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Molecular Biology and Genetics |
The Albert Einstein Cancer Center, Departments of Developmental and Molecular Biology [B. B., M. F., A. I., R. G. P.], Medicine [A. I., R. G. P.], and Neurology [A. I.], Albert Einstein College of Medicine, Bronx, New York 10461, and Laboratory of Experimental Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [V. M. F., S. S. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mitogenic and antimitogenic signals selectively regulate components of the cell cycle apparatus (12 , 13) . Orderly progression through G1 phase involves coordinated activation of the cdks, which phosphorylate and inactivate members of the "pocket protein" family. This family includes the product of the retinoblastoma susceptibility gene (the pRB protein) and the related p107 and p130 proteins. The A/B pocket region, which is strongly conserved between these three proteins, binds E2F/DP transcription factors. Pocket protein-E2F complexes bound to DNA repress gene transcription through E2F binding sites, which function as silencer elements (14, 15, 16) . The pocket region of pRB also binds to a HDAC1 (17 , 18) . The NH2-terminal tail domains of core histones contain highly conserved lysines that are posttranslationally modified by acetylation. Acetylation and deacetylation are catalyzed by histone acetyltransferases and HDACs. HDAC1 facilitates the removal of acetyl groups from core histones, enhancing DNA nucleosome interactions and impeding access of transcription factors to their DNA binding sites (19) . It was proposed that recruitment of HDAC1 to pRB contributes to transcriptional repression of target genes (17 , 18 , 20) . However, p107 and p130 were also shown to bind HDAC1 in vitro (21) , raising the fundamental biological question of whether HDAC1-pocket protein associations occur in a selective and regulated manner in vivo.
The biological significance of associations between HDAC and pocket proteins in vivo remained to be determined. Previous findings raised important questions of whether HDAC1 bound pocket-proteins in vivo, whether selective associations occurred between HDAC1 and a particular pocket protein in vivo, and whether these associations may be regulated in a specific manner by mitogens or tumor suppressors. The identification of cell cycle regulatory genes targeted by HDAC complexes in vivo would also provide important support for the emerging theme of chromatin remodeling proteins in oncogenesis (19) . The elucidation of mechanisms regulating selective associations between HDAC1 and particular pocket proteins by TGF-ß1 in vivo may provide insights into the tumor suppressor function of this cytokine. The current studies were performed in vivo using transgenic mice overexpressing hepatic TGF-ß1 to address these questions.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture, DNA transfection, and luciferase assays were performed as described previously (25) . The SAOS2 osteosarcoma cell line, HaCaT keratinocytes, and 293T (BOSC) cells were maintained in DMEM with 10% (v/v) calf serum and 1% penicillin/streptomycin. In transient expression studies, cells were transfected by calcium phosphate precipitation, the medium was changed after 6 h, and luciferase activity was determined after an additional 24 h. The effect of an expression vector was compared with the effect of an equal amount of empty vector cassette. Treatments with TGF-ß1 (200 pM; 24 h) were compared with vehicle. Luciferase content was measured using an AutoLumat LB 953 (EG&G Berthold) by calculating the light emitted during the initial 10 s of the reaction, and the values are expressed in arbitrary light units (26) . Statistical analyses were performed using the Mann-Whitney U test with significant differences established as P < 0.05.
Immunoprecipitations and Western Blotting.
Hepatic cellular extracts were prepared from lysis buffer containing 50
mM HEPES (pH 7.2), 150 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1%
Tween 20, 0.1 mM PMSF, 2.5 µg/ml leupeptin, and 0.1
mM Na3VO4.
