From the Department of Molecular and Cellular
Biology, § Verna and Mars McLean Department of Biochemistry
and Molecular Biology, Baylor College of Medicine, Houston, Texas
77030, the ¶ Department of Molecular Genetics, College of
Medicine, University of Illinois, Chicago, Illinois 60607, the
Institute of Cancer Genetics and Department of Pathology,
College of Physicians & Surgeons, Columbia University, New York, New
York 10032, and the ** Burnham Institute,
La Jolla, California 92037
Received for publication, August 22, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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The candidate tumor suppressor ING1
was identified in a genetic screen aimed at isolation of human genes
whose expression is suppressed in cancer cells. It may function as a
negative growth regulator in the p53 signal transduction pathway.
However, its molecular mechanism is not clear. The ING1
locus encodes alternative transcripts of
p47ING1a, p33ING1b, and
p24ING1c. Here we report differential
association of protein products of ING1 with the mSin3
transcriptional corepressor complex. p33ING1b
associates with Sin3, SAP30, HDAC1, RbAp48, and other proteins, to form
large protein complexes, whereas p24ING1c does
not. The ING1 immune complexes are active in deacetylating core
histones in vitro, and p33ING1b is
functionally associated with HDAC1-mediated transcriptional repression
in transfected cells. Our data provide basis for a p33ING1b-specific molecular mechanism for the
function of the ING1 locus.
Local acetylation and deacetylation of core histones play an
important role in the control of eukaryotic gene expression (1,2). Hyperacetylation of histones increases local accessibility of chromatin
templates, enabling subsequent activation or repression of
transcription by gene-specific factors, while deacetylation is
frequently linked with chromatin condensation and gene silencing. Most
histone acetyltransferases and histone deacetylases
(HDACs)1 are enzymes that do
not bind DNA directly; instead, they are recruited to chromatin through
association with distinct proteins in multiprotein complexes (3). One
of the conserved proteins that serve as an organizer for the assembly
of histone deacetylases with multiple polypeptides in yeast and
mammalian cells is Sin3 (4-7). A biochemically purified mammalian Sin3
complex includes HDAC1 and HDAC2, RbAp48 and RbAp46, SAP30, and SAP18
(6-8). The abundance and relative stability of both Sin3 and HDAC1
proteins have led to the proposal that the "core" Sin3 repressor
complexes are pre-assembled and available for recruitment by transient
association with gene-specific transcription factors, including Mad,
MeCP2, Ikaros, p53, PLZF, nuclear hormone receptors, and yeast Ume6
whose abundance and activities are regulated (3).
The ING1 (inhibitor of growth
1) gene was recently identified as a candidate tumor
suppressor in a genetic screen aimed at isolation of human genes whose
expression is suppressed in cancer cells (9). The ING1 gene
was localized to chromosome 13q33-34 (10,11), a region that has been
implicated in the progression of various tumors (12). Deregulated
expression and mutations of ING1 gene were found in breast
carcinomas (11) and in squamous cell carcinomas (13), respectively.
Ectopic expression of the originally isolated ING1 cDNA
or suppression of the ING1 gene expression by antisense RNA
demonstrated that ING1 is a negative regulator of cell
proliferation involved in the p53 growth regulatory pathway (9,14).
It has been subsequently found that the ING1 gene encodes
several differentially initiated and spliced mRNAs, which have
common 3' exon and encode at least two distinct proteins in mouse (15), and possibly three distinct proteins in human cells
(p47ING1a, p33ING1b, and
p24ING1c) (13,16,17). All the known or
anticipated ING1 protein isoforms share an identical C-terminal domain
with a conserved PHD finger motif. The PHD finger motif was thought to
facilitate DNA binding of proteins otherwise unrelated to ING1 (18),
suggesting that ING1 proteins might directly interact with DNA.
Significantly, missense mutations were detected within the PHD finger
and the nuclear localization motif of ING1 in some head and neck
squamous cell carcinomas with allelic loss at the 13q33-34 region,
suggesting that the PHD finger and the nuclear function of ING1 is
important for its tumor suppressor function (13).
All functional analysis of the biological effects of ectopically
expressed ING1 was so far done only with the cDNA encoding p24ING1c due to the lack of information on the
alternative forms of ING1. Owing to a cloning error, the cDNA that
suppressed cell growth was incorrectly termed
p33ING1 in the original studies (9,14). One
candidate mechanism that was proposed to be responsible for the growth
suppressor function of the ING1 locus is cooperation with the p53 tumor
suppressor (14). Neither p53 nor ING1 can cause growth inhibition when the other one is suppressed, and the p24ING1c
expression has been shown to be required for transcriptional activation
of the p21WAF1 promoter, a key mechanism of
p53-mediated growth control. Recent analysis of mouse ING1
gene structure and function suggests that the shortest of ING1 protein
isoforms, the mouse equivalent of human
p24ING1c, is required for the activation of
p53-responsive genes. In contrast, overexpression of the longer form
p37ING1, an equivalent of the human
p33ING1b protein, interferes with the activation
of p53-dependent promoters when p53 is stabilized after DNA
damage (15). It appears that isoforms of ING1 protein may have
different roles in growth control and that their unique N-terminal
sequences may determine differences in their function.
In a search for mechanisms of function of the ING1 protein, we explored
ING1 associated proteins in human cells. We found differential
association of p33ING1b and
p24ING1c with nuclear proteins.
p33ING1b resides in a complex of ~1-2 MDa,
whereas p24ING1c does not. Among
p33ING1b-associated proteins are known
components of the mSin3 transcriptional corepressor complex, including
HDAC1. Consistently, p33ING1b is functionally
associated with HDAC-dependent transcriptional repression,
in reporter gene expression assays in vivo, and in histone
deacetylation assays in vitro. We demonstrate that the mSin3-mediated HDAC1-dependent transcriptional repression
requires the unique N-terminal 99-amino acid sequence characteristic of the p33ING1b protein, therefore defining a new,
p33ING1b-specific mechanism for the function of
the ING1 locus.
Antibodies--
Rabbit anti-ING1 antibodies were generated using
recombinant His-epitope-tagged human p33ING1b
protein prepared from Escherichia coli. Goat anti-ING1
antibodies were from Santa Cruz (sc-7566). Mouse monoclonal anti-RbAp48
antibodies were from GeneTex (MS-RBP14-PX1), and rabbit anti-Sin3A
antibodies were from Santa Cruz (sc-767). Rabbit anti-HDAC1 and
anti-SAP30 antibodies were generous gifts of Dr. Glen Humphrey and Dr.
Robert Eisenman, respectively. Anti-FLAG M2-agarose affinity gel was from Sigma (A-1205).
Purification of the p33ING1b Complexes--
The FLAG
epitope-tagged p33ING1b used for mammalian
expression were constructed by subcloning the full-length cDNA with
the tagged sequence into the pCIN4 vector (19). H1299 cells (1 × 106) were transfected by calcium phosphate precipitation on
a 10-cm plate essentially as previously described with minor
modifications. Five µg of pCIN4-Flag-ING1 expression plasmid with 15 µg of carrier DNA (pGEM-3) were used for transfections on each plate.
Thirty hours after transfection, the cells were transferred to the same Dulbecco's modified Eagle's medium containing 1000 µg/ml G418 (Life
Technologies, Inc.) for selection. After 2 months' selection, single
colonies were picked and expanded for Western blot analysis.
The tagged cells were grown in Dulbecco's modified Eagle's medium
with 10% fetal bovine serum and 1000 µg/ml G418, and nuclear extracts were prepared as described previously. Forty milliliters of
the nuclear extract prepared from different cell lines was adjusted to
200 mM NaCl and 0.2% Nonidet P-40 by addition of 5 M NaCl and 10% Nonidet P-40, and incubated with 300 µl
of M2-agrose beads (Sigma) at 4 °C for 10 h by rotation. After
five washes with BC200 with 0.2% Nonidet P-40, proteins were eluted
from beads by incubation at 4 °C for 30 min with 300 µl of BC100
with 0.2% Nonidet P-40 plus 0.2 mg/ml FLAG peptide.
