Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 23 (21), 7719-31

Histone Deacetylation of RB-responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Affiliations

Histone Deacetylation of RB-responsive Promoters: Requisite for Specific Gene Repression but Dispensable for Cell Cycle Inhibition

Hasan Siddiqui et al. Mol Cell Biol.

Abstract

The retinoblastoma tumor suppressor protein (RB) is targeted for inactivation in the majority of human tumors, underscoring its critical role in attenuating cellular proliferation. RB inhibits proliferation by repressing the transcription of genes that are essential for cell cycle progression. To repress transcription, RB assembles multiprotein complexes containing chromatin-modifying enzymes, including histone deacetylases (HDACs). However, the extent to which HDACs participate in transcriptional repression and are required for RB-mediated repression has not been established. Here, we investigated the role of HDACs in RB-dependent cell cycle inhibition and transcriptional repression. We find that active RB mediates histone deacetylation on cyclin A, Cdc2, topoisomerase IIalpha, and thymidylate synthase promoters. We also demonstrate that this deacetylation is HDAC dependent, since the HDAC inhibitor trichostatin A (TSA) prevented histone deacetylation at each promoter. However, TSA treatment blocked RB repression of only a specific subset of genes, thereby demonstrating that the requirement of HDACs for RB-mediated transcriptional repression is promoter specific. The HDAC-independent repression was not associated with DNA methylation or gene silencing but was readily reversible. We show that this form of repression resulted in altered chromatin structure and was dependent on SWI/SNF chromatin remodeling activity. Importantly, we find that cell cycle inhibitory action of RB is not intrinsically dependent on the ability to recruit HDAC activity. Thus, while HDACs do play a major role in RB-mediated repression, they are dispensable for the repression of critical targets leading to cell cycle arrest.

Figures

FIG. 1.
FIG. 1.
PSM-RB (active RB) represses a set of cell cycle genes and induces cell cycle arrest. (A) A5-1 cells were cultured in the presence or absence of Dox for 16 h prior to BrdU labeling for an additional 8 h. The percentage of BrdU-positive cells was determined from three independent experiments. (B) The cloned rat regulatory regions ofdifferent RB target genes analyzed in the present study. (C) The A5-1 parental cell line was stably transfected with rat promoter constructs for the indicated genes or empty vector (pGL2-B) driving expression of the firefly luciferase gene. Three clones were selected for each construct, maintained in the presence or absence of Dox for 18 h, and then assayed for luciferase activity. (D) A5-1 cells were cultured in the presence or absence of Dox for 18 h. Total RNA was then extracted and reverse transcribed into cDNA. This cDNA was subjected to linear PCR amplification with specific primers for the indicated genes. (E) A5-1 cells were cultured in the presence (lane 1) or absence (lane 2) of Dox for 24 h. Total protein was isolated, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and immunoblotted with antibodies as indicated.
FIG. 2.
FIG. 2.
Active RB induces histone deacetylation at promoters of specific cell cycle genes. (A) Total chromatin was isolated from A5-1 cells cultured in the presence of Dox, and increasing amounts of chromatin (0 to 4 μl) were subjected to PCR in the presence of [α-32P]dCTP and primers specific for the cdc2 promoter. Production of PCR product was quantified by using a phosphorimager. (B) A5-1 cells were cultured in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of Dox for 24 h and cross-linked with formaldehyde, and ChIP assays were performed as described in Materials and Methods. Residency of acetylated histone H4 at the indicated gene promoters was determined by carrying out the ChIP assay with antibodies specific to acetylated histone H4 (lanes 3 and 4). Input (lanes 1 and 2) refers to PCR containing 1% of the total chromatin used in IP. IP with Dbf-4 (lanes 5 and 6) is a negative control. PCR products were detected by autoradiography. HPRT, hypoxanthine-guanine phosphoribosyltransferase. (C) Cells were cultured as described for panel B, except that immunoprecipitation was performed with antibodies specific for Dbf-4 (lanes 1 and 2), E2F4 (lanes 3 and 4), and HDAC1 (lanes 5 and 6). The mock represents a ChIP assay that was performed without the inclusion of chromatin substrate. PCR products were detected by autoradiography.
FIG. 3.
FIG. 3.
RB-induced deacetylation at specific promoters is mediated by HDACs. (A) A5-1 cells were maintained in media with (lanes 1 to 4) or without (lanes 5 to 8) Dox and the HDAC inhibitor TSA (0, 50, 100, and 200 nM) for 24 h. Proteins were resolved by SDS-PAGE and immunoblotted with antibodies as indicated. (B) A5-1 cells were cultured in the presence or absence of Dox and TSA (100 nM). Cells were cross-linked after 24 h, and the ChIP assay was performed with an acetylated histone H4-specific antibody (lanes 13 to 16). Inputs (lanes 2 to 5) represent PCR with 1% of chromatin utilized in immunoprecipitations. Controls include H2 O (PCR control, lane 1), Mock (IP without chromatin, lane 6), Dbf-4 (nonspecific antibody, lanes 7 to 10), and No Ab (IP without antibody, lanes 11 to 12). (C) PCR products from Fig. 3B were quantified by using the phosphorimager, and data were normalized to account for variation in inputs.
FIG. 4.
FIG. 4.
HDAC activity is dispensable for RB-mediated cell cycle arrest. (A) A5-1 cells were cultured in the presence or absence of Dox and TSA for 16 h prior to BrdU labeling for an additional 8 h. Cells were fixed and stained with anti-BrdU antibody and Hoechst to identify proliferating cells and nuclei, respectively. (B) The percentage of BrdU-positive cells was determined from three independent experiments. (C) A5-1 cells were cultured in the presence or absence of Dox and TSA for 24 h. Cells were harvested, fixed with ethanol, and stained with propidium iodide (PI). Cell cycle distribution was then determined by flow cytometry. DNA content (PI intensity) is plotted against cell number. The percentage of cells in G0-G1, S, and G2-M phases was determined by ModFit software.
FIG. 5.
FIG. 5.
HDAC-dependent deacetylation is required by RB to repress specific promoters. (A) A5-1-integrated reporter cell lines described in the legend of Fig. 1C were cultured in the presence or absence of Dox and 100 nM TSA for 18 h. Cells were harvested and assayed for luciferase activity. (B) A5-1 cells were cultured in 100 nM TSA in the presence or absence of Dox for 18 h. Total RNA was then extracted and reverse transcribed into cDNA. This cDNA was subjected to linear PCR amplification with specific primers for the indicated genes. (C) A5-1 cells were cultured in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of Dox in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 100 nM TSA for 24 h. Total protein was isolated, resolved by SDS-PAGE, and immunoblotted with antibodies as indicated. (D) Top panel: The cloned regulatory region of the rat RNRII promoter. Left panel: The A5-1 parental cell line was stably transfected with the rat RNRII promoter driving expression of the firefly luciferase gene. Cells were cultured in the presence or absence of Dox with or without 100 nM TSA as indicated, and relative luciferase activity was determined. Center panel: A5-1 cells were cultured in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of Dox and in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of TSA for 18 h. Cells were harvested, and RNRII RNA levels were determined by RT-PCR. Right panel: A5-1 cells were cultured in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of Dox and the absence (lanes 1 and 3) or presence (lanes 2 and 4) of TSA for 24 h. Cells were harvested, and RNRII protein levels were determined by immunoblotting.
FIG. 6.
FIG. 6.
The cyclin A promoter is not subjected to stable gene silencing. (A) A5-1 cells were cultured in the presence or absence of Dox as indicated. Chromatin was isolated and utilized in ChIP assays with antibodies specific for dimethylated K9 histone H3 (lanes 5 and 6). Input (lanes 1 and 2) and Dbf-4 (lanes 3 and 4) controls are shown. Chromatin was amplified with primers specific for the cyclin A and myogenin promoters, and products were detected by autoradiography. (B) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence of 5-aza-2-dC as described in Materials and Methods and then cultured in the absence of Dox for 24 h. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel). (C) A5-1 cells harboring the integrated cyclin A reporter were cultured in the presence or absence of Dox for 24 h. To attenuate PSM-RB, Dox was readministered to the indicated cultures. Relative luciferase activity was determined by reporter assay (left panel), and endogenous protein levels were determined by immunoblotting (right panel).
FIG. 7.
FIG. 7.
RB-mediated repression of the cyclin A promoter involves chromatin remodeling. (A) Parental A5-1 cells (lanes 1 and 2) or A5-1 cells engineered to inducibly coexpress dnBRG-1 and PSM-RB (lanes 3 and 4) were cultured in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of Dox for 24 h. Cells were harvested, and protein and RNA levels were determined by RT-PCR (left panel) or immunoblotting (right panel) as indicated. (B) Left panel: genomic structure of the rat cyclin A locus. Right panel: Southern blot analysis of genomic DNA isolated from A5-1 cells (lanes 1 and 2). Parental A5-1 cells (lanes 3 and 4) or A5-1 cells engineered to inducibly express dnBRG-1 (lanes 5 and 6) were cultured in the presence or absence of Dox as indicated. Permeabilized nuclei were subjected to digestion with EagI, and then isolated genomic DNA was subjected to cleavage with KpnI and EcoRI. Restriction fragments were detected by radioactive Southern blotting.

Similar articles

See all similar articles

Cited by 26 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback