Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Dec;20(24):9192-202.
doi: 10.1128/MCB.20.24.9192-9202.2000.

Retinoblastoma protein disrupts interactions required for RNA polymerase III transcription

J E Sutcliffe  1 , T R Brown, S J Allison, P H Scott, R J White
Affiliations

Retinoblastoma protein disrupts interactions required for RNA polymerase III transcription

J E Sutcliffe et al. Mol Cell Biol. 2000 Dec.

Abstract

The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
TSA stimulates the expression of a Pol III template in vivo. (A) RT-PCR analysis of cDNAs prepared from total RNA extracted from BALB/c 3T3 fibroblasts cultured without TSA (lane 1) or in the presence of 100 nM TSA (lane 2) or 300 nM TSA (lane 3). The cDNAs were PCR amplified using primers specific for DHFR and ARPP P0. (B) Northern blot analysis of the same total RNA (10 μg) samples as were used for panel A, which had been extracted from BALB/c 3T3 fibroblasts cultured without TSA (lane 1) or in the presence of 100 nM TSA (lane 2) or 300 nM TSA (lane 3). The upper panel shows the blot probed with a B2 gene and the lower panel shows the same blot stripped and reprobed with the ARPP P0 gene.
FIG. 2
FIG. 2
Repression of a Pol III template by RB in vivo is maintained in the presence of TSA. (A) RT-PCR analysis of cDNAs prepared from total RNA extracted from Rb−/− fibroblasts or matched wild-type fibroblasts that were cultured in the presence or absence of 300 nM TSA. The cDNAs were PCR amplified using primers specific for DHFR and ARPP P0. (B) Northern blot analysis of the same total RNA (10 μg) samples as in panel A, which had been extracted from Rb−/− fibroblasts or matched wild-type fibroblasts that were cultured in the presence or absence of 300 nM TSA. The upper panel shows the blot probed with a B2 gene, and the lower panel shows the same blot stripped and reprobed with the ARPP P0 gene.
FIG. 3
FIG. 3
RB can repress Pol III transcription of a transfected VA1 gene in the presence of TSA. SAOS2 cells were transfected with pVA1 (0.5 μg), pCAT (4 μg), and 8 μg of pSG5L-HA-RB(wt), encoding wild-type RB (bars 2 and 4), or 8 μg of pSG-RB;567L, encoding an inactive RB null mutant (bars 1 and 3). Cells depicted by bars 3 and 4 were maintained in 100 nM TSA for 24 h prior to harvesting. VA1 and CAT RNA levels were assayed by primer extension and then quantitated using a phosphorimager. Values shown represent the signal for VA1 normalized to the levels of CAT expression to correct for transfection efficiency; they are expressed relative to the value obtained in the absence of TSA or functional RB (bar 1), which is designated 100%, and are means from two experiments.
FIG. 4
FIG. 4
Recombinant RB represses Pol III transcription in vitro despite the presence of TSA. (A) pVA1 (250 ng) was transcribed using 10 μg of HeLa cell extract that had been preincubated for 15 min at 30°C with 250 ng of GST or GST-RB. Lanes 3 and 4 also contained 165 nM TSA and lanes 5 and 6 contained 330 nM TSA. (B) pLeu (250 ng) was transcribed using 10 μg of HeLa cell extract that had been preincubated for 15 min at 30°C without addition (lanes 1, 2, and 11), with 250 ng of GST (lanes 3 through 6), or with 250 ng of GST-RB (lanes 7 through 10). Lanes 4 and 8 contained 165 nM TSA, lanes 5 and 9 contained 331 nM TSA, and lanes 2, 6, and 10 contained 496 nM TSA.
FIG. 5
FIG. 5
RB disrupts the interaction between TFIIIB and TFIIIC2. (A) Reticulocyte lysate (15 μl) containing in vitro-translated BRF was immunoprecipitated (IP) in the presence of buffer or 150 μg of HeLa nuclear extract using anti-TFIIIC2 antibody 4286. Recombinant histidine-tagged RB (100 ng) was included in lane 4. Proteins retained after extensive washing were resolved on a sodium dodecyl sulfate (SDS)–7.8% polyacrylamide gel and then visualized by autoradiography. Lane 1 shows 10% of the input reticulocyte lysate containing in vitro-translated BRF. (B) Reticulocyte lysate (15 μl) containing in vitro-translated BRF was immunoprecipitated in the presence of 150 μg of HeLa nuclear extract (lanes 2 to 4) using anti-TFIIIC2 antibody 4286. Lanes 2, 3, and 4 contained 200 ng of GST, GST-RB, and GST-RBΔ21, respectively. Proteins retained after extensive washing were resolved on an SDS–7.8% polyacrylamide gel and then visualized by autoradiography. Lane 1 shows 10% of the input reticulocyte lysate containing in vitro-translated BRF.
FIG. 6
FIG. 6
RB disrupts the interaction between TFIIIB and endogenous TFIIIC2 both in vitro and in vivo. (A) HeLa cell extract (150 μg) was immunoprecipitated (IP) using antiserum 4286 against TFIIIC2 (lanes 3 through 5) or the corresponding preimmune serum (lane 2). Lanes 3, 4, and 5 contained, respectively, 200 ng of GST, GST-RB, and GST-RBΔ21. Precipitated material was resolved on a sodium dodecyl sulfate (SDS)–7.8% polyacrylamide gel and then analyzed by Western blotting with anti-TBP antibody SL30. Lane 1 shows 10% of the input nuclear extract. (B) C33A cells were transfected with pcDNA3HA.BRF (2 μg) along with 2 μg of pSG5L vector (lanes 1 and 4), pSG5L-HA-RB(wt) (lane 2), or pSG-RB;567L (lane 3). Transfected cells were immunoprecipitated using anti-HA antibody (Ab) F-7 (lanes 1 to 3) or control antibody M-19 against TAFI48 (lane 4). Immunoprecipitated material was resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western blotting with antibody 4286 against the TFIIIC110 subunit of TFIIIC2 and antibody F-7 against the HA tag on transfected BRF.
FIG. 7
FIG. 7
Binding of TBP to BRF is maintained in the presence of RB. (A) Anti-TBP antibody SL30 was used to immunoprecipitate (IP) reticulocyte lysate (15 μl) containing in vitro-translated TBP and/or in vitro-translated BRF. Histidine-tagged recombinant RB (100 ng) was added to lane 4. Proteins retained after extensive washing were resolved on a sodium dodecyl sulfate (SDS)–7.8% polyacrylamide gel and then visualized by autoradiography. (B) HeLa cell extract (150 μg) was immunoprecipitated using antiserum 128 against BRF (lanes 3 to 5) or the corresponding preimmune serum (lane 2). Lanes 4 and 5 received 200 ng of GST and GST-RB, respectively. Precipitated material was resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western blotting with antibody SL30 against TBP. Lane 1 shows 10% of the input nuclear extract.
FIG. 8
FIG. 8
RB represses a U6 snRNA gene promoter in vivo, even in the presence of TSA. SAOS2 cells were transfected with pU6/Hae/RA.2 (0.5 μg), pCAT (4 μg), and 8 μg of pSG5L-HA-RB(wt), encoding wild-type RB (lanes 2 and 4), or 8 μg of pSG-RB;567L, encoding an inactive RB null mutant (lanes 1 and 3). In lanes 3 and 4, the cells were maintained in 200 nM TSA for 24 h prior to harvesting. Levels of RNA derived from pU6/Hae/RA.2 and pCAT were assayed by primer extension and then quantitated using a phosphorimager. Values shown represent the signal for U6 normalized to the levels of CAT expression to correct for transfection efficiency; they are expressed relative to the value obtained in the absence of TSA or functional RB (lane 1), which is designated 100%, and are means from two experiments.
FIG. 9
FIG. 9