Large scale immunoprecipitation for mass spectrometric analysis was
carried out with 10 mg of crude or fractionated nuclear extracts and
100 µg of affinity-purified rabbit anti-ING1 antibodies, with an
excess of the purified p33ING1b antigen as a
negative control. Immune complexes were isolated by binding to 100 µl
of Sepharose-Protein A beads, washed five times with 100 volumes of
NETN buffer, eluted, and separated by SDS-PAGE.
Identification of Proteins with Mass Spectrometry--
Protein
sequencing using mass spectrometry was carried out as described (20).
Tryptic peptides that were recovered from in-gel digested protein bands
were analyzed using an electrospray ion trap mass spectrometer (LCQ,
Finnigan MAT, San Jose, CA) coupled on-line with a capillary high
performance liquid chromatography (Magic 2002, Michrom BioResources,
Auburn, CA). Data derived from the mass spectrometry/mass spectrometry
spectra were used to search a compiled protein data base that was
composed of the protein data base NR and a six-reading frame translated
expressed sequence tag data base to identify the protein using the
program PROWL, which is publicly available on the World Wide Web.
Immunoprecipitation and Immunoblotting--
Immunoprecipitations
were done by incubating 1 mg of HeLa nuclear protein extracts prepared
according to the Dignam method, with 5-10 µg of the appropriate
antibodies for 2 h at 4 °C, followed by isolation of the immune
complexes on Protein A beads (Amersham Pharmacia Biotech.). Immune
complexes were washed three times with 1 ml of NETN buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.5 mM dithiothreitol) prior to
SDS-PAGE and immunoblotting. Immunoblottings were done after low
voltage protein transfer (30 V, 12 h) from polyacrylamide gels to
nitrocellulose in Tris-glycine buffer, pH 8.0, with 5% methanol.
Membranes were blocked with 5% milk in Tris-buffered saline with Tween
20 buffer (100 mM Tris, pH 8.0, 150 mM NaCl,
0.05% Tween 20), incubated with 1:200-1:1000 dilution of the primary
antibodies for 1 h at room temperature, washed, and incubated with
1:25000 dilution of horseradish peroxidase-conjugated secondary
antibodies. Antibody detection was with ECL (Amersham Pharmacia
Biotech), using horseradish peroxidase-conjugated antibodies from Santa Cruz.
Histone Deacetylase Assays--
Histone deacetylase activity was
measured using acid-soluble histones that were isolated from
[3H]acetate-labeled HeLa cells, by a published procedure
(21, 22). Immune complexes were incubated for 3 h at 37 °C with
40,000 cpm of 3H-labeled histones (~2000 cpm/µg) in a
total volume of 200 µl of HD buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 2.5% glycerol). Reactions were stopped by
addition of 50 µl of STOP buffer (1 M HCl, 0.16 M acetic acid), extracted with two volumes of ethyl acetate, and the supernatant was counted in a scintillation counter. Assays were carried out with or without 10 mM sodium
butyrate, as indicated in the figure legends.
Transcriptional Repression Assays--
NIH3T3 cells (1 × 106) were transfected using LipofectAMINE Plus (Life
Technologies, Inc.) with a combination of 1 µg of luciferase reporter
plasmids 2Py-Luc or 6AP-Luc and 100 ng of the LacZ reporter construct
(pCMV-LacZ) to normalize transfection efficiency. One hundred ng of
ras expression vector (v-Ha-ras cDNA cloned
in pLXSN vector under the control of the LTR promoter) and 50 ng of
each of the test expression vectors FNE2DBD, pCINE2DBD:En,
FN-lINGE2DBD, and FN-sINGE2DBD (shown in Fig. 4A as
1, 2, 3, and 4,
respectively) were cotransfected with the reporter plasmids. Cells were
harvested for luciferase and
Polymerase chain reaction-derived DNA fragments encoding either human
p33ING1 or p24ING1 were
fused individually to the N terminus of the GAL4 DNA-binding domain
(amino acids 1-94). 293T cells (1 × 106, in 2.5-cm
plate), were transiently cotransfected using GenePORTER (Gene Therapy
Systems) with 0.5 µg of appropriate pING1-GAL4 expression vector and
1.5 µg of reporter plasmid. The reporter plasmid carried secreted
alkaline phosphatase (SEAP) gene under the control of the
constitutively active SV40 early promoter with five Gal4 binding sites.
