FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091239660> ?p ?o ?g. }

• W2091239660 endingPage "20123" @default.
• W2091239660 startingPage "20116" @default.
• W2091239660 abstract "The SWI/SNF chromatin remodeling complex plays a critical role in the coordination of gene expression with physiological stimuli. The synthetic enzymes ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase are coordinately regulated to ensure appropriate deoxyribonucleotide triphosphate levels. Particularly, these enzymes are actively repressed as cells exit the cell cycle through the action of E2F transcription factors and the retinoblastoma tumor suppressor/p107/p130 family of pocket proteins. This process is found to be highly dependent on SWI/SNF activity as cells deficient in BRG-1 and Brm subunits fail to repress these genes with activation of pocket proteins, and this deficit in repression can be complemented, via the ectopic expression of BRG-1. The failure to repress transcription does not involve a blockade in the association of E2F or pocket proteins p107 and p130 with promoter elements. Rather, the deficit in repression is due to a failure to mediate histone deacetylation of ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase promoters in the absence of SWI/SNF activity. The basis for this is found to be a failure to recruit mSin3B and histone deacetylase proteins to promoters. Thus, the coordinate repression of deoxyribonucleotide triphosphate metabolic enzymes is dependent on the action of SWI/SNF in facilitating the assembly of repressor complexes at the promoter. The SWI/SNF chromatin remodeling complex plays a critical role in the coordination of gene expression with physiological stimuli. The synthetic enzymes ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase are coordinately regulated to ensure appropriate deoxyribonucleotide triphosphate levels. Particularly, these enzymes are actively repressed as cells exit the cell cycle through the action of E2F transcription factors and the retinoblastoma tumor suppressor/p107/p130 family of pocket proteins. This process is found to be highly dependent on SWI/SNF activity as cells deficient in BRG-1 and Brm subunits fail to repress these genes with activation of pocket proteins, and this deficit in repression can be complemented, via the ectopic expression of BRG-1. The failure to repress transcription does not involve a blockade in the association of E2F or pocket proteins p107 and p130 with promoter elements. Rather, the deficit in repression is due to a failure to mediate histone deacetylation of ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase promoters in the absence of SWI/SNF activity. The basis for this is found to be a failure to recruit mSin3B and histone deacetylase proteins to promoters. Thus, the coordinate repression of deoxyribonucleotide triphosphate metabolic enzymes is dependent on the action of SWI/SNF in facilitating the assembly of repressor complexes at the promoter. SWI/SNF is a multisubunit complex that regulates transcription through its ability to remodel chromatin (1Imbalzano A.N. Kwon H. Green M.R. Kingston R.E. Nature. 1994; 370: 481-485Crossref PubMed Scopus (520) Google Scholar, 2Khavari P.A. Peterson C.L. Tamkun J.W. Mendel D.B. Crabtree G.R. Nature. 1993; 366: 170-174Crossref PubMed Scopus (533) Google Scholar, 3Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 4Peterson C.L. Workman J.L. Curr. Opin. Genet. Dev. 2000; 10: 187-192Crossref PubMed Scopus (381) Google Scholar). This complex contains either BRG-1 or Brm as the central ATPase. Additionally, the complex is composed of accessory proteins termed BRG-1-associated factors (5Wang W. Xue Y. Zhou S. Kuo A. Cairns B.R. Crabtree G.R. Genes Dev. 1996; 10: 2117-2130Crossref PubMed Scopus (565) Google Scholar). Together, these subunits form a 2-MDa complex that utilizes the energy from ATP hydrolysis to catalyze changes in chromatin structure. Given this central role for SWI/SNF in transcriptional control, the complex is associated with multiple biological processes including differentiation, hormonal signaling, and cell cycle control. The SWI/SNF complex is important for modulating transcriptional