RB disrupts the interaction between TFIIIB and Pol III. (A) Reticulocyte lysate (15 μl) containing in vitro-translated BRF was immunoprecipitated (IP) in the presence of buffer (lane 2) or 150 μg of HeLa nuclear extract (lanes 3 to 5) using antiserum against the Pol III-specific subunit BN51. Lanes 4 and 5 contained 200 ng of GST and GST-RB, respectively. Proteins retained after extensive washing were resolved on a sodium dodecyl sulfate (SDS)–7.8% polyacrylamide gel and then visualized by autoradiography. Lane 1 shows 10% of the input reticulocyte lysate containing in vitro-translated BRF. (B) Reticulocyte lysate (15 μl) containing in vitro-translated BRF was immunoprecipitated in the presence of buffer (lane 2) or 150 μg of HeLa nuclear extract (lanes 3 to 5) using antiserum against the Pol III-specific subunit BN51. Lanes 3, 4, and 5 contained 200 ng of GST, GST-RB, and GST-RBΔ21, respectively. Proteins retained after extensive washing were resolved on an SDS–7.8% polyacrylamide gel and then visualized by autoradiography. Lane 1 shows 10% of the input reticulocyte lysate containing in vitro-translated BRF.
FIG. 10
FIG. 10
RB disrupts the interaction between TFIIIB and endogenous Pol III both in vitro and in vivo. (A) HeLa cell extract (150 μg) was immunoprecipitated (IP) using antiserum 128 against BRF (lane 2), anti-BN51 antiserum against Pol III (lanes 3 and 4), or the corresponding preimmune serum (lane 3). Recombinant histidine-tagged RB (100 ng) was included in lane 5. Precipitated material was resolved on a sodium dodecyl sulfate (SDS)–7.8% polyacrylamide gel and then analyzed by Western blotting with anti-TBP antibody SL30. Lane 1 shows 10% of the input nuclear extract. (B) HeLa cell extract (150 μg) was immunoprecipitated using antiserum 128 against BRF. Lanes 2 and 3 contained, respectively, 200 ng of wild-type (WT) GST-RB and GST-RB carrying an inactivating substitution at residue 706. After extensive washing, coprecipitated Pol III was detected using the random polymerization assay. Values shown are means of four experiments; error bars indicate the standard deviation. (C) C33A cells were transfected with pcDNA3HA.BRF (2 μg) along with 2 μg of pSG5L vector (lanes 1 and 4), pSG-RB;567L (lane 2), and pSG5L-HA-RB(wt) (lane 3). Transfected cells were immunoprecipitated (IP) using anti-HA antibody (Ab) F-7 or control antibody BF683 against cyclin A. Immunoprecipitated material was resolved on an SDS–7.8% polyacrylamide gel and then analyzed by Western blotting with antiserum 4286 against TFIIIC2 (top panel), anti-BN51 antiserum against Pol III (middle panel), and antibody F-7 against the HA tag on transfected BRF (bottom panel).
FIG. 11
FIG. 11
Model to explain the repression of Pol III transcription by RB. Specific initiation at class III genes is dependent on the interaction between TFIIIB and Pol III; in most cases TFIIIB must be recruited to promoters through binding to TFIIIC2. RB associates with TFIIIB and prevents it from interacting with Pol III and TFIIIC2. In this way, RB is able to disrupt preinitiation complex formation and inhibit transcription.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alzuherri H M, White R J. Regulation of a TATA-binding protein-associated factor during cellular differentiation. J Biol Chem. 1998;273:17166–17171. - PubMed
    1. Bernues J, Simmen K A, Lewis J D, Gunderson S I, Polycarpou-Schwarz M, Moncollin V, Egly J-M, Mattaj I. Common and unique transcription factor requirements of human U1 and U6 snRNA genes. EMBO J. 1993;12:3573–3585. - PMC - PubMed
    1. Brehm A, Kouzarides T. Retinoblastoma protein meets chromatin. Trends Biochem Sci. 1999;24:142–145. - PubMed
    1. Brehm A, Miska E A, McCance D J, Reid J L, Bannister A J, Kouzarides T. Retinoblastoma protein recruits histone deacetylase to repress transcription. Nature. 1998;391:597–601. - PubMed
    1. Cairns C A, White R J. p53 is a general repressor of RNA polymerase III transcription. EMBO J. 1998;17:3112–3123. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources