Distinct mechanisms for repression of RNA polymerase III transcription by the retinoblastoma tumor suppressor protein

Abstract

The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAP(C) and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.

FIG. 1.

RB selectively occupies U6 snRNA promoters in vivo and specifically represses U6 snRNA transcription in vitro. (A) RB occupies a human snRNA promoter in vivo. ChIP experiments from human 184B5 cells were performed with anti-RB (lane 5), anti-SNAP43 (lane7), and nonspecific IgG (lane 6) antibodies. Precipitated DNAs were analyzed by PCR for enrichment of U6 snRNA, U1 snRNA, and U2 snRNA promoters or GAPDH exon 2 as a negative control. Lanes 1 to 4 show a 10-fold serial dilution of input chromatin from 10 to 0.01%. (B) RB occupies the U6 promoter in a variety of cell types. ChIPs from nontumorigenic human mammary epithelial cells (184B5), normal human fibroblast cells (NFSL83), human mammary epithelial adenocarcinoma cells (MCF-7), and human cervical carcinoma cells (HeLa) were performed with preimmune sera (lane 4) or anti-SNAP43 (lane 5) or anti-RB (lane 6) antibodies. Enrichment of U6 snRNA promoter, U2 snRNA promoter, or GAPDH exon 2 DNA in each immunoprecipitation reaction mixture was determined by PCR with a primer combination specific to each gene. Lanes 1 to 3 illustrate a 10-fold serial dilution (1 to 0.01%) of input chromatin. (C) Schematic representation of selected RNA polymerase II and RNA polymerase III promoters used for in vitro transcription assays. (D) RB represses U6 snRNA gene transcription by RNA polymerase III but not U1 snRNA transcription by RNA polymerase II. In vitro transcription assays for the RNA polymerase II and RNA polymerase III-transcribed genes were performed with HeLa cell nuclear extract as described previously (15, 16). Lanes 2 and 3 were treated with 200 and 800 ng of GST-RB (379-928), respectively. Lanes 4 and 5 were treated with 200 and 800 ng of GST, respectively, as a negative control.

FIG. 2.

The A/B pocket domain alone is insufficient for efficient repression of RNA polymerase III transcription. (A) Schematic representation of the truncated RB proteins used in these experiments. (B) Analysis of truncated GST-RB proteins. GST-RB (379-928) (lane 2), GST-RB (379-870) (lane 3), GST-RB (379-772) (lane 4), GST-RB (379-577) (lane 5), and GST (lane 6) were expressed in E. coli and purified by affinity chromatography with glutathione-agarose beads and competitive elution with glutathione. Purified proteins were dialyzed against Dignam buffer D, and aliquots were separated by SDS-PAGE and visualized by staining with Coomassie blue. Lane 1 contains a protein molecular size standard. (C, top) Schematic representation of the human U6 snRNA and Ad VAI promoters. (Bottom) GST-RB (379-928) and GST-RB (379-870) repress human U6 snRNA and VAI transcription by RNA polymerase III. Approximately 2 μl of HeLa cell nuclear extract (approximately 7.5 μg/μl) was incubated with 250 and 1,000 ng of GST-RB (379-928) (lanes 2 and 3), GST-RB (379-870) (lanes 4 and 5), GST-RB (379-772) (lanes 6 and 7), GST-RB (379-577) (lanes 8 and 9), or GST protein (lane 10). With separate reaction mixtures, human U6 snRNA or Ad VAI transcription in the treated extracts was performed at 30°C for 30 min. Lane 1 shows the level of transcription for each gene supported by the untreated extract. Sample handling was monitored by the presence of a nonspecific RNA handling control. (D) Characterization of the RB regions required for interactions with the RNA polymerase III basal machinery. GST pulldown analysis was performed to determine the region of RB that can interact with each component of the RNA polymerase III basal machinery. SNAP43, SNAP50, TBP, Brf1, Brf2, Bdp1, and Oct-1 were expressed in vitro and labeled with [³⁵S]methionine. Ten percent of each in vitro-expressed protein added to the reaction mixtures is shown in lane 1. Each protein was incubated with the various truncated GST-RB proteins or with GST, and stable protein complexes were purified with glutathione-Sepharose. The beads were extensively washed, and bound proteins were separated by SDS-PAGE. Associated proteins were visualized by autoradiography.

FIG. 3.

RB occupies a human U6 snRNA but not Ad VAI promoter during repression. (A) Affinity purification of GST-RB proteins and associated promoter-containing DNAs from in vitro RB repression assays. Portions of the human U6 snRNA and Ad VAI transcription assays shown in Fig. 2C were cross-linked with formaldehyde prior to affinity purification with glutathione-Sepharose to recover the GST-RB proteins. The concomitant recovery of associated promoter plasmid DNA was then measured by PCR. The recovered samples were analyzed for the presence of the U6 reporter construct (pU6/Hae/RA.2) or the Ad VAI reporter construct (M13-Ad VAI) with primers specific to the promoters contained in these plasmids. (B) RB occupies an endogenous human U6 snRNA (class 3) promoter but not a tRNA^Lys (class 2) promoter in vivo. ChIP experiments were performed similarly to those described previously. A 10-fold serial dilution of input chromatin, from 10 to 0.01%, is shown in lanes 1 to 4. Immunoprecipitation reaction mixtures were prepared with IgG (lane 5), anti-SNAP43 (lane 6), anti-TBP (lane 7), or anti-RB (lane 8) antibodies, and any recovered DNA was analyzed with primers specific for the U6 snRNA, U2 snRNA, and tRNA^Lys promoters or GAPDH exon 2 as a negative control.

FIG. 4.

RB co-occupies the same endogenous U6 snRNA promoter as SNAP_C, TFIIIB, and RNA polymerase III. Sequential ChIPs were performed from human 184B5 cells with anti-RB (top part), anti-SNAP43 (second part), anti-RNA polymerase II (third part), and IgG (bottom part) for the first round of immunoprecipitation (IP), followed by a second immunoprecipitation with the indicated antibodies. Chromatin recovered after the second immunoprecipitation was analyzed by PCR for enrichment of U6 and U2 promoter DNA or GAPDH exon 2 DNA as a negative control. Lane 1 shows 10% of the input chromatin.

FIG. 5.

RB and RNA polymerase III co-occupy the same repressed U6 snRNA promoter in vitro and in vivo. (A) In vitro U6 snRNA transcription reaction mixtures were prepared with untreated HeLa cell nuclear extracts (NE; lane 1) or extract treated with 500 ng of GST-RB (379-928) or GST (lanes 2 and 3, respectively). The reaction mixtures shown in lane 4 contained no added extract. (B) Sequential cross-linked immunoprecipitations (IP) from the transcription reaction mixtures from part A were performed with anti-RNA polymerase III antibodies for the primary immunoprecipitation and IgG (lane 2), anti-RNA polymerase III (lane 3), and anti-RB (lane 4) antibodies for the secondary immunoprecipitation. PCR analysis of the recovered DNA was performed with primers specific to the U6/Hae/RA.2 reporter or pUC119, which was included as a negative control in these assays. (C) Sequential ChIPs from human 184B5 cells were performed with anti-RNA polymerase III antibodies for the primary immunoprecipitation and anti-RNA polymerase III (lane 2) or anti-RB (lane 3) antibodies for the secondary immunoprecipitation. A portion of chromatin recovered after the second round of immunoprecipitation was analyzed for enrichment of U6 snRNA promoter or GAPDH exon 2 DNA by PCR (top parts). The remaining portion of chromatin was treated with DNase I. Associated RNA was examined by PCR in the presence (+ RT) or absence (− RT) of reverse transcriptase with U6 snRNA-specific primers (bottom parts).