programs related to cell cycle control, and aberrations in SWI/SNF subunits are progressively associated with tumorigenesis (6Hendricks K.B. Shanahan F. Lees E. Mol. Cell Biol. 2004; 24: 362-376Crossref PubMed Scopus (134) Google Scholar, 7Reisman D.N. Sciarrotta J. Wang W. Funkhouser W.K. Weissman B.E. Cancer Res. 2003; 63: 560-566PubMed Google Scholar). Specifically, SNF5 is a tumor suppressor involved in rhabdoid tumors (8Imbalzano A.N. Jones S.N. Cancer Cell. 2005; 7: 294-295Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 9Klochendler-Yeivin A. Fiette L. Barra J. Muchardt C. Babinet C. Yaniv M. EMBO Rep. 2000; 1: 500-506Crossref PubMed Scopus (331) Google Scholar, 10Roberts C.W. Galusha S.A. McMenamin M.E. Fletcher C.D. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13796-13800Crossref PubMed Scopus (332) Google Scholar), while sporadic loss of multiple other subunits is observed in a litany of tumor types (11Wang L. Baiocchi R.A. Pal S. Mosialos G. Caligiuri M. Sif S. Mol. Cell Biol. 2005; 25: 7953-7965Crossref PubMed Scopus (64) Google Scholar). In general, the loss of these factors has been implicated in deregulation of target genes associated with cell cycle control, particularly those regulated by the RB/E2F signaling axis (12Bosco E.E. Wang Y. Xu H. Zilfou J.T. Knudsen K.E. Aronow B.J. Lowe S.W. Knudsen E.S. J. Clin. Investig. 2007; 117: 218-228Crossref PubMed Scopus (155) Google Scholar, 13Hallstrom T.C. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10848-10853Crossref PubMed Scopus (133) Google Scholar, 14Mayhew C.N. Perkin L.M. Zhang X. Sage J. Jacks T. Knudsen E.S. Oncogene. 2004; 23: 4107-4120Crossref PubMed Scopus (38) Google Scholar).The retinoblastoma tumor suppressor (RB) 2The abbreviations used are: RB, retinoblastoma tumor suppressor; dNTP, deoxyribonucleotide triphosphate; RNRII, ribonucleotide reductase subunit II; TS, thymidylate synthase; DHFR, dihydrofolate reductase; GFP, green fluorescent protein; HDAC, histone deacetylase; TSA, trichostatin A; ChIP, chromatin immunoprecipitation. 2The abbreviations used are: RB, retinoblastoma tumor suppressor; dNTP, deoxyribonucleotide triphosphate; RNRII, ribonucleotide reductase subunit II; TS, thymidylate synthase; DHFR, dihydrofolate reductase; GFP, green fluorescent protein; HDAC, histone deacetylase; TSA, trichostatin A; ChIP, chromatin immunoprecipitation. was identified based on loss in the pediatric eye tumor of the same name (15Bartek J. Bartkova J. Lukas J. Exp. Cell Res. 1997; 237: 1-6Crossref PubMed Scopus (229) Google Scholar, 16Bartkova J. Lukas J. Bartek J. Prog. Cell Cycle Res. 1997; 3: 211-220Crossref PubMed Scopus (79) Google Scholar, 17Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4945) Google Scholar, 18Wang J.Y. Knudsen E.S. Welch P.J. Adv. Cancer Res. 1994; 64: 25-85Crossref PubMed Google Scholar). Subsequently, the gene product has been extensively analyzed and demonstrated to function as a transcriptional modulator. Specifically, RB and related pocket proteins p107/p130 bind to members of the E2F family of transcription factors and mediate transcriptional repression (19Cam H. Dynlacht B.D. Cancer Cell. 2003; 3: 311-316Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 20Cobrinik D. Oncogene. 2005; 24: 2796-2809Crossref PubMed Scopus (486) Google Scholar, 21Dimova D.K. Dyson N.J. Oncogene. 2005; 24: 2810-2826Crossref PubMed Scopus (584) Google Scholar, 22Frolov M.V. Dyson N.J. J. Cell Sci. 2004; 117: 2173-2181Crossref PubMed Scopus (330) Google Scholar, 23Morris E.J. Dyson N.J. Adv. Cancer Res. 2001; 82: 1-54Crossref PubMed Scopus (296) Google Scholar). These effects on transcription are mediated by the co-recruitment of a myriad of co-repressors to E2F-regulated promoters (24Rastogi S. Joshi B. Dasgupta P. Morris M. Wright K. Chellappan S. Mol. Cell Biol. 2006; 26: 4161-4171Crossref PubMed Scopus (79) Google Scholar, 25Taylor-Harding B. Binne U.K. Korenjak M. Brehm A. Dyson N.J. Mol. Cell Biol. 2004; 24: 9124-9136Crossref PubMed Scopus (56) Google Scholar). In this context, SWI/SNF activity is critically important, and deficiency of SWI/SNF activity has been associated with deregulated E2F-dependent gene expression (26Nagl Jr., N.G. Zweitzig D.R. Thimmapaya B. Beck Jr., G.R. Moran E. Cancer Res. 2006; 66: 1289-1293Crossref PubMed Scopus (97) Google Scholar).A critical gene expression program involved in cell cycle control is the regulation of dNTP synthetic enzymes. As cells progress into S-phase there is a coordinated stimulation in the levels of ribonucleotide reductase subunit II (RNRII), thymidylate synthase (TS), and dihydrofolate reductase (DHFR). It is believed that the up-regulation of such synthetic enzymes is important for expanding the pool of dNTPs for DNA synthesis (27Angus S.P. Wheeler L.J. Ranmal S.A. Zhang X. Markey M.P. Mathews C.K. Knudsen E.S. J. Biol. Chem. 2002; 277: 44376-44384Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 28Hakansson P. Hofer A. Thelander L. J. Biol. Chem. 2006; 281: 7834-7841Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Consistent with this overall idea, multiple DNA tumor viruses harbor oncoproteins that up-regulate the same dNTP metabolic enzymes, presumably to promote viral replication (29Bebenek K. Roberts J.D. Kunkel T.A. J. Biol. Chem. 1992; 267: 3589-3596Abstract Full Text PDF PubMed Google Scholar). Importantly, these oncoproteins disrupt the function of RB, and it is known that expression of each of these genes is regulated via the E2F family of transcription factors. Conversely, as cells exit cell cycle these genes are repressed by the action of RB and related proteins. Here we define the mechanism through which SWI/SNF participates in the regulation of RNRII, TS, and DHFR in the context of RB-mediated repression.EXPERIMENTAL PROCEDURESCell Culture, Plasmids, Infections, and Transfections—SW13 and TSUPr-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units of penicillin-streptomycin, 2 mml-glutamine at 37 °C in 5% CO2. Plasmids encoding β-galactosidase, p16ink4a, CMV-Neobam, and BRG-1 have been previously described (30Strobeck M.W. Fribourg A.F. Puga A. Knudsen E.S. Oncogene. 2000; 19: 1857-1867Crossref PubMed Scopus (66) Google Scholar, 31Strobeck M.W. Knudsen K.E. Fribourg A.F. DeCristofaro M.F. Weissman B.E. Imbalzano A.N. Knudsen E.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7748-7753Crossref PubMed Scopus (209) Google Scholar). The reporter constructs RNRII-luc, TS-luc, and DHFR-luc have been previously described (30Strobeck M.W. Fribourg A.F. Puga A. Knudsen E.S. Oncogene. 2000; 19: 1857-1867Crossref PubMed Scopus (66) Google Scholar, 31Strobeck M.W. Knudsen K.E. Fribourg A.F. DeCristofaro M.F. Weissman B.E. Imbalzano A.N. Knudsen E.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7748-7753Crossref PubMed Scopus (209) Google Scholar).Adenoviruses encoding GFP, p16ink4a, and BRG-1 have been previously described (32Angus S.P. Fribourg A.F. Markey M.P. Williams S.L. Horn H.F. DeGregori J. Kowalik T.F. Fukasawa K. Knudsen E.S. Exp. Cell Res. 2002; 276: 201-213Crossref PubMed Scopus (34) Google Scholar, 33Ni Z. Karaskov E. Yu T. Callaghan S.M. Der S. Park D.S. Xu Z. Pattenden S.G. Bremner R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14611-14616Crossref PubMed Scopus (72) Google Scholar). Infections were performed at a multiplicity of infection of 50–100 for ∼95–100% infection efficiency after 24–36 h as determined by GFP fluorescence.Reporter Assays, Immunoblotting, Reverse Transcriptase PCR, and Quantitative PCR—Immunoblotting was performed using standard techniques. The following antibodies were used, purified mouse anti-human Rb (catalogue number 554136; BD Biosciences), p107 (SC-318; Santa Cruz Biotechnology), p130 (SC-317; Santa Cruz Biotechnology), RNRII (SC-10848), TS (gift from Masakazu Fukushima), DHFR (610696; BD Transduction Laboratories), p16ink4a (SC-759; Santa Cruz), E2F4 (SC-1082; Santa Cruz), Lamin B (SC-6217; Santa Cruz). All of the immunoblots were repeated at least two times with independent samples.The reporter assays were performed as described previously (34Siddiqui H. Solomon D.A. Gunawardena R.W. Wang Y. Knudsen E.S. Mol. Cell Biol. 2003; 23: 7719-7731Crossref PubMed Scopus (69) Google Scholar). The data reflect the average of at least three independent experiments.Reverse Transcriptase PCR was performed as described previously (34Siddiqui H. Solomon D.A. Gunawardena R.W. Wang Y. Knudsen E.S. Mol. Cell Biol. 2003; 23: 7719-7731Crossref PubMed Scopus (69) Google Scholar). The following primer pairs were utilized: human RNRII 5′-CCT CTC CAA GGA CAT TCA GC-3′ and 5′-GGC AAT TTG GAA GCC ATA GA-3′, human TS 5′-TTC AGG ACA GGG AGT TGA CC-3′ and 5′-CAT GTC TCC CGA TCT CTG GT-3′, human DHFR 5′-GGT TCG CTA AAC TGC ATC GT-3′ and 5′-TGA GCT CCT TGT GGA GGT TC-3′, human glyceraldehyde-3-phosphate dehydrogenase 5′-TGG AAA TCC CAT CAC CAT CT-3′ and 5′-TTC ACA CCC ATG ACG AAC AT-3′. The experiments were performed at least three times, and representative data are shown. Reverse transcriptase PCR was also performed on RNA extracted from TSUPr-1 cells that had been treated with 400 nm histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (Sigma) for 18–24 h in the presence of adenovirus encoding GFP and p16ink4a.Real-time PCR—Real-time PCR was performed using SYBR Green PCR master mix (Applied Biosystem). Absolute DNA quantities were measured by using the Applied Biosystem Fast Real-time PCR, and data were normalized to account for variation in inputs to determine the relative quantities. The following primers were used to amplify regions of the human RNRII, 5′-GGG AGA TTT AAA GGC TGC TGG AGT GA-3′ and 5′-ACA CGG AGG GAG AGC ATA GTG GA-3′, TS5′-TAA GAC TCT CAG CTG TGG CCC TG-3′ and 5′-ACG GAG GCA GGC GAA GT-3′, DHFR 5′-TCG CCT GCA CAA ATA GGG ACG A-3′ and 5′-ACG ATG CAG TTT AGC GAA CCA ACC-3′. The experiments were repeated three times with independent samples, with the following conditions: 95 °C, 10 min; 59 °C, 1 min; 60 °C, 1 min, for 40 cycles.Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed as previously described (34Siddiqui H. Solomon D.A. Gunawardena R.W. Wang Y. Knudsen E.S. Mol. Cell Biol. 2003; 23: 7719-7731Crossref PubMed Scopus (69) Google Scholar). The following antibodies were utilized for ChIP: p107 (SC-318; Santa Cruz Biotechnology), p130 (SC-317; Santa Cruz), E2F4 (SC-1082; Santa Cruz), anti-HDAC1 (05–614; Upstate Biotechnology, Inc.), Brm (15597; Abcam), acetylated histone H4 (06–866; Upstate Biotechnology, Inc.), Dbf4 (SC-11354; Santa Cruz), and mSin3B (SC-768; Santa Cruz). The following primers were used to amplify regions of the human RNRII 5′-GAG GCA TGC ACA GCC AAT-3′ and 5′-GAC ACG GAG GGA GAG CAT AG-3′, TS5′-TCC GTT CTG TGC CAC ACC-3′ and 5′-TGG ATC TGC CCC CAG GTA CT-3′, DHFR 5′-ACA TGG CAG GGC AAG GAT-3′ and 5′-CCC GCT CGT TAC AGC AGA-3′. The ChIP assays were repeated twice with independent samples.RESULTSThe RB Pathway Regulates RNRII, TS, and DHFR Gene Expression in a SWI/SNF-dependent Manner—The dNTP metabolic genes RNRII, TS, and DHFR are tightly regulated to ensure the appropriate coordination of dNTP production with cell cycle control. To define the mechanisms through which this regulation occurs, we challenged the response of such enzymes to activation of the RB pathway. For these analyses we initially utilized the SW13 cell line, which is deficient in BRG-1 and Brm ATPase requisite for SWI/SNF activity, and TSUPr-1, which is proficient in Brm (35Strobeck M.W. Reisman D.N. Gunawardena R.W. Betz B.L. Angus S.P. Knudsen K.E. Kowalik T.F. Weissman B.E. Knudsen E.S. J. Biol. Chem. 2002; 277: 4782-4789Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). To activate the endogenous RB pathway, SW13 and TSUPr-1 cells were infected with adenoviruses encoding either GFP (control) or p16ink4a. Expression of p16ink4a inhibits cell cycle progression by disrupting the cyclinD-cdk4 kinase complex, resulting in the dephosphorylation/activation of RB as well as related pocket proteins p107 and p130 (17Sherr C.J. Science. 1996; 274: 1672-1677Crossref PubMed Scopus (4945) Google Scholar, 36Knudsen E.S. Wang J.Y. J. Biol. Chem. 1996; 271: 8313-8320Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). As expected, overexpression of p16ink4a led to the dephosphorylation of RB, p107, and p130 in both cell types (Fig. 1A). It should be noted that the overall levels of p107 and pRB are also decreased, which is consistent with previously published models of p16ink4a-mediated cell cycle arrest (32Angus S.P. Fribourg A.F. Markey M.P. Williams S.L. Horn H.F. DeGregori J. Kowalik T.F. Fukasawa K. Knudsen E.S. Exp. Cell Res. 2002; 276: 201-213Crossref PubMed Scopus (34) Google Scholar, 37Alevizopoulos K. Vlach J. Hennecke S. Amati B. EMBO J. 1997; 16: 5322-5333Crossref PubMed Scopus (192) Google Scholar). The expression of p16ink4a resulted in highly significant diminution of the RNA levels of RNRII, TS, and DHFR in TSUPr-1 cells (Fig. 1B, compare lanes 3 and 4). In contrast, we observed no attenuation of endogenous RNA levels of RNRII, TS, and DHFR in SW13 cells infected with p16ink4a, even though the pocket proteins were amply dephosphorylated (Fig. 1B, compare lanes 1 and 2). These observations were further strengthened by analyses of RNRII, TS, and DHFR protein levels in SW13 and TSUPr-1 cells. Consistent with RNA analyses, RNRII, TS, and DHFR protein levels were significantly attenuated in TSUPr-1 cells in the presence of p16ink4a (Fig. 1C, lanes 3 and 4), in contrast to SW13 cells (Fig. 1C, lanes 1 and 2). These results indicate that RNRII, TS, and DHFR expressions are regulated by the RB pathway in a manner that is dependent on SWI/SNF activity.Having confirmed that RNRII, TS, and DHFR genes are regulated via the RB pathway, the specific impact on their respective promoters was investigated. SW13 and TSUPr-1 cells were cotransfected with RNRII, TS, and DHFR promoter plasmid, respectively, and either vector control or p16ink4a-encoding plasmids. Overexpression of p16ink4a significantly repressed the promoter activity of RNRII, TS, and DHFR in SWI/SNF-proficient TSUPr-1 cells. In contrast, p16ink4a failed to repress promoter activity in SWI/SNF-deficient SW13 cells (Fig. 2A). To functionally demonstrate the requirement for SWI/SNF activity, BRG-1 expression was restored in SW13 cells in combination with p16ink4a, and RNRII, TS, and DHFR promoter activity was analyzed through reporter analysis. Similar to our earlier observation, p16ink4a alone failed to repress RNRII, TS, and DHFR promoter activity. However, cotransfection of BRG-1 and p16ink4a significantly repressed these promoters in SWI/SNF-deficient SW13 cells (Fig. 2B). Consistent with reporter assays, we observed attenuation of endogenous RNA (Fig. 2C) and protein levels (Fig. 2D) of RNRII, TS, and DHFR upon restoring p16ink4a and BRG-1 in SWI/SNF-deficient SW13 cells. In summary, these data demonstrate that SWI/SNF activity is required for RB-mediated repression of RNRII, TS, and DHFR promoters and attenuation of its RNA and protein levels.FIGURE 2SWI/SNF is required for RB-mediated repression of RNRII, TS, and DHFR expression. A, SW13 and TSUPr-1 cells were cotransfected with cytomegalovirus β-galactosidase and RNR11-Luc, TS-Luc, and DHFR-Luc reporter plasmids and either vector or p16ink4a expression plasmids. Relative luciferase activity was normalized to β-galactosidase activity for transfection efficiency, and the vector control was set to 100. B, SW13 cells were cotransfected with cytomegalovirus β-galalactosidase, RNRII-Luc, TS-Luc, or DHFR-Luc reporter plasmids and either vector, p16ink4a, or BRG-1 expression plasmids. The relative luciferase activity was normalized to β-galactosidase activity for transfection efficiency, and vector control was set to 100. Error bars represent average relative luciferase activity and S.D. Results are representative of three independent experiments. C, SW13 cells were infected with either GFP (lane 1), p16ink4a (lane 2), BRG-1 (lane 3), or p16ink4a and BRG-1 (lane 4)-encoding adenoviruses. Total RNA was extracted 36 h post-infection and subjected to linear reverse transcriptase PCR amplification with primers specific for the indicated genes. D, SW13 cells were infected as in panel C. Cells were harvested 36 h post-infection, total protein was resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SW1/SNF Is Dispensable for E2F4 and RB-related Pocket Protein Association at RNRII, TS, and DHFR Promoters—The inability of the RB pathway to repress RNRII, TS, and DHFR promoters in the absence of SWI/SNF activity could be a consequence of multiple levels of dysfunction in repression. Initially the requirement of SWI/SNF for E2F family members to access the respective promoters was determined. Specifically, the ability of E2F proteins to bind the RNRII, TS, and DHFR promoters was evaluated by chromatin immunoprecipitation assays (38Wells J. Boyd K.E. Fry C.J. Bartley S.M. Farnham P.J. Mol. Cell Biol. 2000; 20: 5797-5807Crossref PubMed Scopus (207) Google Scholar). The E2F family of transcription factors consists of at least seven members (E2F1–7), of which E2F4 and E2F5 are transcriptional repressors (39Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1962) Google Scholar, 40Gaubatz S. Lindeman G.J. Ishida S. Jakoi L. Nevins J.R. Livingston D.M. Rempel R.E. Mol. Cell. 2000; 6: 729-735Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 41Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 11-20Crossref PubMed Scopus (958) Google Scholar). SW13 and TSUPr-1 cells were infected with either GFP or p16ink4a-encoding adenoviruses, and protein-DNA complexes were cross-linked with formaldehyde and immunoprecipitated with antibodies specific for E2F4 and Dbf4 (nonspecific control). Approximately equal recruitment of E2F4 at RNRII, TS, and DHFR promoters was observed in the presence or absence of SWI/SNF (Fig. 3A). Thus, SWI/SNF activity is apparently not required for the recruitment of E2F4 to these promoters.FIGURE 3SWI/SNF is dispensable for RB-related pocket proteins and E2F4 association at RNRII, TS, and DHFR promoters. All experiments utilized SW13 (lanes 1 and 2) and TSUPr-1 (lanes 3 and 4) cells infected with either GFP (lanes 1 and 3) or p16ink4a (lanes 2 and 4)-encoding adenoviruses. ChIP assays were performed with antibodies for E2F4 and Dbf4 (A) (nonspecific), p107 and Dbf4 (B), p130 and Dbf4 (C), and Brm and Dbf4 (D). Input and immunoprecipitated DNA were amplified by PCR with primers specific for the RNRII, TS, and DHFR promoters.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As the subsequent step in transcriptional repression, pocket proteins are believed to be recruited to promoters to facilitate the transcriptional repression. For these analyses, SW13 and TSUPr-1 cells were infected with either GFP or p16ink4a to activate endogenous pocket proteins, and recruitment of p107 and p130 to the promoters was then determined by chromatin immunoprecipitation. We observed differential recruitment of p107 and p130 to the respective promoters. Specifically, there was a general trend toward loss of p107 with p16ink4a expression (Fig. 3B), which is countered by enhanced p130 recruitment to the promoters (Fig. 3C). These findings are consistent with the differential involvement of p107 and p130 in promoter regulation in quiescent versus proliferative cells (42Balciunaite E. Spektor A. Lents N.H. Cam H. Te Riele H. Scime A. Rudnicki M.A. Young R. Dynlacht B.D. Mol. Cell Biol. 2005; 25: 8166-8178Crossref PubMed Scopus (103) Google Scholar, 43Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar). Importantly, this differential recruitment occurred in both SWI/SNF-proficient and -deficient cells, indicating that p107/p130 recruitment to chromatin is SWI/SNF-independent.Lastly, SWI/SNF ATPase recruitment to the promoters was determined. As shown in Fig. 3D, Brm protein present in TSUPr-1 cells is associated with all of the promoters analyzed. As expected, Brm was absent at the promoters in SW13 cells, consistent with the documented lack of Brm expression in this model (35Strobeck M.W. Reisman D.N. Gunawardena R.W. Betz B.L. Angus S.P. Knudsen K.E. Kowalik T.F. Weissman B.E. Knudsen E.S. J. Biol. Chem. 2002; 277: 4782-4789Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Strikingly, Brm is present at the promoters of RNRII, DHFR, and TS irrespective of p16ink4a expression (Fig. 3D, lanes 3 and 4). This finding suggests that SWI/SNF complexes are contributing to the physiological regulation of the promoters both during unperturbed cell cycle progression and during active repression and are not required for the recruitment of E2F or pocket proteins to the promoters.RB-induced Deacetylation at RNRII, TS, and DHFR Promoters Is Mediated by HDACs in the Presence of SWI/SNF—Pocket proteins are believed to repress transcription by facilitating alterations in chromatin structure that are mediated via the recruitment of additional co-repressors (10Roberts C.W. Galusha S.A. McMenamin M.E. Fletcher C.D. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13796-13800Crossref PubMed Scopus (332) Google Scholar, 44Angus S.P. Solomon D.A. Kuschel L. Hennigan R.F. Knudsen E.S. Mol. Cell Biol. 2003; 23: 8172-8188Crossref PubMed Scopus (29) Google Scholar, 45Ferreira R. Magnaghi-Jaulin L. Robin P. Harel-Bellan A. Trouche D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10493-10498Crossref PubMed Scopus (224) Google Scholar, 46Gunawardena R.W. Siddiqui H. Solomon D.A. Mayhew C.N. Held J. Angus S.P. Knudsen E.S. J. Biol. Chem. 2004; 279: 29278-29285Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, the extent to which these multiple chromatin-modifying enzymes act in concert to mediate transcription is not well documented. To initially investigate chromatin modifications and corresponding dependence on SWI/SNF for this process, we examined histone deacetylation. Chromatin immunoprecipitation assays were performed utilizing anti-acetyl histone H4 antibody (Fig. 4A). As expected, infection with p16ink4a resulted in significant histone deacetylation at the RNRII, TS, and DHFR promoters in SWI/SNF-proficient TSUPr-1 cells. In contrast, p16ink4a infection failed to induce histone H4 deacetylation at the promoters in SWI/SNF-deficient SW13 cells. Thus, these data suggest that whereas recruitment of E2F and pocket proteins to these promoters is SWI/SNF-independent, the histone deacetylation of these promoters is SWI/SNF-dependent.FIGURE 4RB-induced deacetylation at RNRII, TS, and DHFR promoters is mediated by HDACs and dependent on SWI/SNF. A, SW13 and TSUPr-1 cells were infected with GFP or p16ink4a-encoding adenoviruses. ChIP assays were performed with antibody specific for acetylated histone H4. Real-time PCR was performed as described under “Experimental Procedures,” and the GFP control was set to 100. Error bars represent the mean ± S.D.; n = 3. B, TSUPr-1 cells were infected with GFP (lane 1), GFP+ TSA (lane 2), p16ink4a (lane 3), or p16ink4a and TSA (lane 4) for 18–24 h. Total RNA was extracted and reversed-transcribed into cDNA. This cDNA was subjected to linear PCR amplification with specific primers for the indicated genes. C, TSUPr-1 cells were infected and treated with TSA as indicated in panel B. Total protein was resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To address the functional role of histone deacetylation, we suppressed histone deacetylase activity using the pharmacological inhibitor of HDAC, TSA. For these analyses, SWI/SNF-proficient TSUPr-1 cells were infected with adenoviruses encoding GFP or p16ink4a in the presence or absence of TSA for 18–24 h. The administration of TSA alone did not have a significant effect on target gene expression. However, TSA was able to partially reverse the transcriptional repression induced by p16ink4a, as observed at both the RNA (Fig. 4B) and protein levels (Fig. 4C). These results indicate that HDAC activity contributes to the SWI/SNF-mediated transcriptional repression of these target promoters.Recruitment of HDAC1 and mSin3B at the RNRII, TS, and DHFR Promoters Is Dependent on SWI/SNF Activity—Multiple HDAC complexes have been implicated in RB-mediated transcriptional repression, many of which contain the Sin3 subunits (47Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (955) Google Scholar, 48Lai A. Kennedy B.K. Barbie D.A. Bertos N.R. Yang X.J. Theberge M.C. Tsai S.C. Seto E. Zhang Y. Kuzmichev A. Lane W.S. Reinberg D. Harlow E. Branton P.E. Mol. Cell Biol. 2001; 21: 2918-2932Crossref PubMed Scopus (164) Google Scholar, 49Rayman J.B. Takahashi Y. Indjeian V.B. Dannenberg J.H. Catchpole S. Watson R.J. te Riele H. Dynlacht B.D. Genes Dev. 2002; 16: 933-947Crossref PubMed Scop" @default.
• W2091239660 created "2016-06-24" @default.
• W2091239660 creator A5008791492 @default.
• W2091239660 creator A5020501989 @default.
• W2091239660 creator A5029801038 @default.
• W2091239660 creator A5070465876 @default.
• W2091239660 date "2007-07-01" @default.
• W2091239660 modified "2023-10-14" @default.
• W2091239660 title "SWI/SNF Activity Is Required for the Repression of Deoxyribonucleotide Triphosphate Metabolic Enzymes via the Recruitment of mSin3B" @default.
• W2091239660 cites W1486122524 @default.
• W2091239660 cites W1520197177 @default.
• W2091239660 cites W1600943063 @default.
• W2091239660 cites W1709607181 @default.
• W2091239660 cites W1964318436 @default.
• W2091239660 cites W1972164727 @default.
• W2091239660 cites W1972455404 @default.
• W2091239660 cites W1976903076 @default.
• W2091239660 cites W1985626007 @default.
• W2091239660 cites W1987428672 @default.
• W2091239660 cites W1997466838 @default.
• W2091239660 cites W2003239757 @default.
• W2091239660 cites W2004415585 @default.
• W2091239660 cites W2007012438 @default.
• W2091239660 cites W2009726317 @default.
• W2091239660 cites W2012914676 @default.
• W2091239660 cites W2016475398 @default.
• W2091239660 cites W2017203551 @default.
• W2091239660 cites W2025721764 @default.
• W2091239660 cites W2026368142 @default.
• W2091239660 cites W2029080338 @default.
• W2091239660 cites W2029925836 @default.
• W2091239660 cites W2032645409 @default.
• W2091239660 cites W2034277114 @default.
• W2091239660 cites W2038649631 @default.
• W2091239660 cites W2046841127 @default.
• W2091239660 cites W2047509634 @default.
• W2091239660 cites W2052605603 @default.
• W2091239660 cites W2058886425 @default.
• W2091239660 cites W2060333115 @default.
• W2091239660 cites W2061304668 @default.
• W2091239660 cites W2065343028 @default.
• W2091239660 cites W2065906581 @default.
• W2091239660 cites W2066680166 @default.
• W2091239660 cites W2069023233 @default.
• W2091239660 cites W2070677152 @default.
• W2091239660 cites W2072139657 @default.
• W2091239660 cites W2075750487 @default.
• W2091239660 cites W2078111498 @default.
• W2091239660 cites W2085458327 @default.
• W2091239660 cites W2090842851 @default.
• W2091239660 cites W2093647324 @default.
• W2091239660 cites W2099848412 @default.
• W2091239660 cites W2102067723 @default.
• W2091239660 cites W2104285292 @default.
• W2091239660 cites W2104968595 @default.
• W2091239660 cites W2105706734 @default.
• W2091239660 cites W2107350929 @default.
• W2091239660 cites W2107627035 @default.
• W2091239660 cites W2116183942 @default.
• W2091239660 cites W2117273898 @default.
• W2091239660 cites W2117521220 @default.
• W2091239660 cites W2120871109 @default.
• W2091239660 cites W2126087647 @default.
• W2091239660 cites W2132641868 @default.
• W2091239660 cites W2133799137 @default.
• W2091239660 cites W2139477475 @default.
• W2091239660 cites W2145348333 @default.
• W2091239660 cites W2147131744 @default.
• W2091239660 cites W2147921995 @default.
• W2091239660 cites W2149706478 @default.
• W2091239660 cites W2151155001 @default.
• W2091239660 cites W2152616419 @default.
• W2091239660 cites W2159058669 @default.
• W2091239660 cites W2161225294 @default.
• W2091239660 cites W2167000545 @default.
• W2091239660 cites W2171756101 @default.
• W2091239660 cites W2335019470 @default.
• W2091239660 cites W2791873134 @default.
• W2091239660 cites W1974472930 @default.
• W2091239660 doi "https://doi.org/10.1074/jbc.m701406200" @default.
• W2091239660 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17510060" @default.
• W2091239660 hasPublicationYear "2007" @default.
• W2091239660 type Work @default.
• W2091239660 sameAs 2091239660 @default.
• W2091239660 citedByCount "16" @default.
• W2091239660 countsByYear W20912396602012 @default.
• W2091239660 countsByYear W20912396602013 @default.
• W2091239660 countsByYear W20912396602014 @default.
• W2091239660 countsByYear W20912396602016 @default.
• W2091239660 countsByYear W20912396602017 @default.
• W2091239660 countsByYear W20912396602018 @default.
• W2091239660 crossrefType "journal-article" @default.
• W2091239660 hasAuthorship W2091239660A5008791492 @default.
• W2091239660 hasAuthorship W2091239660A5020501989 @default.
• W2091239660 hasAuthorship W2091239660A5029801038 @default.
• W2091239660 hasAuthorship W2091239660A5070465876 @default.
• W2091239660 hasConcept C104317684 @default.
• W2091239660 hasConcept C150194340 @default.

• next