3, and HDAC8. Members of the Class II
HDACs include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10. These
HDACs appear to be more similar to yeast protein Hda1 (6-9). A third
class of HDACs exists, which unlike the other classes of HDACs,
requires an NAD cofactor for activity. The members of this class are
homologs of the yeast Sir2 protein (10-12).
Previous biochemical analysis revealed the association of
transcriptional corepressor Sin3 with a multiprotein complex containing histone deacetylase activity (13-15). This complex was shown to contain HDAC1,2 and act as a transcriptional corepressor for a number
of DNA-binding repressors including Mad, the nuclear hormone receptors,
and the RE1-binding silencer
protein, REST (also called NRSF) (14, 16-19). In addition, a number of
groups reported the isolation and characterization of a complex termed
NuRD (also NURD and NRD) that not only contains histone deacetylases 1 and 2 but also a DNA-dependent ATPase subunit (20-22).
Here, we report the isolation of a new family of HDAC1,2 complexes that
also contain the FAD-binding protein BHC110 (23). Unique to this family
of corepressor complexes is the presence of a distinct structural
DNA-binding subunit defining different HDAC1/2-containing complexes.
Immunoaffinity Purification of the
BHC110/HDAC2-containing Complex--
HeLa nuclear extract
was fractionated according to the protocol described above using P11.
Anti-BHC110 and anti-HDAC2 antibodies (500 µg each) were cross-linked
to Protein A-Sepharose (1 ml, Repligen) using standard techniques for
affinity purification. The P11 0.3 M KCl fraction was
incubated with 1 ml of antibody-Protein A beads for 4-5 h at
4 °C. The beads were washed first with 1 M KCl in buffer
A (20 mM Tris-HCl, pH 7.9, 0.2 mM EDTA, 10 mM Affinity Purification of FLAG-XFIM--
FLAG-XFIM and a
selectable marker for puromycin resistance were co-transfected into 293 human embryonic kidney cells by calcium phosphate co-precipitation.
Transfected cells were grown in the presence of 10 µg/ml puromycin,
and individual colonies were isolated and analyzed for FLAG-XFIM
expression. A cell line expressing FLAG-tagged XFIM, F-XFIM, was used
for the affinity purification of the XFIM-containing complex as
previously described for the FLAG-BRAF35 cell line (23).
Chromatographic Purification of TFII-I Complex from HeLa Nuclear
Extract--
HeLa nuclear extract (3 g) was loaded on a 500-ml column
of phosphocellulose (P11, Whatman) and fractionated stepwise by the indicated KCl concentration in buffer A. The P11 0.3 M KCl fraction (700 mg) was loaded on a 80-ml DEAE-Sephacel
column (Amersham Biosciences) and eluted with 0.35 M
KCl in buffer A. The 0.35 M KCl elution (500 mg) was
dialyzed to 10 mM KxPO4 in buffer B (5 mM Hepes, pH 7.6, 1 mM dithiothreitol,
0.5 mM PMSF, 10 µM CaCl2, 10%
glycerol, 40 mM KCl) and loaded on a 70-ml Bio-Gel HT
column (hydroxyapatite, Bio-Rad). The column was resolved by using a linear 10-column volume gradient of 50-500 mM
KxPO4. A pool of fractions 28-30 was dialyzed to
700 mM NH4SO4 in Buffer HB (20 mM HEPES, pH 7.6, 4 mM dithiothreitol, 0.5 mM EDTA, 10% glycerol, 0.5 mM PMSF) and loaded
on a butyl-Sepharose column (Amersham Biosciences). The column
was resolved using a linear 10-column volume gradient of 700 to
0 mM NH4SO4 in Buffer HB.
TFII-I-containing fractions 12-16 were dialyzed to 100 mM
KCl in Buffer A and loaded on Heparine-5PW (TosoHaas). The column was
resolved using a linear 20-column volume gradient of 100-500
mM KCl in Buffer A. TFII-I containing fractions 12-16 was
fractionated on a Superose 6 HR 10/30 column (Amersham
Biosciences) equilibrated in 0.5 M KCl in buffer A. Superose 6 was calibrated using molecular weight standards from
Amersham Biosciences. The void was determined according to the
manufacturer's guidelines (one-third of column volume or 7 ml).
Fractions 16-20 and 24-28 were used for immunoaffinity purification
of the TFII-I-containing complexes.
Mass Spectrometric Peptide Sequencing--
Excised bands were
subjected to in-gel reduction, carboxyamidomethylation, and tryptic
digestion (Promega). Multiple peptide sequences were determined in a
single run by microcapillary reverse-phase chromatography (a custom New
Objective 50-µm column terminating in a nanospray 15-µm tip),
directly coupled to a Finnigan LCQ Deca quadrupole ion trap mass
spectrometer. The ion trap was programmed to acquire successive sets of
three scan modes consisting of: full scan MS over alternating ranges of
395-800 m/z or 800-1300 m/z, followed by two data-dependent
scans on the most abundant ion in those full scans. These dependent
scans allowed the automatic acquisition of a high resolution (zoom)
scan to determine charge state and exact mass and MS/MS spectra for
peptide sequence information. MS/MS spectra were acquired with a
relative collision energy of 30%, an isolation width of 2.5 daltons,
and dynamic exclusion of ions from repeat analysis. Interpretation of
the resulting MS/MS spectra of the peptides was facilitated by programs
developed in the Harvard Microchemistry Facility (24) and by data base correlation with the algorithm SEQUEST (25).
Immunoblot Analysis--
Anti-BHC110, anti-BHC80, and
anti-BRAF35 antibodies were described previously (23). Anti-HDAC2
antibodies were obtained from Zymed Laboratories Inc..
Anti-TFII-I and XFIM antibodies were developed to a peptide
corresponding to the amino acids IKETDGSSQIKQEPDPTW and DPLTLPEKPLAGDLP
for TFII-I and XFIM, respectively. Immunoblotting was performed
with alkaline phosphatase.
HDAC Assays--
HDAC assays were performed as described
(23).
Chromatin Immunoprecipitation (ChIP)--
ChIPs were performed
as described (23). PCR of the c-fos promoter was performed
on immunoprecipitated chromatin using oligonucleotides C-FOS/A
(5'-AGCAGTTCCCGTCAATCC-3') and C-FOS/B
(5'-TGAGCATTTCGCAGTTCC-3').
Transient Transfection--
293 cells were transfected
with 5XGAL4UAS-Tk-luciferase reporter in the presence of
effector plasmids (GAL4(BD), fusion vectors GAL4(BD)/TFII-I,
GAL4(BD)/SAP30, GAL4(BD)/HP1 RNAi and Transfections--
The small interfering RNA (RNAi)
sequence targeting TFII-I (AA GUU ACU CAG CCA AGA ACG A) or the control
RNAi was purchased from Dharmacon. Transfections were performed on
2 × 106 HeLa cells with a final concentration of 200 mM small interfering RNA duplex using Oligofectamine
reagent (Invitrogen) according to the manufacturer's
guidelines. After two rounds of RNAi treatment followed by
12 h of starvation in 1% serum medium, cells were subsequently
stimulated with 50 nM epidermal growth factor (EGF) (Sigma) and left for the times indicated before harvesting (see Fig.
6b).
BHC110 Defines a New Family of HDAC-containing Complexes--
We
recently reported the isolation from HeLa nuclear extract of a
BRAF35-HDAC complex (BHC) containing flavin adenine dinucleotide (FAD)-binding subunit, BHC110 (23). This complex contains subunits similar to the CoREST complex described previously (26, 27). To
determine whether there are other BHC110-containing complexes in HeLa
cells, we developed anti-BHC110 antibodies and affinity-purified the
BHC110-containing complexes following the scheme in Fig.
1. The anti-BHC110 affinity eluate was
subjected to ion trap mass spectrometric sequencing. In addition to
other components of the BHC complex (23), this analysis revealed the
stable association of BHC110 with
ZNF261/XFIM, a candidate gene for X-linked
mental retardation in Xq13.1 (28, 29),
ZNF198/FIM, a gene related to XFIM that is
associated with myeloproliferative disorder that involves
myeloid hyperplasia and eosinophilia (29, 30), KIAA0182, a proline-rich
protein of unknown function, and TFII-I, the initiator binding protein
and a transcriptional coactivator (31, 32) (Fig. 1, lane 2,
and Fig. 2). The association of these
polypeptides and BHC110 is specific as the affinity eluate from a
control antibody column was devoid of their presence (Fig. 1,
lane 3).
To determine whether these polypeptides are also stable components of
an HDAC-containing complex, we affinity-purified HeLa HDAC2-containing
complexes from the 0.3 M KCl eluate of phosphocellulose chromatography (P11) (Fig. 1). Ion trap mass spectrometric sequencing of the anti-HDAC2 affinity eluate revealed that, in addition to components of the previously described complexes of Mi2 (20-22), Sin3
(13-15), and BHC (23, 26, 27), the anti-HDAC2 eluate contained
ZNF261/XFIM, ZNF198/FIM,
KIAA0182, and TFII-I (Fig. 1, lane 1, and Fig. 2). These
results indicate that these novel subunits are associated with both
HDAC2 and BHC110, although these polypeptides most likely represent
multiple distinct HDAC2/BHC110-containing complexes. To test this
hypothesis we embarked on isolating other BHC110-containing complexes
that are distinct from the BHC complex.
TFII-I Is a Component of an XFIM Complex--
To isolate other
BHC110-containing complexes, we developed a 293-derived cell line
stably expressing a FLAG-tagged XFIM (F-XFIM). FLAG-XFIM was
affinity-purified from F-XFIM nuclear extract using anti-FLAG
antibodies followed by elution of bound material with FLAG peptide
(Fig. 3a). A combination of
ion trap mass spectrometry and Western blot analysis demonstrated the
presence of TFII-I, BHC110, and HDAC1/2 polypeptides (Fig.
3a). These polypeptides were absent in affinity-purified
eluate of the parent 293 cell line (Fig. 3a). Further
analysis of the XFIM complex by Superose 6 gel filtration
confirmed the association of XFIM, TFII-I, BHC110, and HDAC2 as a
component of a single complex of about 1 MDa, although a small
percentage of HDAC2 and TFII-I eluted at a smaller molecular mass (Fig.
3c). This complex was termed the XFIM complex (Fig. 3a). Moreover the XFIM complex displayed HDAC activity
toward core histones (Fig. 3c). It is noteworthy that
analysis of the XFIM protein following gel filtration by colloidal
staining revealed a higher stoichiometry for XFIM (~4 XFIMs per
complex) to other subunits of the complex. Taken together, these
results establish BHC110 and HDAC2 as common subunits of at least two
distinct (BHC and XFIM) histone deacetylase complexes (23).
To determine the fraction of TFII-I that associates with BHC110 we
purified TFII-I by conventional column chromatography (Fig. 4a). Analysis of TFII-I on the
gel filtration, the last step of purification, revealed the predominant
peak of immunoreactivity at fraction 24, whereas a small portion
(5-10%) of TFII-I eluted at a larger molecular mass coincident with
BHC110 and HDAC2 (Fig. 4a). Affinity purification using
anti-TFII-I antibodies revealed the association of BHC110 and HDAC2
only with the TFII-I derived from the larger molecular mass fraction
(Fig. 4a, see fractions 18-20). Furthermore,
immunoprecipitation using anti-HDAC2, anti-BHC110, and anti-TFII-I
antibodies demonstrated the specific association of TFII-I with HDAC2
and BHC110 (Fig. 4, b and c). These results indicate that although TFII-I is predominantly monomeric, a fraction of
TFII-I is in a stable complex with BHC110 and HDAC2.
XFIM Complex Is Recruited to the c-fos Promoter--
Because
TFII-I was reported as a transcriptional coactivator for serum response
factor (SRF) at the c-fos promoter (33), we analyzed the
c-fos promoter as a target of the XFIM complex. We first
confirmed the responsiveness of the c-fos promoter to inhibitors of histone deacetylation. Consistent with previous reports
c-fos displayed an increased transcription level
following either sodium butyrate or trichostatin A treatment (34) (Fig. 5a). Importantly, ChIP
experiments established the presence of the components of the XFIM
complex at the c-fos promoter in its basal state (Fig.
5b). In response to mitogens and growth factors such as
serum or EGF, c-fos displays classical immediate-early gene
activation kinetics, where it is induced within 15 min of stimulation,
followed by return to basal levels within 2 h of stimulation.
Therefore, three phases of c-fos expression can be identified: an initial repressed state, an activated state, and a
return to a repressed state (34) (Fig. 5c). To understand the role of the XFIM complex in each phase of c-fos
responsiveness, we used ChIP to examine the c-fos promoter
following EGF stimulation. Similar to results in Fig. 5b,
analysis of the promoter in the initial repressed state revealed the
presence of BHC110, HDAC2, and TFII-I in addition to that of the SRF
protein (Fig. 5d). However, although the SRF levels at the
promoter were enhanced 30 min following the stimulation of
c-fos transcription, HDCA2 and BHC110 were no longer
detectable (Fig. 5d). BHC110 and HDAC2 were returned to the
promoter as the repressed state was reestablished (Fig. 5d,
60 and 90 min). Interestingly, TFII-I occupancy of the promoter was
unchanged following EGF stimulation (Fig. 5d). Moreover,
although the histone H3 acetylation state is not affected by EGF
stimulation, there is an increase in acetylated histone H4 coincident
with the absence of HDAC1,2 complex 30 min following EGF stimulation. These results suggest a role for the XFIM complex as a corepressor at
the c-fos promoter and are consistent with the contention
that TFII-I may play a dual role in that it participates as a component of a corepression complex in the basal repressed state of the promoter,
but once the gene is activated it remains bound to the promoter to form
a stable complex with the activator as previously described (33,
34).
Recruitment of TFII-I to the Promoter Results in
Transcriptional Repression--
To directly assess the role of
TFII-I in transcription, we tethered TFII-I to the GAL4 DNA-binding
domain and tested its activity using a promoter containing five
GAL4-binding sites (Fig. 6a). Interestingly, although GAL4-VP16 resulted in a potent activation of
transcription from this promoter, GAL4-TFII-I caused a moderate (~50%) repression of transcription (Fig. 6a). However,
the transcriptional repression by Gal4-TFII-I was smaller than that
obtained with either GAL4-SAP30 or GAL4-HP1
To further assess the role of TFII-I in transcription of an endogenous
c-fos promoter, we utilized small interfering RNA-mediated depletion (RNAi) specific for TFII-I to inhibit its synthesis. Two
rounds of RNAi treatment were necessary to see a substantial (larger
than 80%) decrease in TFII-I mRNA levels (Fig. 6b).
Analysis of c-fos transcription following TFII-I RNAi
indicated a pronounced and specific de-repression of basal
transcription in the absence of TFII-I (Fig. 6b). Moreover,
the EGF-mediated activation of c-fos promoter still
persisted. It is difficult to assess the change in the -fold
stimulation following TFII-I RNAi because there is no basal activity in
the absence of the RNAi treatment. These results point to a role for
TFII-I in the maintenance of the basal repressed state of the
c-fos promoter.
We identify a new family of HDAC1,2-associated complexes
containing BHC110. Moreover, we define the polypeptide composition of a
novel member of this family containing the candidate gene for X-linked
mental retardation XFIM and the initiator-binding protein
TFII-I. A unique feature of HDAC1,2/BHC110-containing complexes is the
presence of specific DNA-binding subunits as a component of each
individual complex. Indeed, the majority of the novel subunits
identified are either known DNA-binding proteins such as BRAF35 (23)
and TFII-I (this work) or are proteins with a putative role in DNA
binding such as ZNF261/XFIM, ZNF198/FIM, and ZNF217 (Fig. 2).
The presence of specific and different DNA-binding subunits in each
corepressor complex also allows for specificity in targeting each
complex to a unique promoter. For example, the TFII-I-containing corepressor complex is recruited to promoters that either contain TFII-I-binding sites and/or DNA-binding proteins such as SRF, which can
form a cooperative interaction with TFII-I (31, 34). However, upon
activation and a consequent increase in the activator concentration at
the promoter, the association of TFII-I with the activator prevails
over that of the corepressor leading to a function for TFII-I in the
coactivation process. The DNA-binding subunit of the corepressor
complexes may also increase the cooperative binding of
sequence-specific repressor to their regulatory sites.
Although a number of the new HDAC1,2-associated subunits such as TFII-I
and BHC80 are unique to mammalian species, others are evolutionarily
conserved. Among these BHC110, ZNF261/XFIM, ZNF198/FIM, and BRAF35 have
close homologs in Drosophila melanogaster, indicating that
similar corepressor complexes may also be involved in gene-specific
repression in D. melanogaster. Finally, the close association of a number of HDAC-associated subunits with specific disease states (Fig. 2) attests to the importance of this family of
corepressor complexes in human health.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, 10% glycerol, 0.2 mM PMSF) followed by a wash with 0.5 M KCl in
buffer A with 0.5% Tween 20. The beads were then washed with 100 mM KCl in buffer A, and the proteins were eluted with 0.1 M glycine, pH 2.5, and neutralized with 0.1 volume of 1.0 M Tris-HCl, pH 8.0.
, and GAL(BD)-VP16). Transfection
efficiencies were normalized using
-galactosidase assay.
Transfections were repeated at least six times in at least two separate
experiments. Cell extraction, luciferase assays, and
-galactosidase
assays were performed according to the manufacturer's instructions
(Promega).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HDAC2 and BHC110 are components of multiple
complexes. The purification scheme is shown. HeLa nuclear
extract was fractionated by chromatography as described under
"Materials and Methods." The 0.3 M KCl elution of P11
was fractionated using antibody columns as indicated. The bound
proteins were washed with buffer containing 1 M KCl and
eluted using 0.2 M glycine, pH 2.5. The affinity-purified
-HDAC2 (lane 1),
-BHC110 (lane
2), and
IgG (lane 3) were
separated on an SDS-polyacrylamide gel (4-12%), and proteins were
visualized by colloidal blue staining. Molecular masses of
marker proteins (kDa) are indicated on the left, and the
proteins analyzed by ion trap mass spectrometry for each complex are
indicated. NE, nuclear extract; FT,
flow-through.
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Fig. 2.
Diagrammatic depiction of the novel
HDAC1,2/BHC110-associated proteins and description of their
reported domain structure and association with human
disease. aa, amino acids. PHD, plant
homology domain; SANT, SWI3, ADA2,
N-COR, and TFIIIB B" domain.
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Fig. 3.
Purification of the XFIM complex.
a, nuclear extract from F-XFIM cell line or the parent cell
line (mock) was fractionated using an anti-FLAG M2 affinity column. The
anti-FLAG affinity eluate was analyzed by SDS-PAGE followed by silver
staining. FT, flow-through. b, Western
analysis of the XFIM FLAG eluate fractionated by Superose 6 gel
filtration using antibodies indicated to the left of the
figure. c, HDAC assay was performed with HeLa
nuclear extract and the affinity-purified FLAG-XFIM complex using core
histones purified from HeLa cells. NE, nuclear
extract.
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Fig. 4.
A fraction of TFII-I is in a stable complex
with BHC110. a, conventional chromatographic purification of
TFII-I followed by anti-TFII-I affinity as discussed under "Materials
and Methods." The numbers delineate salt concentrations in
molar. NE, nuclear extract. b, Western
analysis of the affinity-purified -HDAC2,
-BHC110, and
-BHC80 eluates using anti-TFII-I and anti-HDAC2 antibodies.
c, Western analysis of the affinity-purified
-TFII-I or
control
TRAP220 eluates using anti-BHC110 and anti-HDAC2
antibodies. NE, (nuclear extract).
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Fig. 5.
Analysis of the transition of the
c-fos promoter from basal repression to activation
following EGF stimulation. a, RT-PCR analysis of
c-fos after treatment with sodium butyrate or trichostatin A
(TSA) (18 h). b, ChIP analysis of the
c-fos SRE promoter in 293 cells using -HDAC2,
-BHC110,
-XFIM,
-TFII-I,
-IgG preimmune, or beads (controls).
c, RT-PCR analysis of c-fos and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
transcription following the indicated times after stimulation by EGF in
293 cells. d, ChIP analysis of the c-fos SRE
promoter in 293 cells using
-SRF,
-TFII-I,
-BHC110,
-HDAC2,
-acetylated histone H4, and
-acetylated histone H3. Total cell
extracts were taken at the indicated times after EGF stimulation.
Following immunoprecipitation of formaldehyde cross-linked lysates, PCR
of eluted DNA using oligonucleotides specific for the c-fos
SRE promoter was performed. Total chromatin extracts were used for
input control. As a negative control, protein A-precipitated lysate was
used.
, two previously
characterized transcriptional corepressors (35, 36). Taken together,
our results point to a role for TFII-I in transcriptional
repression.
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Fig. 6.
TFII-I repress transcription in
vivo. a, transient transfection assay using
GAL4 (BD), GAL4(BD)/TFII-I, GAL4(BD)/SAP30, GAL4(BD)/HP1 , and
GAL4(BD)-VP16 as described under "Materials and Methods."
b, administration of TFII-I RNAi results in de-repression of
c-fos transcription. RNAi was administered twice followed by
serum starvation for 12 h. Cells were then stimulated by EGF, and
RT-PCR was used to determine levels of TFII-I and c-fos
RNA.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Brent H. Cochran for TFII-I antibodies and David Shultz for plasmids. Many thanks to T. Beer for expertise in HPLC and mass spectrometry and Daniel Bochar for HDAC assays. We thank the National Cell Culture Center (Minneapolis, MN) for propagation of HeLa cells and the Wistar Institute protein microchemistry/mass spectrometry facility.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors made equal contributions to this work.
¶ Supported by a postdoctoral fellowship from Association pour la recherche sur le cancer (Paris, France).
** Supported by National Institutes of Health Grant GM61204 and by a grant from the American Cancer Society. To whom correspondence should be addressed: Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104. Tel.: 215-898-3896; Fax: 215-898-3986; E-mail: shiekhattar@wistar.upenn.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M208992200
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ABBREVIATIONS |
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The abbreviations used are: HDAC, histone deacetylase; PMSF, phenylmethylsulfonyl fluoride; MS, mass spectroscopy; ChIP, chromatin immunoprecipitation; EGF, epidermal growth factor; BHC, BRAF35-HDAC complex; SRF, serum response factor; RT, reverse transcription.
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