Transforming Growth Factor-β1 Recruits Histone Deacetylase 1 to a p130 Repressor Complex in Transgenic Mice in Vivo¹ Free

The TGF³-βs are members of a superfamily that regulate cell growth and function (1). The TGF-βs are widely expressed inhibitors of cellular proliferation strongly implicated as components of a tumor suppressor pathway in different organ systems (2, 3, 4, 5). TGF-β1 is the most abundant of the three TGF-β mammalian isoforms. Analysis of mice homozygously deleted of the Tgfb1 gene suggested TGF-β1 functions as a tumor suppressor with true haploid insufficiency in the heterozygous animals (6). Mice heterozygous for the Tgfb1 gene expressed reduced TGFβ-1 levels and demonstrated enhanced hepatic tumorigenesis to chemical carcinogens compared with litter mate controls (6). Transgenic mice overexpressing TGF-β1 in the liver under control of the albumin promoter (Tg-TGFβ-1) have reduced DNA synthesis induced after PH, providing in vivo evidence for the antiproliferative function of TGF-β1 (7). The mechanism by which TGF-β1 inhibits the cell cycle apparatus are highly cell type and context dependent. Experiments conducted in cultured cells demonstrated that TGF-β1 can inhibit growth by inducing expression of the Cdk inhibitors p15^(INK4B/MTS2) and p21^Cip1, through altering the distribution of p27^Kip1 from cdk4/6 to cdk2 (8) and through inducing inhibitory Cdk tyrosine phosphorylation(9). The cdc25 phosphatases activate the cdks by dephosphorylating their inhibitory tyrosine and threonine phosphorylated residues (10, 11). In tissue culture experiments, TGF-β1 increases cdk tyrosine phosphorylation through repression of the cdk-activating tyrosine phosphatase cdc25A(9). Repression by TGF-β1 was blocked by the addition of the histone deacetylase inhibitor trichostatin A, suggesting a role for HDAC in TGF-β function (9). Analysis of the molecular mechanisms of TGF-β1 cell cycle inhibitory function in vivo have been limited.

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-β1 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-β1 to address these questions.

The reporter constructions derived from the Cdc25A promoter(22) NPGL2 −460/+129, the E2F site mutant of the cdc25A promoter, NPGL2mE2F-A (23), the expression vectors encoding pCMV-p130 (24), and HDAC1FLAG (18)in pBJ5 (from Dr. S. Schreiber) were described previously.

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-β1 (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.

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-β1 (anti-TGF-β1 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 were performed using[³H]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[³H]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 × 10⁷) either untreated or treated with TGF-β1 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 500μl of cell lysis buffer [5 mm1,4-piperazinediethanesulfonic acid (pH 8.0), 85 mm KCl, 0.5% NP40, 1 mmPMSF, 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 1× 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.

Analysis of liver regeneration were performed on male Alb-TGF-β1 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.

Transgenic mice in which TGF-β1 was overexpressed under control of the albumin promoter (Tg-TGF-β1) expressed hepatic TGF-β1 levels a mean of 10-fold greater than nontransgenic strain-matched controls (Wt;Fig. 1,A) 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-β1 livers (Fig. 1,A, lower panel). p130 abundance was increased in the Tg-TGFβ-1 mice in association with higher TGF-β1 levels (Fig. 1,A) and was increased an average 2-fold as compared with Wt mice (Fig. 1,B). The Tg-TGF-β1 mice expressed increased total hepatic p130, and the fraction of hypophosphorylated p130 compared with phosphorylated was higher than in Wt mice (Fig. 1,B). TGF-β1 treatment also increased the relative amount of hypophosphorylated p130 (Fig. 1 C) in HaCaT keratinocytes.

Pocket proteins, including p130, associate in vitro with HDAC1, contributing to their transcriptional repressor function(17, 18, 21). To examine further the mechanisms by which TGF-β1 regulated p130 function and cdc25A abundance, we examined the possibility that p130 associated with HDAC1 in vivo. Immunoprecipitation of Tg-TGF-β1 hepatic extracts using nonsaturating amounts of p130-specific antibody to ensure equal amounts of p130 in the IP as indicated in the figure legend with sequential HDAC1 Western blotting demonstrated that a p130/HDAC1 complex formed in vivo. The relative abundance of this complex was increased in the Tg-TGF-β1 transgenic mice (Fig. 2,A). The reciprocal IP, in which Tg-TGF-β1 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-β1 livers (Fig. 2,B). Thus, in the Tg-TGFβ1 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-β1 transgenic mice (Fig. 2,C, upper panel). The amount of p130 bound to HDAC1 showed a tendency to be increased in mice expressing higher levels of TGF-β1 (Fig. 2 C).

In previous studies transfecting expression vectors into cultured cells, each of the pocket proteins was capable of binding HDAC1. These studies raised the question of whether specific associations may form in vivo and thereby coordinate signal transduction specificity (17, 18, 21). The Wt and Tg-TGF-β1 transgenic mice hepatic extracts were therefore subjected to IP with specific pocket protein antibodies, and sequential Western blotting was performed using either HDAC1 antibody or pocket protein antibody (Fig. 2,D). 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-β1 samples with the same pocket protein antibody. In the p130 IP, HDAC1 binding was greater in the Tg-TGF-β1 livers compared with the Wt hepatic samples (compare the fourth lane with the first lane in Fig. 2,D). In the p107 IP, the relative abundance of p107 associated with HDAC1 was increased in the Wt compared with the Tg-TGF-β1 livers (Fig. 2,D). 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-β1 transgenic mice (Fig. 2 D). Together, these findings suggest that in the Tg-TGF-β1 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-β1 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-TGFβ-1 hepatic extracts (for n = 4 separate animals; Fig. 2 E). 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.

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-β1 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-β1 mice to determine whether TGF-β1 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-β1(Fig. 3). The data were expressed for the relative amount of cdc25A and TGF-β1 based on Western blotting determined in arbitrary densitometric units. cdc25A protein levels were reduced in the Tg-TGF-β1 mice livers (Fig. 3).

The current studies showed that p130 was overexpressed in the Tg-TGF-β1 livers, p130/HDAC1 abundance was increased, and cdc25A levels were reduced. In previous studies performed in cultured cells,TGF-β1 inhibited the cdc25A promoter (23). These studies raised the possibility that p130/HDAC may inhibit cdc25A expression. Because previous studies had examined HDAC function in 293T (31, 32), COS, or Saos2 cells (20), we examined the possibility that p130/HDAC1 could directly repress cdc25A expression using 293T cells. p130 overexpression inhibited the cdc25A promoter linked to a luciferase reporter gene (Fig. 4,A). 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. 4,A), suggesting that the inhibition by p130 was DNA sequence dependent. Exogenous HDAC1 did not affect cdc25A promoter activity(Fig. 4,B); however, p130 repressed and HDAC1 enhanced p130-mediated repression of cdc25A (Fig. 4 C). 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.

To determine whether p130 formed part of the complex binding to the cdc25A E2F site in the context of its native chromatin structure, we used a modification of a reversible formaldehyde cross-linking procedure (33). Chromatin IP assays were performed using extracts from untreated and TGF-β1-treated cells. Immunoprecipitation was performed using antibodies specific for their cognate protein. After immunoprecipitation and reversal of the cross-linking, the endogenous cdc25A promoter was enriched by PCR amplification using primers specific for the cdc25A E2F site (Fig. 4,D). A cdc25A-specific PCR product was observed with antibodies to E2F-4 and p107 but not p130 (Fig. 4,D) or IgG (data not shown) in several separate experiments. Sequence analysis confirmed the PCR product as the human cdc25A promoter. In TGF-β1-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. 4 D). These findings indicate that in the presence of TGFβ-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-β1, contributes to a transcriptional repressor complex at the cdc25A promoter E2F site.

PH, a potent proliferative stimulus to the liver, induces delayed cellular proliferation in Tg-TGF-β1 mice with a reduced maximal rate of DNA synthesis (7). Because hepatic TGF-β1 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-β1 mice, increasing to 7% at 36 h [Fig. 5,A (n = 26) versus Wt hepatic BrdUrd 16% at 36 h (n = 9)]. Hepatic cdc25A protein levels in the Tg-TGF-β1 mice increased 2.5-fold at 18 h to 4-fold at 42 h, approaching levels found in Wt liver (Fig. 5,B). p130-IP with sequential HDAC1 Western blotting showed p130 associated HDAC1 decreased by 20% at 18 h and by 40% at 24 h (Fig. 5,C). p130/HDAC1 complex formation did not decrease in the Wt liver at these time points after PH (Fig. 5,C). After PH, p130 protein does not change at time 0 and 36 h, in both Wt and Tg-TGF-β1 mice (Fig. 5 C,right panel). These findings suggest that the decrease in HDAC1 binding to p130 upon hepatectomy observed in the Tg-TGF-β1 mice was dependent upon the transgenic overexpression of TGF-β1.

In previous studies, hepatic TGF-β1 haploid insufficiency predisposed to hepatic carcinogenesis (6), and transgenic TGF-β1 overexpression inhibited hepatic cellular proliferation(7), providing further support for the current understanding that TGF-βs function as important components of a tumor suppressor pathway (2, 3, 4, 5). The current studies were performed in vivo because of recent evidence that important differences in TGF-β1 signaling occur in cultured cells compared with whole animal analyses (6, 34, 35, 36). Thus, although p21^Cip1, p15^Ink4b, and cdk4 were identified previously as targets of TGF-β1 regulation in cultured cells (34, 35, 36), the abundance of these proteins was unaffected by altered TGF-β1 levels in vivo(6). In vivo the abundance of p27^Kip1 and c-myc were changed in the direction opposite to that predicted from in vitro activities(6), likely reflecting a complex paracrine interplay required for TGF-β1 tumor suppressor function (37). In the current studies, we demonstrate the impact of hepatic TGF-β1 overexpression on selective critical components of the pRB-tumor suppressor pathway in vivo.

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-β1 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-TGFβ1 samples (Fig. 2,A). 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-TGFβ1 transgenic mice; however, the amount of p130 was unchanged at the same time points from 0 to 36 h (Fig. 5 C). 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-β1 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. 5 C), was regulated by both cytostatic and proliferative stimuli in vivo. This“disengagement” of HDAC1 from p130 after PH in the Tg-TGFβ-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-β1 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-β1 levels and HDAC/p130 association may provide mechanistic insights into the dose dependency of TGF-β1 tumor suppressor function. The current studies demonstrate that TGF-β1 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-β1 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 TGFβ-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-β1 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-β1 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.

We thank Drs. E. Harlow, W. Kaelin, S. Schreiber, and G. Vairo for plasmids, chemicals, and antibodies. We thank Drs. P. Farnham and J. Wells for advice on the chromatin IP assays.