From the Laboratory of Molecular Growth Regulation,
NICHD, National Institutes of Health, Bethesda, Maryland 20892-2753, the § Departments of Biochemistry and Cell Biology, Baylor
College of Medicine, Houston, Texas 77030, and the ¶ Dana-Farber
Cancer Institute, Boston, Massachusetts 02115
Received for publication, August 14, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Human histone deacetylases I (HDAC1) and
II (HDAC2) are homologous proteins (84% identity) that catalyze
release of acetyl groups from modified N-terminal lysines of core
histones. Histone deacetylation is correlated with both transient and
persistent states of transcriptional inactivity (i.e.
silencing) in many eukaryotes. In this study, we analyzed complexes
containing HDAC1 and HDAC2 to identify the proteins most stably
associated with these deacetylases. Complex cI (9.5 S) contained
transcriptional corepressor CoREST/kiaa0071 and a protein homologous to
FAD-dependent oxidoreductases, kiaa0601. Complex cII (15 S)
contained Core histones are subject to reversible acetylation at selected
lysine residues in their N-terminal domains. Histone acetylation is
correlated with transcriptional activity or competence such that
hyperacetylation within chromatin domains favors transcriptional competency, whereas hypoacetylation favors transcriptional silencing. Acetylation neutralizes positive charges on lysine Acetylation states are maintained by the coordinate activity of histone
acetyltransferases and histone deacetylases
(HDACs).1 Histone
acetyltransferases are oxidoreductases, transferring an acetyl group
from acetyl-CoA, whereas HDACs remove acetyl groups by hydrolysis (4,
5). Most HDACs belong to a superfamily of zinc metalloenzymes with a
conserved ~380-residue catalytic domain (6, 7). The class I HDACs
(~500 residues in length) are the best characterized with respect to
function. These HDACs are involved in maintaining both short-term
(induction/repression) and long-term (epigenetic) patterns of gene
activity (8-10). HDAC mutants of Rpd3 in Saccharomyces
cerevisiae and Clr6 in Schizosaccharomyces pombe affect
telomeric silencing (11, 12); similarly, HDAC mutants in
Drosophila, RPD3 (13), and Caenorhabditis
elegans, HDA-1 (14), affect silencing of developmentally regulated genes.
Three class I HDACs have been identified in human. HDAC1 (482 residues)
(15) and HDAC2 (488 residues) (16) share 84% identity, whereas HDAC3
(428 residues) (17, 18) is more divergent, sharing 51% identity with
HDAC1/2. It is unclear whether HDAC1 and HDAC2 have distinct functions,
but they appear to play complementary roles in transcriptional
repression. For example, in GH3 pituitary cells,
thyroid-stimulating hormone- Immunopurification of either HDAC1 or HDAC2 from human cell lines
typically yields multiprotein complexes containing both HDAC1 and
HDAC2. These include HDAC complexes containing transcriptional corepressor mSin3A (20-22) and chromatin-remodeling ATPase Mi-2 (NURD
complex) (23-28). To gain further insight into the mechanism by which
HDAC1 and HDAC2 complexes may be independently recruited to specific
sites in chromatin, we have analyzed core complexes containing either
HDAC1 or HDAC2. Two major HDAC1/2 complexes were identified in HeLa
cells: cI is a novel complex containing transcriptional corepressor
CoREST/kiaa0071 (29) and protein kiaa0601, inferred to be an
FAD-dependent enzyme; cII is similar to the Mi-2 remodeling
complex. Under native conditions, cI and cII may contain HDAC1, HDAC2,
or both, but these can be dissociated by a mild chaotrope to
core complexes containing only HDAC1 or HDAC2. This behavior suggests a
general model for interaction between HDAC1 and HDAC2 via dimerization
of cI- or cII-specific components.
We identified a potential HDAC-targeting factor,
mCpG-binding protein MBD2 (30), as an integral component of
the HDAC1 cII complex. MBD2 was not detected in HDAC2 cII complexes,
implying that targeting of HDAC2 may occur via dimerization with HDAC1. Finally, we note that a region of sequence similarity exists between cI
component CoREST/kiaa0071 and cII component Mta-L1 (31), containing
SANT domains (32). SANT domain proteins are also components of HDAC3
core complexes (33) and may play a general role in HDAC complex assembly.
Cell Culture and Transduction--
HeLa S3 cells were grown in
suspension culture as previously described (34). Cells were
transduced with a retroviral construct (pOZ) expressing a bicistronic
mRNA encoding FLAG-tagged HDAC1 protein linked to an
interleukin-2 receptor subunit surface marker, and the
transduced population was purified by repeated cycles of affinity cell
sorting (34). A cDNA construct encoding human HDAC1 with a
C-terminal FLAG tag, used to construct the retroviral vector, was
generously provided by Dr. Stuart L. Schreiber. A cDNA encoding
human HDAC2 (accession number NP_001518) was isolated by
performing polymerase chain reaction using gene-specific primers located in the 5'- and 3'-untranslated regions with a
CLONTECH Marathon cDNA library; this cDNA
clone was used to prepare an expression construct with a C-terminal
FLAG tag (see above). For detection of FAD, 2.8 × 108
cells were radiolabeled with 1 mCi of [3H]riboflavin (25 Ci/mmol; American Radiolabeled Chemicals) in 350 ml of
Dulbecco's modified Eagle's medium (minus riboflavin) for 16 h
prior to harvest.
Antibodies--
Rabbit antisera were raised against 16-mer
peptides representing the C termini of human HDAC1 (15),
CoREST/kiaa0071 (29), kiaa0601 (35), Mta-L1 (31), MBD2 (30), and
Finb (36). Immune IgG was affinity-purified using a peptide
column and was covalently coupled to protein A-agarose (Pierce) using
dimethyl pimelidate (37). Mouse anti-FLAG mAb M2 and anti-FLAG
antibody-agarose were obtained from Eastman Kodak Co. Mouse mAbs 13D10
and 15G12, raised against human RbAp48, were obtained from GeneTex
(38). Affinity-purified polyclonal antibodies to HDAC2 (sc-6296) and mSin3A (sc-767) were obtained from Santa Cruz Biotechnology.
Horseradish peroxidase-conjugated second antibodies for Western
blotting were obtained from Pierce.
Isolation of HDAC1/2 Complexes--
In view of reports that
histone deacetylases may be resistant to extraction from isolated
nuclei (39), we employed a sequential extraction procedure to
facilitate recovery of soluble complexes from the transduced cells. For
preparation of nuclei, cells were washed twice in phosphate-buffered
saline, resuspended in hypotonic buffer (20 mM MES (pH
6.5), 10 mM NaCl, 1.5 mM MgCl2, and
1 mM DTT), and lysed by Dounce homogenization (40),
yielding nuclei and cytosol. The nuclear pellet was washed in isotonic
sucrose buffer (STM: 250 mM sucrose, 50 mM Tris
(pH 7.4), 5 mM MgCl2, and 1 mM
DTT), yielding STM wash, and the nuclear envelope was removed by
addition of 1% Triton X-100, yielding Triton wash. Nuclei were
sequentially extracted with low magnesium buffer (10 mM
Tris (pH 7.4), 0.2 mM MgCl2, and 1 mM DTT), yielding low magnesium wash, and then with low
magnesium buffer plus 0.5 M NaCl (41, 42), yielding high
salt extract and residual nuclei. All buffers contained Complete
protease inhibitor (Roche Molecular Biochemicals).
Most of HDAC1/2 was obtained in the high salt extract (see Fig.
1A). The high salt extract was dialyzed in ion-exchange
chromatography buffer (20 mM Tris (pH 8.0), 100 mM NaCl, 1 M urea, 1 mM DTT, 10%
glycerol, and 0.005% CHAPS), centrifuged at 27,000 × g, and applied to a Q-Sepharose column (Amersham Pharmacia
Biotech) equilibrated with chromatography buffer. The column was eluted
with a gradient of 100-750 mM NaCl, and the
HDAC1-containing fractions (~250-350 mM NaCl) were
pooled and dialyzed in immunopurification buffer (20 mM
Tris (pH 8.0), 150 mM NaCl, 5 mM
MgCl2, 10% glycerol, and 0.1% Tween 20). The Q-Sepharose
pool was mixed with either anti-FLAG or anti-HDAC1 antibody-agarose for
3 h on a rotator, packed into a column, and washed in ~50
volumes of immunopurification buffer. Bound proteins were eluted from
anti-FLAG antibody-agarose by incubation with immunopurification buffer
containing FLAG peptide (50 µg/ml) or from anti-HDAC1
antibody-agarose with 100 mM glycine (pH 2.5) and 0.1%
Tween 20. (Note that removal of urea by dialysis was required to
efficiently bind the FLAG epitope; endogenous HDAC1 could be
immunopurified without dialysis. The same proteins co-immunopurified
with endogenous HDAC1 in the presence or absence of 1 M
urea.)
To resolve HDAC1/2 complexes, 200 µl of anti-FLAG eluate was
centrifuged in a 3.7-ml 10-35% glycerol gradient for 5.5 h at 55,000 rpm using an SW 60Ti rotor (Beckman Instruments) (43). Twenty
200-µl fractions were collected. Marker proteins (ovalbumin (4.54 S),
aldolase (7.3 S), catalase (11.3 S), and thyroglobulin (19 S)) were
centrifuged in parallel gradients.
Histone Deacetylase Enzyme Assay--
Histone deacetylase
activity was measured using human core histones radiolabeled with
[3H]acetate in vivo as substrate (44).
Aliquots (1-10 µl) were incubated with 10 µg of
[3H]histone in 100 µl of assay buffer (20 mM Tris (pH 8.0), 75 mM NaCl, and 1 mM DTT) for 20 min at 37 °C. The reaction was quenched by addition of 20 µl of 12 N HCl and extracted with 0.5 ml of ethyl acetate. Acetate release was measured by liquid
scintillation counting of the ethyl acetate (organic) phase. As a
specificity control, release of [3H]acetate was inhibited
by either 0.1 µM trichostatin A or 10 mM
sodium butyrate.
Identification of Proteins by Mass Spectrometry--
Protein
identification by mass spectrometry was carried out as described (34)
with minor modifications. An electrospray ion trap mass spectrometer
(LCQ, Finnigan MAT, San Jose, CA) coupled on line with a Magic 2002 capillary HPLC (Michrom BioResources, Auburn, CA) was used to acquire
tandem mass spectra. A 0.1 × 50-mm Magic MS
C18 column (5-µm particle diameter, 200-Å pore size) with mobile phases A (methanol/water/acetic acid, 5:95:1) and B
(methanol/water/acetic acid, 85:15:1) was used with a gradient of
2-98% mobile phase B over 2.5 min, followed by 98% mobile phase B
for 2 min. Specific protein bands from Coomassie Blue-stained SDS-polyacrylamide gels were excised, destained, and digested with
trypsin, and the resulting peptides were extracted. 20-50% of the
sample was used to obtain liquid chromatography/tandem mass
spectra. The tandem mass spectra were used to search the compiled NCBI
nonredundant protein and expressed sequence tag data bases with the
program PepFrag to identify the proteins.
Purification of HDAC1/2 Complexes--
HDAC1 and HDAC2 were
extracted from isolated HeLa cell nuclei with 0.5 M NaCl
(high salt extract) (see Fig.
1A (HS) and
"Experimental Procedures"). Endogenous HDAC1 and HDAC2 were
isolated using specific immunoadsorbents recognizing their
(nonconserved) C-terminal 16 residues. When HDAC1 was isolated directly
from the high salt extract under non-stringent conditions, some HDAC2
co-immunopurified with it (Fig. 1B), as in previous studies
(22, 25, 27, 45, 46). We examined chaotropic agents as a means to fully
dissociate HDAC1 from HDAC2. Histone deacetylase enzymatic activity was
stable indefinitely in 1 M urea, but was irreversibly lost
at higher concentrations (2-4 M). HDAC1 and HDAC2 were
fully dissociated by Q-Sepharose chromatography in 1 M
urea. The proteins coeluted in the Q-Sepharose pool (0.25-0.35
M NaCl), but bound independently to the immunoadsorbents
(Fig. 1B).
Expression of Epitope-tagged HDAC1/2 in HeLa Cells--
To
facilitate physical characterization of purified, native HDAC1 and
HDAC2 multiprotein complexes, we expressed recombinant proteins with a
C-terminal FLAG epitope tag (HDAC1-F and HDAC2-F) in HeLa S3 cells.
Cell lines with stable expression of either HDAC1-F or HDAC2-F were
obtained by transduction with a bicistronic retroviral vector and
repeated cycles of magnetic affinity cell sorting (see "Experimental
Procedures"). Endogenous and recombinant HDAC1/2 proteins expressed
in these cells were quantified by Western blotting using
affinity-purified antibody specific to the C terminus of either HDAC1
or HDAC2, respectively, and mouse anti-FLAG mAb M2. Levels of
endogenous and recombinant HDAC1 proteins were compared using a
baculovirus-expressed HDAC1 protein with an N-terminal FLAG tag as a
standard; approximately equal amounts of endogenous and recombinant
HDAC1 proteins were expressed in the transduced cells (Fig.
1A).
Endogenous and FLAG-tagged HDAC1 proteins were isolated from the
Q-Sepharose pool (see above) using anti-HDAC1 or anti-FLAG immunoadsorbents, respectively. The polypeptides copurifying with either endogenous or FLAG-tagged HDAC1 from the Q-Sepharose pool were
compared by SDS-PAGE (Fig. 2). The
polypeptide composition of the anti-FLAG eluate was generally similar
to that of the anti-HDAC1 eluate, except for the presence of several
additional bands. These additional bands were also present in the
control anti-FLAG eluate, indicating that they were contaminants; they
were removed by further purification of the HDAC1-F complexes (see
below).
The polypeptides common to both endogenous and FLAG-tagged HDAC1
proteins were identified by mass spectrometry (34), revealing components characteristic of deacetylase chromatin-remodeling complexes: Mi-2 (p240, ATPase), Mta-L1 (p78, a SANT domain and zinc
finger protein, also termed Mta2), RbAp48/46 (p56/p53, WD40 repeat
proteins), and MBD3 (p36/p33, mCpG-binding domain
homology) (23, 25, 27, 28). In addition, we identified MBD2 (p50/p33, a
mCpG-binding protein) (30), kiaa0601 (p115, a protein
homologous to FAD-dependent enzymes) (see Fig. 8), and an
additional SANT domain protein (kiaa0071) (see Fig. 9), comigrating
with HDAC1 (p63) (35). The p63 and p61 bands both contained HDAC1; no
HDAC2-specific peptides were detected in either band.
Sedimentation Analysis of HDAC1 and HDAC2 Complexes--
The
variable staining intensities of the HDAC1-associated polypeptides
suggested that they might represent a mixture of different multiprotein
complexes. We employed velocity sedimentation in a glycerol density
gradient to separate HDAC1-F complexes differing in size and shape.
Histone deacetylase enzyme activity sedimented as a 15 S peak with a
9.5 S shoulder (Fig. 3, upper
panel). The distribution of HDAC1-F by Western blotting (Fig. 3,
lower panel) was similar to the activity profile, indicating
similar specific activities for all sedimenting species of HDAC1.
Different polypeptides co-sedimented with HDAC1-F in the 9.5 S and 15 S
regions of the gradient (Fig. 3, middle panel), indicating that these fractions represent different core complexes, designated cI
(9.5 S) and cII (15 S). The polypeptide components of cI (fractions 6 and 7) and cII (fractions 9 and 10) were analyzed by mass spectrometry (see above). The p63 band in both cI and cII contained HDAC1 and kiaa0071. The p115 band in cI contained kiaa0601. cII contained the
remodeling complex components Mi-2 (p240), Mta-L1 (p78), RbAp48/46 (p55/p53), and MBD3 (p36/p33) and, in addition, MBD2 (p50/p30). Additional bands were identified in cII: p70 is a human homolog of the
Xenopus laevis HDAC-associated zinc finger protein p66 (24);
p59 and p38 represent truncated forms of Mta-L1. The narrow width of
the 15 S peak (same as the sedimentation standards) suggests that cII
represents a single multiprotein complex. (This idea is further
supported by immunopurification studies described below.) The variation
in staining intensity of the bands could be due either to different
stoichiometries (i.e. 3-5 mol of p240/p78/p63/p55/p53 per
mol of p50/p36/p33) or to differences in the amount of Coomassie Blue
dye bound per mol of each protein.
To obtain specific probes for cI and cII, affinity-purified
anti-peptide antibodies were prepared against the C-terminal 16 residues of kiaa0601, kiaa0071, Mta-L1, and MBD2. kiaa0601 and kiaa0071
were found predominantly in cI, whereas Mta-L1 and MBD2 were found
exclusively in cII (Fig. 3, lower panel). The data from
Coomassie Blue staining and Western blotting suggest that cI is a
multiprotein complex composed of kiaa0601 (p115), kiaa0071 (p63), HDAC1
(p63), and p37, which is consistent with the predicted molecular mass
for a 9.5 S complex, ~200 kDa. The predicted molecular mass for a 15 S complex, ~400 kDa, would appear to be an underestimate for cII.
Assuming only 1 mol of each visible band in the multiprotein complex
would indicate a molecular mass of at least 1 MDa. However, sedimentation coefficients are a function of both the molecular mass
and frictional ratio, the latter being related to particle asymmetry
(43). In view of the wide size range of the proteins in cII, it is
likely to have a greater frictional ratio than the sedimentation
standards; thus, 400 kDa would represent only a minimum estimate.
FLAG-tagged HDAC2 was isolated using the same procedures employed for
HDAC1 (see above). In contrast to HDAC1, HDAC2 deacetylase activity
sedimented as a single broad peak centered around 15 S (Fig.
4). This HDAC2 core complex contained the
same major components as the HDAC1 cII complex: Mi-2 (p240), Mta-L1
(p78), p70, RbAp48/46 (p55/p53), and MBD3 (p36); but did not contain
MBD2, as judged by Western blotting.
Endogenous HDAC1 and HDAC2 Are in Distinct cI and cII Core
Complexes--
To determine whether endogenous HDAC1 and HDAC2 in
normal (untransduced) HeLa cells exist as distinct cI and cII core
complexes, we employed antibody adsorbents specific for cI
(anti-kiaa0601 and anti-kiaa0071) and cII (anti-Mta-L1 and anti-MBD2)
components. Binding was performed using the native high salt extract
(no urea treatment) under non-stringent conditions (see "Experimental
Procedures"). After extensive washing, the adsorbents were eluted
with pH 2.5 buffer, and the eluates were analyzed by SDS-PAGE (Fig.
5A). As expected, all
adsorbents (except the control) bound HDAC1. (Protein recovery from the
anti-MBD2 adsorbent was relatively low.) Also as predicted, the cI
components kiaa0601 and kiaa0071 bound to anti-HDAC1, anti-kiaa0601,
and anti-kiaa0071 adsorbents, but not to anti-Mta-L1 and anti-MBD2
adsorbents; conversely, the cII components Mta-L1 and MBD2 bound to
both anti-Mta-L1 and anti-MBD2 adsorbents, but not to anti-kiaa0601 and
anti-kiaa0071 adsorbents. This demonstrates that cI and cII are
distinct HDAC1 complexes in the native high salt extract. Blotting
indicated that mSin3A was not associated with either cI or cII.
HDAC2 was also bound by both cI- and cII-specific adsorbents (Fig.
5A), as might be expected, since some HDAC2 is associated with HDAC1 (Fig. 1). To examine whether HDAC2 exists as cI or cII
complexes independently of HDAC1, the fraction of endogenous HDAC2 not
associated with HDAC1 (Fig. 1) was isolated from the anti-HDAC1
flow-through (i.e. depleted of HDAC1) using an anti-HDAC2 adsorbent. Both cI components kiaa0071 and kiaa0601 and the cII component Mta-L1 co-immunopurified with HDAC2 (Fig. 5A).
However, MBD2 was not detected and appears not be associated with
endogenous HDAC2 cII, consistent with our analysis of FLAG-tagged HDAC2
(Fig. 4).
Further insight into the composition and potential significance of cI
and cII can be gleaned from examination of the polypeptide composition
of the eluates (Fig. 5B). The anti-kiaa0071 eluate is
similar to the cI region of the glycerol gradient, except for the
additional p61 band (which is characteristic of endogenous HDAC1) (Fig.
2), supporting our interpretation that kiaa0601, kiaa0071, HDAC1, and
p37 compose a multiprotein complex. The absence of any major
polypeptides in the anti-kiaa0071 eluate besides the cI components
indicates that cI represents the quantitatively major form of kiaa0071
in the high salt extract. The anti-kiaa0601 eluate is similar to the
anti-kiaa0071 eluate, except for a few additional polypeptides, notably
p140 and p160. These polypeptides did not appear to cross-react with
the anti-kiaa0601 antibody in Western blotting; we favor the
interpretation that they are components of other multiprotein complexes
containing kiaa0601, but not kiaa0071 or HDAC1/2.
When the polypeptide composition of the anti-Mta-L1 eluate was
examined, it proved to be similar to the cII region of the gradients
(Figs. 3 and 4). The absence of any other major polypeptides in the
anti-Mta-L1 eluate besides the cII components indicates that cII
represents the quantitatively major form of Mta-L1 in the high salt
extract. The anti-MBD2 eluate is similar to the anti-Mta-L1 eluate.
Despite the large difference in staining intensity between Mta-L1 (p78)
and MBD2 (p50), these polypeptides appeared in a fixed ratio in all
preparations containing cII, supporting our interpretation that cII
represents a single molecular species (see above). Overall, our
analysis has demonstrated that the majority of HDAC1 and HDAC2 in HeLa
cells exist in two distinct types of multiprotein complexes, cI and
cII, which may contain HDAC1, HDAC2, or both. cI and cII can be
dissociated to core complexes containing only HDAC1 or HDAC2 in
association with other enzyme and targeting factors. We propose a
general model for interaction between HDAC1 and HDAC2 via dimerization
of cI- or cII-specific components (see "Discussion" and Fig.
7).
Complex cI Contains Riboflavin--
The cI component kiaa0601
exhibits sequence similarity to a superfamily of
FAD-dependent enzymes (see Fig. 8). To examine whether
kiaa0601 may contain FAD, HeLa cells were radiolabeled in
vivo with [3H]riboflavin (see "Experimental
Procedures"). Riboflavin is converted to FMN and subsequently to FAD.
The immunoadsorbents specific for cI (anti-kiaa0071 and anti-kiaa0601)
both bound ~5% of the radiolabeled flavin in the high salt extract
(Table I), whereas the anti-Mta-L1 and
control adsorbents bound negligible amounts ( Subcellular Localization of cI and cII Components--
To gain
further insight into the possible role of cI and cII components as
targeting factors for HDACs, we examined their subcellular localization
in human skin fibroblasts. The cI components kiaa0071 and kiaa0601 were
distributed similarly to HDAC1, in the nucleoplasm and excluded from
the nucleoli (Fig. 6). The cII component
Mta-L1 was localized to the nucleoplasm and was also detected in the
juxtanuclear Golgi complex (47). MBD2 exhibited a punctate distribution
in nuclei, differing from HDAC1 and Mta-L1. This may be explained by
the fact that only a minor fraction of MBD2 (~30%) was extractable
with the HDACs in 0.5 M NaCl; possibly the majority of MBD2
is in different complexes, tightly bound to chromatin.
Two Distinct HDAC1/2 Core Complexes--
HDAC1 and HDAC2 are
closely related mammalian histone deacetylases that appear to mediate
complementary functions in transcriptional regulation at specific sites
in chromatin (see the Introduction). To gain further insight into the
mechanisms by which HDAC1 and HDAC2 interact, we have analyzed
multiprotein complexes from HeLa cell nuclei containing HDAC1 and
HDAC2. Complex cI is composed of kiaa0601 (p116), kiaa0071 (p63), p37
(unidentified), and HDAC, apparently in an equimolar ratio, based on
sedimentation behavior (see "Results" and Fig. 3). Complex cII is
similar to vertebrate deacetylase nucleosome-remodeling (NURD)
complexes (23-28) in that it contains Mi-2 (p240), Mta-L1/Mta2 (p78),
RbAp48/46 (p55/p53), and MBD3 (p36/p33) as well as a homolog of the
Xenopus-specific component p66 (p70). Under native
conditions, cI and cII may contain HDAC1, HDAC2, or both; however, mild
dissociation by 1 M urea yields core complexes containing
only HDAC1 or HDAC2. This behavior suggests a general model for
interaction between HDAC1 and HDAC2 via dimerization of cI- or
cII-specific components (Fig. 7).
Association with specific folding partners appears to be required for
HDAC1/2 function in vivo. When overexpressed in Sf9 insect cells, human HDAC1 and HDAC2 are largely insoluble and have
little enzymatic activity, suggesting incorrect
folding.2 Coexpression with
RbAp48/46 and Mta2 (also termed Mta-L1) is required to assemble
soluble, enzymatically active HDAC complexes (23). The polypeptide
complement required to assemble active HDAC complexes has been termed
the "core complex" (23, 25). The core complex components serve as
folding partners for HDACs and, in addition, may mediate substrate
binding, association with complementary enzymatic functions, and
targeting of HDACs to specific sites in chromatin (e.g. promoters).
A requirement for HDAC-folding partners can also be inferred from the
behavior of epitope-tagged HDAC1/2 stably expressed by our retroviral
vector in transduced cells. The levels of expression of HDAC1/2
obtained using this system in mammalian cells appear to be strictly
limited compared with expression in bacteria or insect cells. At most,
we have been able to obtain only a 4-5-fold excess of recombinant over
endogenous HDAC1 (in normal mouse skin fibroblasts), and this was
accompanied by a decreased level of endogenous HDAC1, amounting to only
a 3-fold increase overall. The absence of significant amounts of free
(monomeric) HDAC1 and HDAC2 implies that they must associate with
specific folding partners for metabolic stability; presumably, excess
HDAC1/2 is turned over and does not accumulate. Comparison of the
relative abundance of cI- and cII-specific components associated with
endogenous versus FLAG-tagged HDAC1 indicates that
recombinant HDAC1 has a greater tendency to form cII complexes (Fig.
2). Similarly, FLAG-tagged HDAC2 formed only cII complexes (Fig. 4),
although endogenous HDAC2 formed both cI and cII complexes (Fig. 5).
This tendency to form cII complexes may be due to a greater
availability of the cII components as folding partners or could be due
to an effect of the FLAG tag.
HDAC1 cII contains additional polypeptides not present in HDAC2 cII
(Figs. 4 and 5). One of these polypeptides was identified as the
mCpG-binding protein MBD2 (30). MBD2 is a transcriptional
corepressor (48) that binds specifically to mCpG residues
in DNA and exhibits a preference for densely methylated regions.
In vivo, MBD2 is localized to heterochromatic regions in
mouse cells (30). MBD2 may direct Mi-2 remodeling complexes to
methylated regions in chromatin, mediating transcriptional silencing
(23, 24). Based on our model (Fig. 7), HDAC1 cII could be recruited
directly to methylated regions in DNA, but recruitment of HDAC2 to
these sites would require heterodimerization of HDAC2 cII with HDAC1 cII.
Significance of Complex cI--
Protein kiaa0071, also termed
CoREST, binds to the RE1 silencing transcription factor (REST) in
vitro and forms a complex with REST in the L6 skeletal muscle cell
line (29). CoREST exhibits transcriptional corepressor activity in a
Gal4 DNA-binding domain-mediated reporter assay, even in cells
lacking REST. It is plausible that CoREST may function as a corepressor
by recruiting HDAC1 to promoters, as has been proposed for MBD2 (see
above) and other deacetylase-associated transcriptional repressors
(48). REST is a transcriptional silencer of neural-specific genes in
non-neural cells; it may serve to target cI complexes to promoters for
neural-specific genes.
Protein kiaa0601 appears to belong to a superfamily of
FAD-dependent enzymes (49, 50) based on sequence similarity
(Fig. 8) and binding radiolabeled flavin
(Table I). Conservation is strong at residues contacting FAD (Fig. 8)
within the three FAD-binding domains of maize polyamine oxidase (51).
Particularly significant is the conservation at Gly57 and
Lys300. These residues in polyamine oxidase position a
water molecule at the site of hydrogen exchange (N-5) in the
isoalloxazine ring and may represent the catalytic center. The
N-terminal one-third of kiaa0601 resembles that of CoREST/kiaa0071 in
having a block of alanines and also has similarity to conserved region
1 of the SWI3-like subunits of the SWI/SNF chromatin-remodeling complex (52). The fact that a large fraction of kiaa0601 exists in a complex
with a histone deacetylase and a transcriptional corepressor (Fig. 5)
suggests that kiaa0601 may be an enzyme with an activity complementary
to that of the deacetylase in transcriptional silencing. The
superfamily of FAD enzymes to which kiaa0601 apparently belongs includes a diverse group of oxidases and dehydrogenases involved mostly
in biosynthetic/degradative pathways (49). In the context of cI, it
seems plausible that kiaa0601 may be an oxidoreductase involved in
covalent modification of chromatin constituents.
Role of Conserved Domains in HDAC Core Complex Assembly--
The
cI component kiaa0071 and the cII component Mta-L1 (Mta2) share
sequence similarity in an ~200-residue segment that is tandemly
repeated in kiaa0071 (Fig. 9). This
region of similarity between kiaa0071 and Mta-L1 contains a SANT domain
(32). Interestingly, the transcriptional corepressors SMRT and NCoR,
which have recently been identified as components of HDAC3 core
complexes (33),3 are likewise
SANT domain proteins, suggesting a general role for SANT domain
proteins as folding partners for HDACs. The ~60-residue SANT domain
(related to the Myb DNA-binding domain) is found in a variety of
transcription factors, present in one to three copies, and may mediate
protein-protein interactions. We further note that the arrangement of
the two SANT domains in kiaa0071, separated by an ~100-residue
spacer, is similar to that in SMRT and NCoR (29). This suggests that
differing configurations of the conserved SANT domains in kiaa0071 and
Mta-L1 may have a role in specific assembly of cI and cII core
complexes.
15 proteins, including CHD3/4 (Mi-2), Mta-L1, RbAp48/46,
and MBD3, characteristic of vertebrate nucleosome-remodeling complexes.
Under native conditions, cI and cII may contain HDAC1, HDAC2 or both;
these can be dissociated to cI and cII core complexes containing only
HDAC1 or HDAC2. The mCpG-binding protein MBD2 was
associated only with the HDAC1 cII core complex. A model is proposed in
which HDAC1 core complexes can be targeted to methylated DNA via MBD2
with recruitment of HDAC2 occurring through formation of HDAC1/2 cII
dimers. We note that the cI component CoREST/kiaa0071 and the cII
component Mta-L1 share a region of homology that includes a SANT
domain; this domain may play a role in complex assembly.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-amino groups and
may alter nucleosome conformation by weakening interactions of the
N-terminal domains with DNA or adjacent nucleosomes. In addition,
acetylation at specific residues may create or abolish binding sites
for non-histone chromosomal proteins (1-3).
expression is negatively regulated by
the thyroid hormone triiodothyronine. This
triiodothyronine-dependent regulation requires HDAC
activity, as indicated by its sensitivity to the inhibitor trichostatin
A. An HDAC1 multiprotein complex binds constitutively to a negative
regulatory element in the thyroid-stimulating hormone-
promoter.
However, chromatin remodeling and transcriptional repression at the
thyroid-stimulating hormone-
promoter require (in addition)
triiodothyronine-dependent recruitment of an HDAC2 complex
to the negative regulatory element (19).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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RESULTS
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ABSTRACT
INTRODUCTION
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Fig. 1.
A, quantitation of endogenous and
FLAG-tagged HDAC1 proteins in HeLa subcellular fractions. Aliquots
(5 × 104 cells) of the cytosol (Cyto), STM
wash, Triton wash (TW), low Mg2+ wash
(LM), high salt extract (HS), and residual nuclei
(Nuc) prepared as described under "Experimental
Procedures" were resolved by SDS-PAGE; proteins were visualized by
Western blotting using rabbit anti-HDAC1 antibody and mouse anti-FLAG
mAb M2. The quantitation standard was a recombinant HDAC1 protein with
an N-terminal FLAG tag expressed in Sf9 cells. B,
immunopurification of endogenous HDAC1. High salt extract (load
(Ld)) was applied either to affinity-purified rabbit
anti-HDAC1 antibody coupled to protein A-agarose
( HDAC1) or to control agarose containing
preimmune IgG (Control). Unbound protein was collected
(flow-through (FT)), and the agarose was extensively washed
and then eluted with 0.1 M glycine (pH 2.5) (eluate
(Elt)). Alternatively, the extract was chromatographed on
Q-Sepharose (Q-S), and a pool of fractions containing HDAC1
was applied to the immunoadsorbents. HDAC1 and HDAC2 in these fractions
were detected by Western blotting.
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Fig. 2.
Polypeptide composition of immunopurified
complexes containing endogenous or FLAG-tagged HDAC1. Left
panel, proteins binding to anti-FLAG M2-agarose from the
Q-Sepharose pool prepared from 5 × 108 untransduced
control cells or from cells expressing FLAG-tagged HDAC1 were eluted
with FLAG peptide. Right panel, proteins from the
Q-Sepharose pool binding to preimmune (PreImm) or anti-HDAC1
( HDAC1) IgG-agarose were eluted in pH 2.5 buffer. Proteins were resolved by SDS-PAGE and visualized by Coomassie
Blue staining. The bands indicated (
) were excised for amino acid
sequence analysis (see "Experimental Procedures").
Dashed lines indicate the same proteins associated
with either epitope-tagged or endogenous HDAC1.
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Fig. 3.
Sedimentation analysis of FLAG-tagged HDAC1
complexes in a glycerol density gradient. The anti-FLAG eluate
(see Fig. 2) was applied to a 10-35% glycerol gradient and
centrifuged at 55,000 rpm for 5.5 h using an SW 60Ti rotor.
Sedimentation standards (ovalbumin (3.5 S), aldolase (7.3 S), catalase
(11.3 S), and thyroglobulin (19 S)) were centrifuged in parallel
gradients. Twenty 0.2-ml fractions were collected. Aliquots (2 µl)
were analyzed for histone deacetylase enzyme activity (upper
panel); fractions representing 9.5 S cI and 15 S cII are
indicated. Total proteins in each fraction were resolved by SDS-PAGE
and visualized by Coomassie Blue staining (middle panel).
The Coomassie Blue-stained bands referred to under "Results"
are indicated ( ). The apparent molecular masses for these bands are
indicated on the left (cI) and right (cII). In the lower
panels are shown the results from Western blotting for
distribution of cI and cII components in the gradient: FLAG-tagged
HDAC1 (anti-FLAG mAb) and endogenous kiaa0601, kiaa0071, Mta-L1, and
MBD2 (see "Results").
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Fig. 4.
Sedimentation analysis of FLAG-tagged HDAC2
complexes in a glycerol density gradient. The anti-FLAG eluate was
centrifuged in a glycerol gradient as described in the legend to Fig.
3. Aliquots were analyzed for histone deacetylase enzyme activity
(upper panel). Total proteins in each fraction were resolved
by SDS-PAGE (lower panel). The apparent molecular masses for
the major components are indicated on the right (see
"Results").
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Fig. 5.
Co-immunopurification of cI and cII
components with endogenous HDAC1 and HDAC2. The high salt
(HS) extract prepared from normal (untransduced) HeLa cells
was applied to antibody-agarose containing either preimmune (Pre
Imm) IgG (control) or affinity-purified antibody to HDAC1, Mta-L1,
MBD2, kiaa0071, or kiaa0601. The flow-through from the anti-HDAC1
adsorbent (depleted of HDAC1) was applied to an anti-HDAC2 adsorbent to
isolate HDAC2 not associated with HDAC1. Bound proteins eluted from the
adsorbents (as described in the legend Fig. 2) were resolved by
SDS-PAGE. A, Western blotting to identify HDAC1, HDAC2,
mSin3A, Mta-L1, MBD2, kiaa0601, and kiaa0071 proteins in the eluates.
B, polypeptide composition of the eluates visualized by
Coomassie Blue staining. Control is an eluate from an
antibody adsorbent to human Finb protein (see "Experimental
Procedures"), which does not bind HDAC complexes. Solid
bars indicate polypeptides copurifying with either cI (kiaa0601
and kiaa0071) or cII (Mta-L1 and MBD2) components. The bands referred
to under "Results" are indicated ( ); a cross-reacting protein in
the anti-MBD2 eluate is shown (×). The p63 and p61 bands contain HDAC1
(indicated on the right); and in addition, the p63 band contains
kiaa0071 in the anti-kiaa0601, anti-kiaa0071, and anti-HDAC1 eluates
(indicated on the left).
0.01%). Since kiaa0601
and kiaa0071 are the major proteins bound specifically by the cI
adsorbents (Fig. 5B), it is reasonable to infer that
kiaa0601 contains the radiolabeled flavin.
Riboflavin in HDAC1/2 complexes
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Fig. 6.
Subcellular localization of cI and cII
components. Shown are the results from the immunofluorescent
staining of human skin fibroblasts using affinity-purified antibodies
to HDAC1, cI components kiaa0071 and kiaa0601, and cII components
Mta-L1 and MBD2, followed by fluorescein isothiocyanate-conjugated
anti-rabbit antibody. Arrows indicate localization of Mta-L1
to the juxtanuclear Golgi complex, in addition to the nucleoplasm (see
"Results").
DISCUSSION
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EXPERIMENTAL PROCEDURES
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Fig. 7.
Conceptual scheme for assembly of higher
order HDAC1/2 complexes from individual cI or cII units. The
formation of a cI or cII heterodimer is illustrated (see
"Discussion").
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Fig. 8.
Predicted amino acid sequence of protein
kiaa0601 and similarity to maize polyamine oxidase. Shown is the
amino acid sequence of protein kiaa0601, predicted from the DNA
sequence of cDNA clone HG0838a (accession number AB011173)
merged with expressed sequence tag clone DKFZp434I0321 (accession
number AL042550), which extends HG0838a 5' by 57 base pairs. The merged
sequence contains two in-frame stop codons located 132 and
150 base
pairs from the first start codon in HG0838a, defining the initiation
codon for kiaa0601 (position 1). The kiaa0601 amino acid sequence
(K; accession number BAA25527) is aligned with similar
sequences in human BAF170 (B; SWI3 homolog; accession number
NP_003066) and maize polyamine oxidase (P; accession
number CAA05249); identities are shaded. The three
FAD and two substrate-binding domains (Sub1 and
Sub2) in the polyamine oxidase crystal structure are
indicated (substrate-binding region 1, omitted, is not similar to
kiaa0601). Asterisks indicate residues contacting FAD in
polyamine oxidase (51).
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Fig. 9.
Sequence similarity among CoREST/kiaa0071,
Mta-L1, and Mta1. A, schematic indicating the region of
Mta-L1/Mta1 similar to the repeated sequence in
CoREST/kiaa0071. The locations of SANT domains are indicated by
open boxes; the locations of zinc fingers in Mta-L1/Mta1 are
indicated by dots. B, sequence alignment of
CoREST (CoRst)/kiaa0071 (accession number AAF01498), Mta-L1
(accession number BAA36562), and Mta1 (accession number Q13330).
Identities between CoREST/kiaa0071 and Mta-L1 or Mta1 are
shaded. Thick lines indicate the SANT
domains; the positions of three conserved aromatic residues are shown
with asterisks.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Stuart L. Schreiber for providing the FLAG-tagged human HDAC1 cDNA construct.
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FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed. Tel.:
301-496-9038; Fax: 301-480-9354; E-mail: howard@helix.nih.gov.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M007372200
2 Y. Wang, G. W. Humphrey, and B. H. Howard, unpublished data.
3 Y. Wang, G. W. Humphrey, and B. H. Howard, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: HDACs, histone deacetylases; mAb, monoclonal antibody; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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