Lysates (5–100 µg as indicated in the text) were immunoprecipitated
with the indicated antibodies (2 µg each) and agarose A beads (Santa
Cruz Biotechnology). Precipitates were washed with lysis buffer and
separated by SDS electrophoresis. Western analysis was performed as
described previously (25)
, using antibodies from Santa
Cruz Biotechnology to p130 (C-20), pRb2-mAb (Transduction
Laboratories), p107 (C18), p105 (XZ55), cdc25A (144), and HDAC1
(Upstate Biotechnology), TGF-ß1 (anti-TGF-ß1 pAb; Promega Corp.,
Madison, WI), and a GDI antibody (Dr. Perry Bickel, Washington
University, St. Louis, MO). Cell homogenates (50 µg) were
electrophoresed in an SDS-12% polyacrylamide gel and transferred
electrophoretically to a nitrocellulose membrane (Micron Separations,
Inc., Westborough, MA). After transfer, the gel was stained with
Coomassie blue as a control for blotting efficiency. The blotting
membrane was incubated for 12 h at 25°C in T-PBS buffer
supplemented with 5% (w/v) dry milk to block nonspecific binding
sites. After a 6-h incubation with primary antibody at a 1:1000
dilution (for p130, p107, p105, and cdc25A) or 1:2500 (GDI) in T-PBS
buffer containing 0.05% (v/v) Tween 20, the membrane was washed with
the same buffer.
HDAC Assays and Chromatin Immunoprecipitation Assays.
HDAC assays were performed using
[3
H]acetate-incorporated histones
(27)
isolated from HeLa cells treated with sodium butyrate
(28)
exactly as described previously. Hepatic extracts
(300 µg) were immunoprecipitated with saturating amounts of pocket
protein antibodies (10 µg) and then incubated with 1 ml of
[3
H]acetate-labeled HeLa histones (10,000 dpm)
for 2 h at 37°C, and acetylase activity was determined as
described previously (27)
.
In vivo cross-linking and chromatin immunoprecipitation were described elsewhere (25) . 293T cells (2 x 107) either untreated or treated with TGF-ß1 for 24 h were fixed by adding formaldehyde directly to the tissue culture media to a final concentration of 1% for 10 min. Cross-linking was then stopped by the addition of glycine to 0.125 M. The cells were rinsed twice with cold PBS and were scraped from the culture dishes. The cells were resuspended in 500 µl of cell lysis buffer [5 mM 1,4-piperazinediethanesulfonic acid (pH 8.0), 85 mM KCl, 0.5% NP40, 1 mM PMSF, 1 mM benzamidine, 10 µg/ml aprotinin, and 1 µg/ml leupeptin] and were incubated on ice for 10 min. The nuclei were collected by centrifugation at 5000 rpm for 5 min and incubated in 500 µl of nuclei lysis buffer [50 mM Tris-Cl (pH 8.0), 10 mM EDTA, 1% SDS, 1 mM PMSF, 1 mM benzamidine, 10 µg/ml aprotinin, and 1 µg/ml leupeptin] for 3–5 h at 4°C. Chromatin was sonicated to an average length of 0.5–1 kb (Vibra-Cell Sonicator, Sonics and Materials, Inc., Danbury, CT) at medium power for four times, 30 s each. The chromatin was precleared by incubation with 10 µl of blocked Staph A cells (300 µg/µl) at 4°C for 15 min. Staph A cells were pelleted by centrifugation at 14,000 rpm for 5 min, and the supernatant was treated with 1 µg of antibody or control IgG for 16 h at 4°C [anti-E2F4 polyclonal antibody (C-104), p130 Ab (C-20), p107 Ab (C18), from Santa Cruz Biotechnology or with rabbit IgG]. The antibody/protein/DNA complexes were immunoprecipitated with 10 µl of blocked Staph A cells for 15 min at room temperature, washed twice with 1.2 ml 1x dialysis buffer [2 mM EDTA, 50 mM Tris-Cl (pH 8.0), and 0.2% Sarkosyl] and four times with 1.2 ml of IP buffer [100 mM Tris-Cl (pH 9.0), 500 mM LiCl, 1% NP-40, and 1% deoxycholic acid].
The complexes were eluted twice with 150 µl of IP elution buffer (50 mM NaHCO3, 1% SDS) at room temperature. The samples were then incubated at 67°C for 5 h with 1 µl of RNase A (10 mg/ml) and 0.3 M NaCl to reverse cross-linking. After incubation, the complexes were precipitated with ethanol, dissolved in 100 µl of TE, and treated with proteinase K (100 µg/ml) at 45°C for 2 h. The samples were extracted once with phenol and once with phenol/chloroform/isoamyl alcohol and then precipitated with ethanol in the presence of 5 µg carrier tRNA. DNA was dissolved in 100 µl of [50 mM Tris, 10 mM EDTA (ph 8.0)], and 5 µl were taken for PCR analysis in a 50-µl reaction. Amplifications were performed using 1 cycle at 95°C for 1 min; 35 cycles at 94°C for 45 s, 55°C for 1 min, and at 72°C for 1 min, followed by further elongation at 72°C for 10 min. Sterile H2O and IP washing buffer were included as negative controls for the PCR reaction. The PCR product was visualized on a 2% agarose gel with ethidium bromide staining.
The oligonucleotides used for PCR of the human cdc25A promoter in the chromatin immunoprecipitation assays were 5'-CTGAGA GCC GAT GAC CTG GCA GAG T, and at the 3' end was TCC CAC CCG CTT GCC CAG CTC C and generated a 256-bp fragment. The PCR product was visualized on a 2% agarose gel with ethidium bromide staining and direct sequenced for verification.
Transgenic Animals and Hepatectomy.
Analysis of liver regeneration were performed on male Alb-TGF-ß1
transgenic mice at 8 weeks of age after standard 70% hepatectomy under
metofane anesthesia (7)
. The monitoring of DNA synthesis
was performed after an i.p. injection of BrdUrd (Boehringer Mannheim;
150 mg/kg of body weight) 1 h prior to sacrifice as described
previously (7)
. Tissues were fixed in Bouins’ fixative
(Polysciences). Proliferation of hepatocytes was assessed as described
(29)
. After staining with hematoxylin, the nuclear DNA
labeling index was determined by counting of BrdUrd-positive nuclei per
3000 hepatocyte nuclei and expressed as the percentage per 100 nuclei.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and varied between animals as described previously
(7
, 29)
. Induction of DNA synthesis involves sequential
phosphorylation and inactivation of "pocket proteins" (pRB and the
related p107 and p130 proteins), each of which display distinct
structural and functional features (30)
. The relative
abundance of hepatic pRB and p107 was unchanged between the Wt and
Tg-TGF-ß1 livers (Fig. 1A
, lower panel). p130
abundance was increased in the Tg-TGFß-1 mice in association with
higher TGF-ß1 levels (Fig. 1A
) and was increased an
average 2-fold as compared with Wt mice (Fig. 1B
). The
Tg-TGF-ß1 mice expressed increased total hepatic p130, and the
fraction of hypophosphorylated p130 compared with phosphorylated was
higher than in Wt mice (Fig. 1B
). TGF-ß1 treatment also
increased the relative amount of hypophosphorylated p130 (Fig. 1C
) in HaCaT keratinocytes.
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). The reciprocal IP, in which Tg-TGF-ß1 hepatic extracts
were precipitated with an HDAC1-specific antibody and examined for
p130, showed that p130 binding to HDAC1 was increased 40–50% in the
Tg-TGF-ß1 livers (Fig. 2B
). Thus, in the Tg-TGFß1
transgenic mice livers, there is an increase in hypophosphorylated p130
and increase in HDAC1 bound p130. The amount of HDAC1 protein assessed
by direct Western blotting of hepatic extracts did not change between
Wt and Tg-TGF-ß1 transgenic mice (Fig. 2C
, upper
panel). The amount of p130 bound to HDAC1 showed a tendency to be
increased in mice expressing higher levels of TGF-ß1 (Fig. 2C
).
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). Although each pocket protein antibody is specific, it
cannot be assumed that the antibodies have identical affinity for their
cognate target. Comparison was therefore made between Wt and
Tg-TGF-ß1 samples with the same pocket protein antibody. In the p130
IP, HDAC1 binding was greater in the Tg-TGF-ß1 livers compared with
the Wt hepatic samples (compare the fourth lane with the first lane in
Fig. 2D
). In the p107 IP, the relative abundance of p107
associated with HDAC1 was increased in the Wt compared with the
Tg-TGF-ß1 livers (Fig. 2D
). There was no HDAC1 associated
with the IgG control as described previously (17
, 18 , 21)
.
As noted above, there was no change in the relative abundance of total
pRB or p107 between the Wt and Tg-TGF-ß1 transgenic mice (Fig. 2D
). Together, these findings suggest that in the
Tg-TGF-ß1 livers compared with Wt, the HDAC1 association with p130 is
enhanced and the HDAC1 association with p107 is reduced.
To determine whether the predominant pocket protein binding to HDAC1
observed in these IP Western blot analysis was associated with in
vivo HDAC activity, HDAC assays were performed as described
previously (5)
. Immunoprecipitation was performed for p107
in the Wt and p130 in the Tg-TGF-ß1 using equal amounts of hepatic
extracts, and HDAC assays were performed (27)
. No HDAC
activity was associated with the IgG control (Lane 1). Both
p107 and p130 IPs under the conditions used in these experiments
demonstrated significant HDAC activity. The total amount of HDAC
activity associated with saturating amounts of p107 in the Wt samples
was 30–40% less than the p130 associated HDAC1 activity in the
Tg-TGFß-1 hepatic extracts (for n = 4
separate animals; Fig. 2E
). These studies indicate that both
p107 and p130 convey HDAC1 activity in vivo. As p107 has
been shown to arrest cells in G1 without
recruiting HDAC (18)
, the significance of the p107/HDAC
binding remains to be determined.
TGF-ß1 Inhibits cdc25A Protein Levels in Vivo.
The cdc25 phosphatases activate the cdks by removing their inhibitory
phosphorylation of tyrosine and threonine residues and can function as
proto-oncogenes in transformation assays (10
, 11)
.
TGF-ß1 treatment inhibited cdc25A expression in HaCaT keratinocytes
(data not shown) as described previously (23)
. We
therefore examined the expression of cdc25A in the hepatic extracts
derived from Wt and Tg-TGF-ß1 mice to determine whether TGF-ß1
inhibited cdc25A levels in vivo. Western blots were analyzed
for cdc25A, and comparison was made with the relative abundance of GDI
as an internal control. Expression of the cdc25A gene showed
a tendency to be reduced in mice expressing higher levels of TGF-ß1
(Fig. 3
). The data were expressed for the relative amount of cdc25A and
TGF-ß1 based on Western blotting determined in arbitrary
densitometric units. cdc25A protein levels were reduced in the
Tg-TGF-ß1 mice livers (Fig. 3
).
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). Point mutation of the recently identified cdc25A promoter
E2F site (23)
reduced basal level activity. The cdc25A
E2Fmut reporter construction was not inhibited by p130 (Fig. 4A
), suggesting that the inhibition by p130 was DNA sequence
dependent. Exogenous HDAC1 did not affect cdc25A promoter activity
(Fig. 4B
); however, p130 repressed and HDAC1 enhanced
p130-mediated repression of cdc25A (Fig. 4C
). The cdc25A
promoter repression observed with HDAC1 (1200 ng) was significant
compared with vector control (P < 0.05).
These results suggest that p130 inhibits the cdc25A promoter and that
in cells transfected with p130, endogenous HDAC1 may be a limiting
factor in cdc25A transcriptional repression.
|
). A
cdc25A-specific PCR product was observed with antibodies to E2F-4 and
p107 but not p130 (Fig. 4D
) or IgG (data not shown) in
several separate experiments. Sequence analysis confirmed the PCR
product as the human cdc25A promoter. In TGF-ß1-treated
extracts, antibodies to E2F-4, p107, and p130 precipitated a complex
bound to the cdc25A-specific E2F site confirmed by direct sequence
analysis (Fig. 4D
). These findings indicate that in the
presence of TGFß-1, a p130/E2F-4 complex binds the cdc25A promoter in
the context of its native chromatin structure. These studies are
consistent with a model in which p130, bound to HDAC1, in the presence
of TGF-ß1, contributes to a transcriptional repressor complex at the
cdc25A promoter E2F site.
Mitogenic Stimulation Dissociates p130/HDAC in Tg-TGF-ß1 Mice
in Vivo.
PH, a potent proliferative stimulus to the liver, induces delayed
cellular proliferation in Tg-TGF-ß1 mice with a reduced maximal rate
of DNA synthesis (7)
. Because hepatic TGF-ß1
overexpression increased p130/HDAC1 abundance and reduced cdc25A
levels, we hypothesized that PH may reduce HDAC1 binding to p130,
thereby inducing cdc25A levels and DNA synthesis. Hepatic BrdUrd
synthesis induced by PH was delayed in the Tg-TGF-ß1 mice, increasing
to 7% at 36 h [Fig. 5A
(n = 26) versus Wt
hepatic BrdUrd 16% at 36 h (n = 9)].
Hepatic cdc25A protein levels in the Tg-TGF-ß1 mice increased
2.5-fold at 18 h to 4-fold at 42 h, approaching levels found
in Wt liver (Fig. 5B
). p130-IP with sequential HDAC1 Western
blotting showed p130 associated HDAC1 decreased by 20% at 18 h
and by 40% at 24 h (Fig. 5C
). p130/HDAC1 complex
formation did not decrease in the Wt liver at these time points after
PH (Fig. 5C
). After PH, p130 protein does not change at time
0 and 36 h, in both Wt and Tg-TGF-ß1 mice (Fig. 5C
,
right panel). These findings suggest that the decrease in HDAC1 binding
to p130 upon hepatectomy observed in the Tg-TGF-ß1 mice was dependent
upon the transgenic overexpression of TGF-ß1.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent studies proposed that pRB-mediated transcriptional repression of
Pol II transcription involved pRB-bound HDAC1, which repressed
transcription by promoting nucleosome formation (17
, 18
, 20)
. Subsequently, the related pocket proteins, p107 and p130,
which have important distinguishable features (30
, 38)
,
were also shown to bind HDAC1 in vitro (21)
. In
the current studies, increased TGF-ß1 levels in transgenic mice
enhanced HDAC1 binding to p130, in contrast with control animals in
which HDAC1 bound p107. Two findings suggest that the association
between p130 and HDAC is not a simple function of cellular mixing. For
a constant amount of p130, the relative amount of HDAC is increased in
the Tg-TGFß1 samples (Fig. 2A
). Thus, the relative amounts
of HDAC bound to p130 and the levels of p130 alter in a discordant
manner. Secondly, mitogenic signals induced by PH reduced HDAC binding
to p130 without altering the levels of p130 (Fig. 5C
). In
the post hepatectomy samples, the amount of HDAC bound to p130
decreased in the Tg-TGFß1 transgenic mice; however, the amount of
p130 was unchanged at the same time points from 0 to 36 h (Fig. 5C
). These findings suggest that the association between
HDAC and p130 and not just the abundance of p130 is regulated in
vivo.
These findings are consistent with a model in which associations
between HDAC and pocket proteins may regulate distinct subsets of
target genes to coordinate TGF-ß1 tumor suppressor function in
vivo. Studies in which the individual pocket proteins were
homozygously deleted in mice demonstrated that pRB and p107/p130 have
overlapping but distinct functions that are required for the normal
expression of different subsets of E2F-responsive genes
(39)
. Furthermore, distinct E2Fs may differentially
regulate specific target genes (40)
. In the current
studies, HDAC1 association with specific pocket proteins, assessed by
p130-IP, HDAC western blotting (Fig. 5C
), was regulated by
both cytostatic and proliferative stimuli in vivo. This
"disengagement" of HDAC1 from p130 after PH in the Tg-TGFß-1 mice
may contribute an additional level of control through which the
individual pocket proteins regulate distinct target genes. In the
current studies, both p107 and p130 were associated with HDAC activity
in vivo. The current studies therefore provide a rational
basis for further analysis comparing relative HDAC activities by each
pocket protein in common tissues, once detailed analysis of relative
affinities of these antibodies is known.
TGF-ß1 functions as a tumor suppressor with true haploid insufficiency and may be paradigmatic of tumor suppressor genes, the function of which is dose dependent (28) . The correlation between TGF-ß1 levels and HDAC/p130 association may provide mechanistic insights into the dose dependency of TGF-ß1 tumor suppressor function. The current studies demonstrate that TGF-ß1 controls target gene expression at several new levels through enhancing HDAC1/particular pocket protein complex formation and by "recruiting" E2F-pocket protein "platforms" to a specific target gene promoter. In the Tg-TGF-ß1 mice, PH induced cdc25A protein levels and DNA synthesis concordant with the dissociation of the p130/HDAC1 repressor complex. The current studies provide evidence that chromatin remodeling proteins may form selective associations in response to both the antimitogenic effect of TGFß-1 and the mitogenic stimulus of PH in vivo. These studies also provide support for an important role for p130 in regulating cdc25A gene expression and complement previous studies using transiently expressed genes. Thus, although HDAC, P/CAF, Brm1, and other chromatin remodeling proteins have been shown to regulate transiently expressed reporter genes, unresolved controversy remains as to the relative role of chromatin in regulating transiently expressed genes (19) . In the current studies, cdc25A was inhibited by p130/HDAC1 through an E2F binding site in the promoter. Chromatin IP analysis suggested that TGF-ß1 treatment recruited p130 to the cdc25A E2F site in the context of its native chromatin structure. Therefore, chromatin remodeling protein complexes directly effect cell cycle control genes at specific DNA sequences in the context of their native chromatin structure.
HDAC recruitment to specific pocket proteins may be a general mechanism invoked by other cytostatic/tumor suppressor signals. Our studies of HDAC1 function provide support for the emerging theme of chromatin remodeling proteins in oncogenesis. HDAC1/NcoR complexes bind nuclear receptors, and HDAC1/mSin3 complexes bind Mad/Mxi complexes in vitro, thereby repressing target gene transcription (41) . In the same manner that HDAC1 recruitment correlates with the anti-oncogenic activity of Mad(Mxi)/Sin3 complexes (42) , TGF-ß1 recruitment of HDAC1 correlated with repression of cdc25A, a potential oncogene (10) . The bmi-1 proto-oncogene product is homologous to Posterior Sex Comb, a member of the Drosophila Polycomb group that stably represses chromatin structure of homeotic genes during development protein. bmi-1, like cdc25A (10) , cooperates with Ras in transformation (43) . bmi-1, however, acts primarily by inhibiting function of the ink4a tumor suppressor locus (43) . Identification of a chromatin regulatory protein’s key transcriptional targets may dictate their tumor suppressor or oncogenic function in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grants
RO1CA77552-01, RO1CA70897, RO1DK53446, RO1CA75503, and P50-HL 56399 (to
R. G. P.). 
2 To whom requests for reprints should be
addressed, at The Albert Einstein Cancer Center, Departments of
Medicine, and Developmental and Molecular Biology, Albert Einstein
College of Medicine, Chanin 302, 1300 Morris Park Avenue, Bronx, NY
10461. Phone: (718) 430-8662; Fax: (718) 430-8674; E-mail: pestell{at}aecom.yu.edu
3 The abbreviations used are: TGF, transforming
growth factor; cdk, cyclin-dependent kinase; HDAC, histone deactylase;
PMSF, phenylmethylsulfonyl fluoride; BrdUrd, bromodeoxyuridine; IP,
immunoprecipitation; PH, partial hepatectomy; Wt, wild type.
Received 2/18/00. Accepted 6/19/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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