From the Gladstone Institute of Virology and
Immunology, University of California, San Francisco, California
94141-9100, the
Cross Cancer Institute, Department of Oncology,
University of Alberta, Edmonton, Alberta T6G 1Z2, Canada, the ** Kazusa
DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan,
and the § Physiological-Chemical Institute, University of
Tuebingen, Tuebingen, D-72076 Germany
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ABSTRACT |
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Histone deacetylases are the catalytic subunits
of multiprotein complexes that are targeted to specific promoters
through their interaction with sequence-specific DNA-binding factors. We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases. Analysis of
the predicted amino acid sequence of HDAC-A revealed an open reading
frame of 967 amino acids containing two domains: a
NH2-terminal domain with no homology to known
proteins and a COOH-terminal domain with homology to known histone
deacetylases (42% similarity to RPD3, 60% similarity to HDA1). Three
additional human cDNAs with high homology to HDAC-A were identified
in sequence data bases, indicating that HDAC-A itself is a member of a
new family of human histone deacetylases. The mRNA encoding HDAC-A
was differentially expressed in a variety of human tissues. The
expressed protein, HDAC-Ap, exhibited histone deacetylase activity and
this activity mapped to the COOH-terminal region (amino acids 495-967)
with homology to HDA1p. In immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that
it might be part of a larger multiprotein complex.
Acetylation of core histones, first described by Allfrey and
co-workers (1), has been correlated with transcription, chromatin assembly, DNA repair, and recombinational events (2-7). Transfer of an
acetyl group from acetyl-CoA onto the Cloning of the first histone acetyltransferase (8) and the first
histone deacetylase (9) has led to the identification of a growing
number of proteins with similar enzymatic activities (reviewed in Refs.
7 and 10). The characterization of the first histone acetyltransferase
and the first histone deacetylase as homologues of Saccharomyces
cerevisiae GCN5 and S. cerevisiae RPD3, respectively,
two factors previously described genetically as transcriptional
regulators, confirmed the long speculated role of histone modification
in eukaryotic transcriptional regulation. Together with the
demonstration that acetylation levels of nucleosomal histones change in
discrete regions associated with certain promoters (11-13), these
results established that chromatin is not only a structural scaffold
responsible for DNA compaction in the eukaryotic nucleus but is also an
active and dynamic participant in transcriptional regulatory mechanisms.
This model has been recently validated by the demonstration that the
enzymatic activity of the yeast histone acetyltransferase GCN5 is
necessary for the transactivational activity of this factor (14, 15).
Similarly, mutation of amino acids critical for the histone deacetylase
activity of RPD3 or HDAC1 reduced partially or totally their repressor
activity (16, 17). Additional evidence for the involvement of histone
acetylation in transcriptional regulation has come from studies with
fungal toxins, such as trichostatin A and trapoxin, that specifically
inhibit histone deacetylases. These inhibitors shift the dynamic
equilibrium between histone acetylation and deacetylation toward the
hyperacetylated state (18). Several reports established a correlation
between hyperacetylation of histones and transcriptional activation;
for example, both trichostatin A and trapoxin increase the
transcriptional activity of the human immunodeficiency virus type 1 promoter in vivo and in vitro (19, 20). However,
other studies have shown that histone deacetylases can also play a
significant role in transcriptional silencing. In mammalian cells,
inhibition of histone deacetylases by trichostatin A or trapoxin
activates or represses a small fraction of cellular genes (21) and
disruption of RPD3 and SIN3 in S. cerevisiae showed that both genes are required to fully activate or repress specific promoters (22-24).
The histone deacetylases identified can be grouped into three families:
S. cerevisiae RPD3 and RPD3-related proteins in higher organisms, such as HDAC1, 2, and 3 (9, 25-28); S. cerevisiae HDA1 and the related S. cerevisiae HOS1, 2, and 3 (29, 30); and HD2 isolated from Zea mays (31), which
presents no homology to the other two families. Biochemical and
molecular biological studies in different systems have established that
histone deacetylases are components of large multiprotein complexes
that are targeted to promoter sites through their interaction with
sequence-specific transcription factors (reviewed in Refs. 32 and
33).
In this report, we describe the identification and cloning of a human
cDNA with a region of homology to yeast HDA1. The
protein encoded by this cDNA, HDAC-Ap, exhibits a catalytically
active histone deacetylase domain in vivo. We also
identified three additional human sequences with strong homology to
HDAC-A that represent additional members of this new family of human
histone deacetylases.
Reagents and Cell Lines--
293 cells were grown in Opti-MEM
medium (Life Technologies) supplemented with 2% fetal bovine serum
(Hyclone), 50 units/ml penicillin, 50 µg/ml streptomycin, and 2 mM glutamine at 37 °C in a humidified 95% air, 5%
CO2 atmosphere. HeLa cells were cultured on glass
coverslips in Dulbecco's modified Eagle's medium, 10% fetal calf
serum using the same supplementations. Trichostatin A was obtained from
Wako Pure Chemical Industries, stored at 10 mg/ml in dimethyl sulfoxide
at Identification and Cloning of HDAC-A--
A human brain cDNA
library was constructed by reverse transcription of human whole brain
poly(A)+ RNA (CLONTECH) with reverse
transcriptase (SuperScript II, Life Technologies) and a (dT)15 primer
carrying the NotI site at the 5' extremity
(5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCC(T)15-3') according to the
supplier's instructions (34). After ligation of the SalI adaptor and NotI digestion, the resulting cDNAs were
separated on 1% low melting point agarose gels, and cDNA fragments
bigger than 9 kb1 were
recovered. These cDNA fragments were then ligated with the SalI/NotI-digested pSPORT1 vector and introduced
into Eschericia coli cells by electroporation (ElectroMax
DH10B cells, Life Technologies). Plasmids were extracted from about
10,000 independent ampicillin-resistant colonies grown on agar plates
by the standard alkaline/sodium dodecyl sulfate (SDS) method. The
isolated plasmids were resolved by agarose gel electrophoresis, and
those corresponding to the original fractionated size were retrieved.
Clones were randomly selected for dideoxy sequencing with ABI PRISM
cycle sequencing and ABI sequencers (Model 373A or 377 DNA,
Perkin-Elmer).
Northern Blot Analysis--
Multiple human tissue Northern blots
and RNA master blots were obtained from CLONTECH. A
32P-labeled probe corresponding to the HDAC-A cDNA was
prepared with the Multiprime DNA labeling system (Amersham).
Prehybridization, hybridization, and washing of blots were performed
using ExpressHyb hybridization solution (CLONTECH)
under high-stringency conditions according to standard methods (35).
Membranes were also hybridized to a probe corresponding to the human
glyceraldehyde-3-phosphate dehydrogenase cDNA to control for the
relative amount of mRNA loaded in each lane. Relative expression
levels were quantitated with a FUJIX BAS1000 PhosphorImager system and
Mac Bas software (Fuji Photo Film Co., Ltd.).
Plasmids--
To generate COOH-terminal epitope-tagged
constructs of different histone deacetylases, we used polymerase chain
reaction (PCR) amplification with a reverse primer containing the
sequence for the FLAG peptide. First the following two primers were
used to amplify HDAC3: forward,
5'-CCGGATCCGAATTCACCATGGCCAAGACCGTGGCC-3'; backward,
5'-GCTCTAGATTACTTGTCATCGTCGTCCTTGTAGTCTCCTCCGAATTCAATCTCCACATCGCTTTCC-3', (restriction sites are underlined; the sequence encoding the FLAG epitope is italic, and the stop codon is bold). The PCR product was
digested with BamHI and XbaI and inserted into
corresponding sites of the pcDNA3.1(+) vector (Invitrogen) using
standard protocols (35, 36). The region coding for HDAC3 was then
replaced with PCR fragments encoding each of the other histone
deacetylases by subcloning into new EcoRI sites that had
been engineered into the PCR primers. The following oligonucleotides
were used for PCR amplification: HDAC1 forward,
5'-CGGAATTCACGATGGCGCAGACGCAGGGCAC-3', backward,
5'-CGGAATTCGGCCAACTTGACCTCCTCCTTG-3'; murine HDAC2 (a gift
from Dr. Ed Seto) forward,
5'-CGGAATTCACCATGGCGTACAGTCAAGGAG-3', backward,
5'-CGGAATTCAGGGTTGCTGAGTTGTTCTG-3'; HDAC-A forward, 5'-CGGAATTCCAGGAGATGCTGGCCATGAAG-3', backward,
5'-CGGAATTCCAGGGGCGGCTCCTCTTC-3'; HDAC-A1-544 forward,
5'-CGGAATTCACCATGCTGGCCATGAAGCACC-3', backward, 5'-CGGAATTCCAGCGTGTCATACACGAGGCC-3'; HDAC-A495-967
forward, 5'-CGGAATTCACCATGGAGGCCGCCGGCATC-3'; HDAC-A544-967 forward,
5'-CGGAATTCACCATGCTGAAGCACCAGTGCACC-3'.
Subcellular Localization of HDAC-A--
HeLa cells growing on
coverslips were transfected using LipofectAMINE (Life Technologies).
After 48-72 h, cells were fixed with 1.0% paraformaldehyde in
phosphate-buffered saline for 5 min at room temperature. Cells were
permeabilized with 0.5% Triton X-100 in phosphate-buffered saline and
stained as described (37). The primary antibody, anti-FLAG (M2, Sigma),
was used at a 1:1000 dilution. Imaging was done on a Leica DM-R
epifluorescence microscope with a 100 × or a 63 × 1.4 N.A. PlanApo lens. Images were collected digitally with a cooled 12-bit
CCD (Princeton MicroMax). Digital deconvolution was performed as
described previously (37).
Immunoprecipitation: Histone Deacetylase Assays--
293 cells
(5 × 107 cells) were transfected with LipofectAMINE.
After 36 h, the cells were harvested and lysed in low stringency lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM
NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40) in the presence of a
protease inhibitor mixture (Boehringer Mannheim). Protein
concentrations of extracts were normalized with a modified Lowry assay
(Bio-Rad). To control for expression of different constructs, Western
blot analysis was performed with the enhanced chemiluminescence
procedure (Amersham) as described (28). Protein G-Sepharose was
preincubated with bovine serum albumin at 10 mg/ml to reduce
nonspecific binding, and extracts were precleared for
immunoprecipitation as described (28). Precleared lysates were
immunoprecipitated by incubation with the M2 anti-FLAG antibody (Sigma)
at 10 mg/ml overnight at 4 °C. As a control, immunoprecipitations
were also performed with the M2 anti-FLAG antibody after a 2-h
preincubation at room temperature with a synthetic peptide
corresponding to the FLAG epitope (100-fold molar excess). Immune
complexes were recovered by adding 20 µl of the preblocked 50%
protein G-Sepharose slurry for 4 h at 4 °C and washing three
times with low stringency lysis buffer, twice with lysis buffer
containing 0.5 M NaCl, and twice with histone deacetylase
buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl,
10% glycerol). For inhibition studies, the immunoprecipitated
complexes were preincubated with trichostatin A (400 nM) in
histone deacetylase buffer for 30 min at 4 °C. Beads were
resuspended in 30 µl of histone deacetylase buffer containing 20,000 cpm of an acetylated H4 peptide. Histone deacetylase activity was
determined after incubation for 2 h at 37 °C, as described
(28).
Coimmunoprecipitation--
Twenty-four hours after transfection,
293 cells (2 × 107) were metabolically labeled with
[35S]EXPRE35S35S protein labeling
mixture (NEN Life Science Products Inc.). Cells were starved by a
20-min incubation in methionine- and cysteine-free Dulbecco's modified
Eagle's medium (Life Technologies). Labeling was done in methionine-
and cysteine-free 90% Dulbecco's modified Eagle's medium and 10%
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum containing 0.15 mCi/ml radiolabeled amino acids
([35S]Met, [35S]Cys). Cells were harvested
after 16 h and lysed as described above. Lysates were divided
after preclearing and immunoprecipitated with M2 antibody or preblocked
M2 antibody (Sigma) for 2 h at 4 °C. Protein G-Sepharose slurry
(50%, 20 µl) was added for 1 h at 4 °C, and
immunoprecipitates were washed three times with low stringency lysis
buffer, three times with the same buffer supplemented with 500 mM NaCl and 0.5% sodium deoxycholate, and once with
phosphate-buffered saline. Immune complexes were eluted from the beads
with phosphate-buffered saline, containing 1% SDS for 15 min at room
temperature and analyzed by SDS-polyacrylamide gel
electrophoresis on 10% gels. After electrophoresis, the gels were fixed, enhanced with Amplify (Amersham), dried, and exposed for autoradiography.
Cloning of a Novel Human cDNA Encoding a Protein with
Similarity to the S. cerevisiae HDA1p--
A cloning strategy was
developed to identify new long cDNAs (34). Random sequencing of a
size-selected (>9 kb) human brain cDNA library identified a new
cDNA encoding a putative protein with striking homology to a yeast
histone deacetylase, HDA1 (see Fig. 1,
A and B). Sequence analysis of this cDNA,
referred to as HDAC-A hereafter, revealed an open reading frame (ORF)
of 2,901 base pairs encoding a putative protein of 967 amino acids
(Fig. 1A). This ORF is preceded by a 1134-nucleotide
untranslated 5' region and followed by a 4415-nucleotide untranslated
domain. The molecular mass of the derived protein is 105 kDa, and the calculated isoelectric point is 6.7. A stop codon (TAA) is located in-frame immediately upstream of the presumed start codon. Although, several potential initiation sites are located in the 5'-untranslated region upstream of the putative translation initiation site (data not
shown), the resulting ORFs are probably too short to yield a protein
in vivo. Since the deduced ORF encodes a functional protein,
this mRNA belongs to a growing number of transcripts that do not
use the first ATG codon to initiate translation (38).
Alignment of HDAC-Ap with other histone deacetylases, such as HDA1p and
RPD3p, showed that HDAC-Ap contains two distinct domains. The
COOH-terminal domain (amino acids 495-967) displayed homology to known
histone deacetylases, whereas the NH2-terminal domain (amino acids 1-494) exhibited no homology to known proteins (Fig. 1B). Comparison of HDAC-Ap with HDA1p and RPD3p revealed a
high degree of homology to HDA1p in the catalytical core region (43% identity and 60% similarity), whereas the homology to RPD3p in a
similar region was significantly lower (25% identity and 43% homology) (Fig. 1C).
HDAC-A Belongs to a Larger Family of Similar Sequences--
Using
the BLAST and TBLASTN algorithms to search for other sequences with
homology to HDAC-A, we identified another human cDNA, NY-CO-9. This
cDNA was recently cloned as an autoantigen from serum from patients
with colon cancer (39). The deduced amino acid sequence of this
cDNA, which we propose to call HDAC-B showed high overall homology
(58% identity, 67% similarity) to the HDAC-A protein, particularly in
the conserved catalytic histone deacetylase domain. The cDNA clone
described by Scanlan and co-workers (independently isolated by us
-KIAA0600) is truncated at the 5' and 3' extremities in comparison to
HDAC-A. However, the genomic sequence of HDAC-B was identified on
chromosome 17 (Human Genome Project, GenBank accession no. AC004150)
and regions corresponding to the 5' and 3' extremities of the HDAC-A
ORF were found. Therefore, we expect the full-length HDAC-B protein to
be similar in size to HDAC-A. Two additional genomic sequences located
on human chromosomes 7p15-7p21 and 12q31, respectively, also showed
high sequence similarity to HDAC-A (Table
I). Expressed sequence tags corresponding
to these genomic clones were identified (Table I), and it is therefore likely that these encode additional HDAC-A homologues. We propose to
call these new cDNAs HDAC-C and HDAC-D.
Differential Expression of the HDAC-A Transcript in Different Human
Tissues--
To explore the expression of HDAC-A in different tissues,
we performed Northern blot analysis (Fig.
2A) with a radiolabeled probe
corresponding to the HDAC-A cDNA. A single species of 8.4 kb was
detected in all tissues examined except testis, where a smaller species
of 3.4 kb was present as well. The size of this transcript is in good
agreement with the size of the cloned cDNA (8459 nucleotides). A
weaker band corresponding to 4.2 kb in many tissues examined might
represent a degradation product of the HDAC-A mRNA. To compare
HDAC-A expression in a wider array of tissues and to obtain more
information on transcript abundance, we used normalized mRNA master
dot blots (CLONTECH). Quantification of the dot
blots showed that HDAC-A was highly expressed in skeletal muscle,
thymus, and small intestine. Heart, colon, brain, ovary, peripheral
blood leukocytes, prostate, pancreas, spleen, and lung showed
intermediate expression, while liver, placenta, and kidney showed very
low expression (Fig. 2B).
HDAC-A Is a Predominantly Nuclear Protein with a Distinct
Subnuclear Localization--
To examine the subcellular localization
of the HDAC-A protein, HeLa cells were transfected with a HDAC-A-FLAG
fusion construct, and indirect immunofluorescence was performed with
the anti-FLAG M2 antibody, as described (37). HDAC-Ap was found
predominantly in the interphase cell nucleus (Fig.
3A). As expected, HDAC-Ap shows diffuse localization during mitosis, when the nuclear compartment is disintegrated (bottom of Fig. 3A1). In the
interphase cell nucleus, HDAC-Ap is excluded from nucleoli and
accumulates in discrete foci (Fig. 3B). Similar
chromatin-depleted foci have been reported for the localization of
endogenous HDAC1p (37). Like HDAC1p, HDAC-Ap is depleted near the
periphery of the cell nucleus and excluded from heterochromatic
territories.
HDAC-A Is a Histone Deacetylase--
To determine whether HDAC-Ap
possesses intrinsic histone deacetylase activity, plasmids expressing
FLAG-tagged fusion proteins were transfected into 293 cells. After
transfection, a new protein of approximately 120 kDa was detected by
Western blots with the anti-FLAG antibody (Fig.
4B). We used the anti-FLAG
antibody to immunoprecipitate HDAC-Ap and assayed for histone
deacetylase activity with a synthetic peptide corresponding to the
NH2-terminal tail of histone H4 as a substrate (28).
Similar immunoprecipitations were performed after transfection of
plasmids encoding FLAG-tagged versions of HDAC1,
mHDAC2, and HDAC3 as controls. Material immunoprecipitated with the anti-FLAG antibody in cells transfected with the HDAC-A-FLAG plasmid demonstrated histone deacetylase activity (Fig. 4A).
In contrast, immunoprecipitated material from cells transfected with the corresponding empty vector plasmid yielded no detectable histone deacetylase activity. No histone deacetylase activity was detected when
immunoprecipitation was performed with the anti-FLAG antibody preincubated with an excess of a synthetic peptide corresponding to the
FLAG epitope. As reported for other histone deacetylases, the enzymatic
activity of HDAC-Ap was inhibited by trichostatin A. These results
demonstrate that HDAC-Ap is a functional enzyme with histone
deacetylase activity in vivo.
To map the catalytical domain of HDAC-A, we generated deletion
constructs of HDAC-A either from the NH2 or COOH terminus
guided by the homology to other histone deacetylases, especially HDA1 (Figs. 1A and 5A). A fragment corresponding to
amino acids 495-967 of HDAC-Ap, the region homologous to HDA1p,
exhibited significant histone deacetylase activity, which was
reproducibly higher than the activity of the full-length protein (Fig.
5B). A fragment of HDAC-Ap
corresponding to amino acids 545-967 exhibited no histone deacetylase
activity, indicating that the short region between amino acids 495 and
545 is critical for enzymatic activity. As expected, the amino-terminal
portion of HDAC-Ap, amino acids 1-544, had no histone deacetylase
activity. The higher histone deacetylase activity of the fragment
containing amino acids 495-967 in comparison to the full-length
protein is consistent with the possibility that the amino-terminal
region of HDAC-Ap contains a negative regulatory element.
HDAC-A Is Part of a Multiprotein Complex--
Both HDAC1p and
HDAC2p are part of a large multiprotein complex, and the S. cerevisiae histone deacetylase HDA1p represents the enzymatic
component of the ~350-kDa HDA complex in yeast (29, 30). To determine
whether HDAC-Ap is also part of a multiprotein complex, we performed
coimmunoprecipitation experiments after transfection of a HDAC-A-FLAG
fusion construct into 293 cells. Analysis of immunoprecipitated
material by SDS-polyacrylamide gel electrophoresis identified a
predominant 119-kDa band, in agreement with the predicted molecular
mass of HDAC-Ap of 105 kDa. Several cellular proteins were found to
coprecipitate with HDAC-Ap. Preincubation of the anti-FLAG antiserum
with the corresponding FLAG peptide before immunoprecipitation showed
that HDAC-Ap specifically interacts with proteins with molecular masses
of 290, 137, 64, 50, 27, 25, 24, and 20 kDa, (Fig.
6). The band corresponding to 95 kDa
seems to be a degradation product of HDAC-A-FLAG, since it was also
detected in Western blot analysis of transfected 293 cells with the
anti-FLAG antibody (Figs. 4B and 5C). The
stringency of the washes used in these immunoprecipitations suggests
that the immunoprecipitated proteins form a stable multiprotein complex in vivo. Preliminary experiments using gel filtration and
density gradient centrifugation analysis to fractionate cellular
extracts indicated that HDAC-Ap segregates as a higher molecular mass
species (~220 kDa on sucrose gradients and ~600 kDa or gel
filtration) than predicted based on its molecular
mass.2 These observations are
consistent with the possibility that HDAC-Ap is part of a multiprotein
complex in the cell nucleus.
We have cloned and analyzed a cDNA encoding the first member
of a new family of human histone deacetylases. The members of this new
family are more closely related to the S. cerevisiae protein
HDA1p than to RPD3p. To distinguish this new family from a previously
identified group of human histone deacetylases with highest homology to
RPD3, HDAC1, 2, and 3 (9, 25-28), we propose to call the newly
identified cDNAs HDAC-A, B, C, and D. HDAC-Ap is expressed in
different tissues and is predominantly localized to the cell nucleus.
HDAC-Ap exhibits histone deacetylase activity in vivo that
maps to a COOH-terminal region of the protein.
HDAC1p and HDAC2p are thought to exert their effects as part of large
multiprotein complexes. These complexes contain mSin3 (22, 40-42), the
corepressors N-Cor and/or SMRT (43-45), RbAp48 and RbAp46 (9), SAP30
and SAP18 (41, 46, 47), and Mi-2 (48-50). They can be recruited to
promoters by sequence-specific DNA-binding proteins such as Mad-Max
(40, 41, 47), the unliganded nuclear hormone receptors (43-45),
DP1-E2F and the Rb family of transcription factors (13, 51-53),
transcriptional repressors containing a BTB/POZ domain (54, 55),
CBF1/RBP-J The HDA complex is ~350 kDa and contains at least three different
proteins with molecular masses of 75, 73/72, and 71 kDa (29, 30). The
only component analyzed in detail so far is HDA1. Owing to the
stringency employed in the purification protocol, it is not clear
whether additional factors are associated with this core complex under
less stringent conditions. Despite their sequence similarity to HDA1,
nothing is known about the biology of the protein products of the HOS genes.
The evolutionary conservation of different histone deacetylase
complexes suggests that they serve distinct and important functions in
transcriptional regulation. As demonstrated for the HDAC1·HDAC2 complex, recruitment by different transcription factors can target the
histone deacetylase activity to distinct promoters under different physiological conditions. In S. cerevisiae, the
RPD3-containing complex specifically targets genes regulated by the
UME6 transcription factor; however, these genes are not affected by a
corresponding deletion of HDA1 (11). It is likely that the HDA1 complex
is targeted to different sites by as yet unidentified sequence-specific DNA-binding factors. These two different histone deacetylase families might also exhibit different specificities in term of their enzymatic targets (different core histones or/and different lysine residues (29)). Differences in substrate use could add another level of
specificity, since it is known that the acetylation states of specific
lysine residues of histone H3 and H4 are associated with distinct
biological functions (for review, see Ref. 32). The growing list of
nonhistone proteins that are also regulated by acetylation, such as p53
(60, 61), EKLF (62), TFIIE, and TFIIF (63), also raises the possibility
that so-called "histone" deacetylases might target nonhistone
proteins for deacetylation. However, the nuclear localization of
HDAC-Ap and its targeting to similar subnuclear granules, as observed
for HDAC1, 2, and 3 (37),4
suggests that these proteins might be functionally related.
Future effort will be devoted to defining the factors that interact
with HDAC-Ap and the enzymatic specificity of HDAC-Ap in comparison to
HDAC1, 2, and 3. It is anticipated that these studies will increase our
emerging understanding of the role of histone deacetylation in
transcriptional regulation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amino group of different lysines residues in the NH2-terminal tail of core histones
is a ubiquitous modification found in all eukaryotic species examined. Histone acetylation levels are controlled by the competing activities of histone acetyltransferases and histone deacetylases.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C, and diluted immediately before use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HDAC-A encodes a putative new histone
deacetylase. A, the nucleotide sequence and the deduced
amino acid sequence corresponding to the longest ORF representing
HDAC-A are shown. An upstream stop codon (TAA) immediately ahead of the
start codon is underlined. The presumed start codon (ATG) is
indicated by a box. Two other ATG codons that were used to
generate NH2-terminal truncations at amino acid positions,
495 and 545, are also indicated by a box. Residues conserved
within different histone deacetylases and critical for enzymatic
activity, as derived from Prodom Release 36, are highlighted
(box, absolutely conserved; circle, very
conserved; underline, histone deacetylase core region).
B, alignment of the HDAC-A, NY-CO-9 (HDAC-B), HDA1, and RPD3
proteins. The sequence of NY-CO-9 is not complete. Proteins were
aligned with PIMA (version 1.4) and printed with BOXSHADE (version
3.21). Identical residues are in black; conserved residues
are in gray. C, schematic representations of HDAC-A, NY-CO-9
(HDAC-B), HDA1, and RPD3 are aligned. Homology values referring to
HDAC-A for two different domains, NH2- and COOH-terminal,
are given in percent. Different regions of each protein are indicated
by the corresponding amino acid positions.
HDAC-A belongs to a new family of human histone deacetylases
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Fig. 2.
Tissue-specific expression of HDAC-A.
A, Northern blot analysis of HDAC-A was performed with
mRNA isolated from different human tisues. Hybridization to human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as
control. The arrow indicates the HDAC-A mRNA; an
additional band in testis and a series of weaker bands possibly
corresponding to a degradation product are also indicated. Molecular
weights are shown on the left. B, quantitative Northern dot
blot analysis for the expression of HDAC-A. Expression levels are
relative to the expression level in the tissue with maximum expression
(skeletal muscle).
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Fig. 3.
HDAC-Ap is a predominantly nuclear protein
with a distinct subnuclear localization. A, a field of
randomly growing HeLa cells that were transfected with an expression
vector encoding FLAG-tagged HDAC-A is shown. Panel A1 shows
staining with the anti-FLAG antibody. Panel A2 shows
staining of DNA using DAPI. Panel A3 shows the corresponding
DIC image. The cell at the bottom is in mitosis and exhibits a diffuse
cellular distribution of HDAC-A-FLAG. The upper two cells are in
interphase and show predominantly nuclear staining. The scale
bar represents 10 µm. B, HDAC-A-FLAG accumulates in
discrete foci within the interchromatin space. The nucleus of a HeLa
cell transiently expressing HDAC-A-FLAG was imaged by collecting
serial z-sections at 0.4-µm intervals. A single optical section near
the center of the cell nucleus is shown. Panel B1 shows
anti-FLAG staining before digital deconvolution. Panel B2
shows the corresponding digitally deconvolved optical section.
Panel B3 shows the corresponding digitally deconvolved DAPI
section. Panel B4 shows a composite image in which DAPI is
green and HDAC-A-FLAG is red. The scale
bar represents 10 µm.
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Fig. 4.
HDAC-A is a functional histone
deacetylase. A, 293 cells were transfected with
plasmids encoding different FLAG-tagged histone deacetylases. Cellular
extracts were immunoprecipitated with a monoclonal antibody recognizing
the FLAG epitope and assayed for deacetylase activity on a
3H-acetylated H4 peptide substrate in the absence or
presence of trichostatin A (TSA, 400 nM). Counts
for released [3H]acetate are given for a representative
experiment. Control immunoprecipitations were performed with anti-FLAG
antibody preincubated with a 100-fold molar excess of a synthetic
peptide corresponding to the FLAG epitope. B, cellular
extracts from 293 cells transfected with FLAG-tagged histone
deacetylases were analyzed by Western blot with an anti-FLAG
antibody.
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Fig. 5.
Histone deacetylase activity maps to a
COOH-terminal domain of HDAC-A. A, schematic
representation of different constructs used to map different domains of
HDAC-A. B, transfections and histone deacetylase
immunoprecipitation analysis was performed as described in the legend
to Fig. 4A. C, Western blot analysis of cellular extracts
with anti-FLAG antibody after transfection of FLAG-tagged HDAC-A
constructs. TSA, trichostatin A.
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Fig. 6.
HDAC-A is part of a multiprotein
complex. 293 cells were transfected with an expression vector
encoding FLAG-tagged HDAC-A or the control empty vector. Cellular
proteins were metabolically labeled with a combination of
[35S]methionine and [35S]cysteine and
immunoprecipitated with monoclonal anti-FLAG antibody in the absence or
presence of an excess of the FLAG-epitope peptide. Immunoprecipitated
proteins were separated by SDS-polyacrylamide gel electrophoresis on
10% gels. Proteins specifically interacting with HDAC-A are indicated
by their molecular weight. Molecular weight markers are indicated on
the right in kilodaltons.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, the mammalian homolog of Drosophila
Suppressor of Hairless (56), CtBP (57), homeodomain-containing
repressors like Rpx, POU domain proteins such as Pit-1 (58), and MeCP2,
a methylcytosine-binding protein involved in DNA methylation (reviewed
in Ref. 59). The transcription factor YY1 interacts directly with
HDAC2p but also with a recently identified new human RPD3 ortholog,
HDAC3p (26). Despite its high degree of homology to HDAC1 and 2, HDAC3p
coimmunoprecipitates with distinct factors and appears to participate
in a different multiprotein
complex.3 In yeast, two
different histone deacetylase complexes (HDA and HDB) have been defined
biochemically (29, 30). The ~600-kDa HDB complex is the yeast
equivalent of the human HDAC1/HDAC2-containing complex, and these two
complexes share several subunits, including RPD3, RbAp48, SIN3, and
SAP30 (22, 46).
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ACKNOWLEDGEMENTS |
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We thank Dr. Yousef Al-Abed for preparing the 3H-acetylated H4 peptide, Dr. Ed Seto for the mouse HDAC2 cDNA and fragments of the human HDAC2 cDNA, Tina Ng for technical assistance, John C. W. Carroll and Neile Shea for graphics, Heather Livesay for manuscript preparation, and Stephen Ordway and Gary Howard for editorial assistance.
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Note Added in Proof |
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After this manuscript was accepted for publication, Verdel and Khochbin (64) reported the identification of related cDNAs in mice. Their cDNA called mHDA1 is highly homologous to HDAC-B reported in this manuscript, whereas their cDNA called mHDA2 does not correspond to either HDAC-A, -B, -C, or -D.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R016M516 and by institutional funds from the Gladstone Institute of Virology and Immunology.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB006626.
¶ Supported by a fellowship from the Boehringer Ingelheim Foundation, Germany.
To whom correspondence should be addressed: Gladstone Institute
of Virology and Immunology, P.O. Box 419100, San Francisco, CA
94141-9100. Tel.: 415-695-3815; Fax: 415-826-8449; E-mail: everdin{at}gladstone.ucsf.edu.
2 W. Fischle and E. Verdin, unpublished observations.
3 W. Fischle, S. Emiliani, and E. Verdin, unpublished observations.
4 M. Hendzel, W. Fischle, and E. Verdin, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: kb, kilobase pair(s); PCR, polymerase chain reaction; ORF, open reading frame.
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REFERENCES |
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