Twenty-four hours after transfection, cells were incubated with 50 ng/ml trichostatin A (TSA), where indicated, and 12 h later cells
were harvested and assayed. Expression of the GAL4 fusion proteins was
determined by Western blotting with the anti-Gal4 antibody.
p33ING1b Associates with Known Components of the mSin3
Corepressor Complex--
To acquire an insight to mechanisms of ING1
function, we sought to isolate ING1-associated proteins in human cells.
We fractionated HeLa nuclear extracted on a DEAE column and
immunoprecipitated the endogenous ING1 protein from the 0.2 M KCl fraction, which contains most of the cellular
p33ING1b protein. We used mass spectrometry to
identify RBP1, Sin3, and HDAC1 along others as ING1-associated proteins
(Fig. 1A.). To facilitate
protein purification and to alleviate the interference of the antibody,
we created an H1299-derived cell line, which stably expressed a FLAG
epitope-tagged p33ING1b (19). The
FLAG-p33ING1b was overexpressed by 5-10-fold
compared with the endogenous p33ING1b protein by
Western. The recombinant FLAG-p33ING1b protein
complex was isolated from nuclear extracts prepared from the stable
line using affinity chromatography. Colloidal Coomassie Blue staining
of a SDS-PAGE gel containing the p33ING1b
complex revealed that ~10 polypeptides specifically copurify with the
FLAG-p33ING1b on the affinity column (Fig.
1B). More bands that were masked by antibody are clearly
detected in the recombinant complex.
We identified proteins that copurified with
p33ING1b in the recombinant
p33ING1b complexes using capillary liquid
chromatography ion trap mass spectrometry (20). Mass spectrometric
analysis of the p33ING1b complexes identified
the mSin3 corepressor and the HDAC1/2 histone deacetylases, as well as
RbAp48, RbAp46, and SAP30. These proteins are components of a
biochemically purified mSin3 complex (6-8). We also identified RBP1
(23) and two novel proteins, p42 and p35, which were not reported as
components of the mSin3 complex. RbAp48, RbAp46, SAP30, and p42 were
masked by antibody in the endogenous ING1 complex. These data
demonstrate that p33ING1b is a component of a
Sin3 containing histone deacetylase complex, thus suggesting a role for
p33ING1b in transcriptional repression.
We confirmed the association of Sin3 and HDAC1 with
p33ING1b by reciprocal immunoblotting of the
endogenous p33ING1b, HDAC1, and Sin3 immune
complexes from HeLa cells (Fig.
2A). Although the polyclonal
rabbit antibodies that we used in these experiments reacted with both
p33ING1b and p24ING1c
proteins (Fig. 2A, lane 3), due to
their identical C-terminal end, only p33ING1b
was detected in the Sin3 and HDAC1 immune complexes (Fig.
2A, lines 1 and 2).
Pre-incubation of the anti-ING1 antibodies with an excess of purified
recombinant His-tagged p33ING1b protein
prevented precipitation of the endogenous
p33ING1b and p24ING1c as
well as Sin3A and HDAC1 (Fig. 2B), demonstrating specificity of the observed associations. Approximately the same amounts of Sin3A,
the Sin3A-directly associated protein SAP30 (7) and p33ING1b were present in either Sin3A or ING1
immune complexes of the endogenous proteins, suggesting that
p33ING1b is a stoichiometric component of the
Sin3A/SAP30 complex in vivo. Moreover, both
p33ING1b and Sin3A immune complexes had similar
amounts of histone deacetylase HDAC1 and the histone H4-binding protein
RbAp48. Gel filtration analysis of partially purified
p33ING1 complexes demonstrated that
p33ING1b, Sin3A, HDAC1, and RbAp48 proteins
coelute in complexes of an apparent size of 1-2 MDa (Fig.
2C), as reported previously for the Sin3 complex (6). It is
not clear whether the previously biochemically purified mSin3 complex
is a stable subcomplex of this larger p33ING1b
complex. It is possible that extensive column fraction may disrupt weaker associations to yield the stable core mSin3 complex.
To establish that p33ING1b associates with
functional HDAC1, we prepared the endogenous ING1 immune complexes from
HeLa cells, as presented on Fig. 2A, and assayed them for
histone deacetylase activity. We found that ING1 complexes were active
in deacetylating 3H-labeled histones in vitro,
and this activity was comparable with the activity of HDAC1 immune
complexes when assays were performed with similar HDAC1 amounts (Fig.
2D, bars 2 and 4). Addition
of 10 mM sodium butyrate, an inhibitor of histone
deacetylase activity (24), inhibited the reaction in both HDAC1 and
ING1 immune complexes to the level of nonenzymatic
[3H]acetyl release (Fig. 2D, bars
3 and 5, compare with bar
1). In contrast, the control immunoprecipitates with various
amounts of rabbit preimmune serum alone did not catalyze histone
deacetylation (Fig. 2D, bars 6 and
7). Therefore, p33ING1b associates
with enzymatically active HDAC1 complexes, suggesting that
p33ING1b may act with Sin3 to mediate
transcriptional repression by a mechanism that involves targeted
recruitment of histone-modifying activity.
The Association with Sin3 Complexes Is Specific to the
p33ING1b Isoform and Is Defined by Its Unique N-terminal
Sequence--
Our analysis of endogenous proteins from HeLa cells
suggest that, although we can detect and immunoprecipitate both
p33ING1b and p24ING1c
isoforms, only p33ING1b associates with
Sin3/HDAC1 complexes (Fig. 2). p24ING1c is
identical to p33ING1b except for lacking the
N-terminal 99 amino acids that are characteristic of the
p33ING1b isoform (16), suggesting that the
N-terminal fragment of p33ING1b controls its
assembly with the Sin3 complex. However,
p24ING1c appears to be less abundant than
p33ING1b in a variety of cell lines that we
tested, and immunodetection of the endogenous
p24ING1c in protein extracts was generally
poorer or negative (Figs. 2A, lane 5 and 3A), unless the extracts
were enriched in p24ING1c by partial
purification (Fig. 2C).
To eliminate the possibility that lower abundance of the
p24ING1c protein rather than its different
protein structure is responsible for the observed differences in the
assembly pattern, we analyzed p24ING1c assembly
under the condition of its overexpression, using stably transfected
HT1080 fibroblasts (14). Quantitative Western blot analysis of nuclear
extracts prepared from the transfected and untransfected HT1080 cells
demonstrates that the recombinant p24ING1c was
at least as abundant as the endogenous p33ING1b
(Fig. 3A, compare lanes 4-6 with
lanes 1-3). However, overproduction did not
force p24ING1c assembly into large protein
complexes (Fig. 3B). Identical results were also obtained
from stably transfected MCF7 cells. Therefore, the association with
Sin3 complexes is specific to the p33ING1b
isoform and is defined by its unique N-terminal sequence.
p33ING1b Is Functionally Associated with
HDAC1-dependent Transcriptional Repression in Vivo--
We
used one reporter system to test whether
p33ING1b is functionally associated with
transcriptional repression in vivo. In this system, the DNA
binding domain (DBD) of the transcription factor Ets2 is fused with the
mouse p37ING1 and
p26ING1, which are human homologues of
p33ING1b and p24ING1c,
respectively. The structures of expression constructs are shown schematically in Fig. 4A. A
previously described fusion protein containing Ets2 DBD and the
repressor domain of the Engrailed protein of Drosophila
melanogaster was used as a positive control (construct
2 in Fig. 4A) (25, 26). The reporter plasmid
contains the luciferase gene under the control of the minimal promoter of the ras-responsive c-fos gene and oncogene
regulatory elements as described previously (27) (Fig. 4B).
The reporter (2Py-luc) contains a tandem repeat of a combination of Ets
and Ap1 binding sites from the enhancer of polyoma virus. The 6AP-Luc
reporter plasmid containing six tandem AP-1 binding sites was used as a control. Both reporters are ras-responsive if cotransfected
with ras-expressing plasmid into NIH 3T3 cells, but 6AP-Luc
is insensitive to Ets. Expression plasmids were coexpressed in NIH3T3
cells with activated ras, serving as an activator of
Ets-directed transcriptional activation of reporter constructs. Protein
levels of the chimeric proteins were normalized using a LacZ
gene reporter. The results of luciferase assays are shown in Fig. 4
(C and D). The long form of ING1 fused with Ets2
DBD works as a potent repressor similar to the positive control of the
Engrailed repressor domain fused to Ets2 DBD (lanes
5 and 4). In contrast, the short form of ING1 exhibits a moderate repression (lane 6), which is
similar to that of the Ets2 DBD alone (lane 3).
This moderate repression may be due to competition with the endogenous
Ets2 protein. Both ING1 fusion proteins and Ets2 DBD are similarly
active in gel mobility shift assays with the oligonucleotide
corresponding to the Ets2 DNA binding site (data not shown). The
repressor effect is specific to Ets2 since none of the plasmids tested
show any effect on the control reporter construct (6AP-luc) lacking
Ets2-binding sequences (Fig. 4D).
We obtained similar results using another reporter system, in which the
activity of SEAP was used as a reporter of transcription from a
constitutively active SV40 promoter that was cloned next to five Gal4
binding sites. In this system, the chimeric
GAL4-p33ING1b fusion protein also mediates
transcriptional repression (Fig. 4E, compare lane
3 and lane 1). The repression is specific to p33ING1b, because the
GAL4-p24ING1c fusion protein has little effect
on the SV40 promoter. Moreover, treatment of transfected cells with
TSA, a specific inhibitor of histone deacetylases, restores the
reporter activity, indicating that the transcriptional repression
mediated by p33ING1b requires active HDAC.
Western blot analysis confirmed that both GAL4-p33ING1b and
GAL-p24ING1c fusion proteins were expressed in
comparable amounts, regardless of the presence or absence of TSA (Fig.
4E). Therefore, tethering p33ING1b to
an artificial promoter in vivo can confer
HDAC-dependent transcriptional repression in reporter gene
expression systems.
Our finding of differential association of the products of the
alternative transcripts of p33ING1b and
p24ING1c with the mSin3 transcriptional
corepressor complex provides a basis for a molecular mechanism of ING1
function as a growth regulator and a candidate tumor suppressor. It
also introduces novel aspects into the understanding of the
Sin3/HDAC1-mediated transcriptional repression, by identifying new
components that might serve as a link to regulation of growth and cell division.
Our data suggest that p33ING1b is the
predominant isoform among ING1 proteins that is associated with the
Sin3/HDAC1-mediated transcriptional repression. This is based on the
observation that the N-terminal 99 amino acids, which are unique to
p33ING1b, are required for: 1) the assembly with
Sin3/HDAC1 complexes, and 2) the HDAC1-dependent
transcriptional corepressor activity in reporter gene assays.
p24ING1c, which is otherwise identical to
p33ING1b except for missing the N-terminal
domain, does not seem to interact with the Sin3/HDAC1 complexes even
when overproduced. The p47ING1a isoform has a
unique N-terminal fragment that is distinct from that of
p33ING1b. Although in this study we did not
rigorously examine the assembly of p47ING1a, the
putative endogenous p47ING1a protein that we can
detect with the affinity purified polyclonal anti-ING1 antibodies does
not coelute with the Sin3/HDAC1 complexes in gel filtration
experiments. Moreover, p47ING1a cannot be
immunoprecipitated by Sin3 and HDAC1 (data not shown). This suggests
that p47ING1a may associate with different
protein partners, but the nature and roles of the
p47ING1a assembly remain to be determined.
The cooperation of ING1 with p53 was the first mechanism that was
proposed to account for the growth suppressor function of ING1 (14).
Recent analysis of the ING1 isoforms in mouse suggests that the
equivalent of the human p24ING1c homologue is
required for the activation of p53-dependent promoters. In
contrast, the mouse equivalent of the human
p33ING1b isoform interferes with the activation
of the p53-dependent responses (15). This result is
consistent with our finding that p33ING1b
functions in transcriptional repression, not activation. Moreover, Sin3-mediated HDAC1 activity was recently indicated in the repression of p53-responsive genes (15). It will be important to test whether p33ING1b plays a role in the negative regulation
of the p53-responsive genes in cooperation with Sin3/HDAC1. A model for
the function of the ING1 locus can be envisaged from our data and
previous studies that the interplay of the ING1 isoforms in
collaboration with p53 sets the transcriptional program that determines
cell proliferation or arrest. We propose that
p33ING1b together with the mSin3 corepressor
machinery represses p53-responsive genes that halt cell cycle
progression and that p24ING1c serves as an
antagonist to relieve this repression. The relative ratio of
p33ING1b and p24ING1c
thus may determine the proliferate potential of the cell.
Work from a number of laboratories demonstrated that Sin3 serves as
scaffold protein for the assembly of multiprotein complexes, which
target histone deacetylase activities to selected genes by interacting
with specific transcription factors. These complexes facilitate
transcriptional repression through a mechanism of induction of local
rearrangements of the chromatin structure (3). In contrast to Sin3 and
HDAC1, which are relatively stable proteins and do not seem to be cell
cycle-regulated, ING1 is cell cycle-regulated. p24ING1c accumulates in cells that are
quiescence or senescence and overexpression of
p24ING1c in primary fibroblasts arrests cells in
G1 phase of the cell cycle (28).
p33ING1b also accumulates in quiescence, but
induction of cell division by addition of mitogens leads to rapid
decline of the p33ING1b
protein.2 In light of our
data presented in this paper, these observations suggest that
p33ING1b may serve as a regulatory subunit of
the mSin3 complex and together with mSin3 might be involved in
repression of some essential cell cycle regulatory genes. The
identities of those genes are not known, but our identification of RBP1
as a presumptive subunit of the p33ING1b/Sin3
complex suggests that among possible candidates are genes that are
regulated by the Rb/E2F pathway through an interaction of RBP1 with Rb.
This intriguing possibility agrees with the observation that HDAC1
interacts with the Rb protein and that the HDAC1 activity is required
for full transcriptional repression of some of the Rb-regulated genes
(29,30). Therefore, our data presented here may also link Sin3/HDAC1 to
cell cycle regulation through the association with
p33ING1b.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase assays 40 h after
transfection. The luceferase and
-galactosidase enzyme activities
from the extracts of transfected NIH3T3 cells were measured according
the Promega protocols.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purification and identification of the
FLAG-p33ING1b complex. A, the endogenous ING1
complex in HeLa cells was purified by large scale immunoprecipitation
(IP) from 0.2 M KCl fraction of the DEAE column
and resolved on 4-20% gradient SDS-PAGE gel. B, the FLAG
epitope-tagged p33ING1b was purified from
nuclear extracts prepared from H1299 cells that stably expressed the
FLAG-p33ING1b protein. As a control, a mock
purification was performed from nuclear extracts prepared from the
parental H1299 cells. The FLAG-peptide elutes were separated by 10%
SDS-PAGE, visualized by staining gels with colloidal Coomassie Blue,
and identified by capillary liquid chromatography electrospray ion trap
mass spectrometry. p42 and p35 are two novel proteins that are
identified from the expressed sequence tag data base.
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Fig. 2.
Endogenous p33ING1b in HeLa cells
associate with Sin3 complexes that contain active HDAC1.
A, coimmunoprecipitation of p33ING1b
with mSin3 complexes. Immunoprecipitations (IP) were
performed with 1 mg of HeLa nuclear protein extracts and 2-10 µg of
rabbit anti-Sin3A (lane 1), rabbit anti-HDAC1
(lane 2), rabbit
anti-p33ING1b (lane 3), or
rabbit pre-immune (lane 4) antibodies. The amount
of individual antibodies was adjusted to obtain approximately the same
amount of p33ING1b in all immune complexes.
B, competition of the purified recombinant His-epitope
tagged p33ING1b protein with
p33ING1b complexes from HeLa extracts.
Immunoprecipitations were done as above, except that the
anti-p33ING1b antibodies were pre-incubated with
an excess of the purified recombinant His epitope-tagged
p33ING1b protein. C, coelution of
p33ING1b with mSin3 complexes in gel filtration
analysis. Partially purified HeLa nuclear protein extracts (0.2 mg, 300 mM KCl elution from CM Sepharose) were separated by gel
filtration on Superose 6 PC3.2/30 column, in a buffer with 50 mM Tris, pH 7.5, 200 mM KCl, and 0.5 mM dithiothreitol. Proteins from the gel filtration
fractions were precipitated with 10% trichloroacetic acid, separated
by SDS-PAGE, and analyzed by immunoblotting, as indicated.
D, histone deacetylase activity associated with HDAC1 and
ING1 immune complexes in vitro. HDAC1 immune complexes used
in histone deacetylation assays (bars 1-3) were
identical with those shown in A (lane 2) and
served as a reference. The ING11 immune complexes (bars
4 and 5) used in deacetylation assays were
isolated with 100 µg of anti-ING1 antibodies, which is 10-fold more
than what was used in A (lane 3), to
obtain comparable amounts of HDAC1 to those present in HDAC1 immune
complexes. Control reactions were done with 10 µg (bar
6) and 100 µg (bar 7) of pre-immune
rabbit serum, or with HDAC1 (bar 3) and ING1
(bar 5) immune complexes that were pre-incubated
with 10 mM sodium butyrate. Bar 1 indicates
level of nonenzymatic [3H]acetyl release observed in
HDAC1 immune complexes incubated at 0 °C.
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Fig. 3.
Effects of overproduction of p24ING1c
on protein complex assembly. A, quantitative Western
blotting of ING1 proteins in cell lines. Different amounts of the ING1
proteins were immunoblotted with anti-ING1 antibody from nuclear
extracts prepared from parental (lanes 1-3),
stable HT1080 cells, that overexpress p24ING1c
(lanes 4-6) and p33ING1b (lanes 7-9) as
well as in HeLa cells. B, overproduction of
p24ING1c does not drive
p24ING1c assembly. Nuclear extracts prepared
from HT1080 cells that overexpress p24ING1c were
analyzed by gel filtration on a Superose 6 column. The ING1 proteins
were detected.
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Fig. 4.
p33ING1b is functionally associated
with HDAC1-mediated transcriptional repression in
vivo. A, schematic structures of the
expression plasmids of the Ets2 DBD fusion systems.
Construct 1, DBD of Ets2 alone;
construct 2, DBD Ets2 fused with the repressor
domain of the Engrailed protein of D. melanogaster (positive
control); constructs 3 and 4, DBD Ets2
fused with mouse homologues of human p33ING1b
and p24ING1c, respectively. B,
schematic structures of promoter regions of the reporter plasmids.
C and D, transcriptional repression results as
measured by the luciferase assays with the indicated systems described
in A and B. The luciferase activity was measured
in lysates of NIH 3T3 cells cotransfected with 1 µg of reporter
plasmid, 100 ng of LacZ gene reporter (to normalize transfection
efficiency), 100 ng of ras expression vector, and 50 ng of
one of the test constructs (1-4). Three independent
experiments yielded similar results. E, transcriptional
repression assays using the GAL4 fusion system. Plasmids containing the
human GAL4-p33ING1b and
GAL4-p24ING1b were transiently transfected in
293T cells, and their ability to repress transcription of a SEAP reporter was assayed. The SEAP reporter construct is under
the control of the constitutively active SV40 promoter and five Gal4
sites. Treatment of transfected cells with TSA (50 ng/ml) restored the
reporter activity. Whole cell lysates from the transfected cells were
also analyzed by Western blot with an antibody against the GAL4
DNA-binding domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Glen Humphrey and Robert Eisenman for the generous gift of antibodies. We thank Fannie Huang for establishing the FLAG-p33ING1b stable cell line, Dr. Irina Grigorian for providing the construct of His-epitope-tagged p33ING1b protein, and Dr. Jin Wang for mass spectrometric sequencing. We thank Dr. Jieming Wong for discussion and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA60730 and CA75179 (to A. V. G.) and CA85533 (to W. G.).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 addressed. Tel.: 713-798-1507;
Fax: 713-798-1625; E-mail: jqin@bcm.tmc.edu.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M007664200
2 K. V. Gurova and A. V. Gudkov, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HDAC, histone deacetylase; ING1, inhibitor of growth 1; DBD, DNA binding domain; PAGE, polyacrylamide gel electrophoresis; TSA, trichostatin A; SEAP, secreted alkaline phosphatase; PHD, plant homeodomain.
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