The Bromodomain Protein Brd4 Stimulates G1 Gene Transcription and Promotes Progression to S Phase

Brd4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. Brd4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of Brd4 in cell growth and transcription using a small hairpin (sh) RNA approach. The Brd4 shRNA vector stably knocked down Brd4 protein expression by ∼90% in NIH3T3 cells and mouse embryonic fibroblasts. Brd4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, Brd4 knockdown cells were arrested at G1, whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G1 in control cells, many of these G1 genes were not up-regulated in Brd4 knockdown cells. Reintroduction of Brd4 rescued expression of these G1 genes in Brd4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that Brd4 was recruited to the promoters of these G1 genes during G0-G1 progression. Furthermore, Brd4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. Brd4 recruitment was low to absent at genes not affected by Brd4 shRNA. The results indicate that Brd4 stimulates G1 gene expression by binding to multiple G1 gene promoters in a cell cycle-dependent manner. Brd4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. Brd4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of Brd4 in cell growth and transcription using a small hairpin (sh) RNA approach. The Brd4 shRNA vector stably knocked down Brd4 protein expression by ∼90% in NIH3T3 cells and mouse embryonic fibroblasts. Brd4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, Brd4 knockdown cells were arrested at G1, whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G1 in control cells, many of these G1 genes were not up-regulated in Brd4 knockdown cells. Reintroduction of Brd4 rescued expression of these G1 genes in Brd4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that Brd4 was recruited to the promoters of these G1 genes during G0-G1 progression. Furthermore, Brd4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. Brd4 recruitment was low to absent at genes not affected by Brd4 shRNA. The results indicate that Brd4 stimulates G1 gene expression by binding to multiple G1 gene promoters in a cell cycle-dependent manner. Brd4 is a ubiquitously expressed 200-kDa nuclear protein that belongs to the BET family (1Jeanmougin F. Wurtz J.M. Le Douarin B. Chambon P. Losson R. Trends Biochem. Sci. 1997; 22: 151-153Abstract Full Text PDF PubMed Scopus (230) Google Scholar, 2Dey A. Ellenberg J. Farina A. Coleman A.E. Maruyama T. Sciortino S. Lippincott-Schwartz J. Ozato K. Mol. Cell. Biol. 2000; 20: 6537-6549Crossref PubMed Scopus (236) Google Scholar). Proteins of this family carry two tandem bromodomains through which they interact with acetylated histones (3Ladurner A.G. Inouye C. Jain R. Tjian R. Mol. Cell. 2003; 11: 365-376Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 4Matangkasombut O. Buratowski S. Mol. Cell. 2003; 11: 353-363Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 5Dey A. Chitsaz F. Abbasi A. Misteli T. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8758-8763Crossref PubMed Scopus (502) Google Scholar, 6Kanno T. Kanno Y. Siegel R.M. Jang M.K. Lenardo M.J. Ozato K. Mol. Cell. 2004; 13: 33-43Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Bromodomains are also present in other chromatin-binding proteins such as histone acetylases and chromatin remodeling factors. They also bind to acetylated histones and are involved in transcriptional regulation of many genes. Recent structural analysis indicates that the bromodomain of Brd2, a factor closely related to Brd4, forms a dimmer to bind to acetyl residues of the histone H4 tails (7Nakamura Y. Umehara T. Nakano K. Jang M.K. Shirouzu M. Morita S. Uda-Tochio H. Hamana H. Terada T. Adachi N. Matsumoto T. Tanaka A. Horikoshi M. Ozato K. Padmanabhan B. Yokoyama S. J. Biol. Chem. 2007; 282: 4193-4201Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Binding of Brd4 and Brd2 to acetylated chromatin persists even during mitosis as well as meiosis when chromatin is highly condensed and transcription is interrupted (2Dey A. Ellenberg J. Farina A. Coleman A.E. Maruyama T. Sciortino S. Lippincott-Schwartz J. Ozato K. Mol. Cell. Biol. 2000; 20: 6537-6549Crossref PubMed Scopus (236) Google Scholar, 5Dey A. Chitsaz F. Abbasi A. Misteli T. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8758-8763Crossref PubMed Scopus (502) Google Scholar, 6Kanno T. Kanno Y. Siegel R.M. Jang M.K. Lenardo M.J. Ozato K. Mol. Cell. 2004; 13: 33-43Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 8Nagashima T. Maruyama T. Furuya M. Kajitani T. Uchida H. Masuda H. Ono M. Arase T. Ozato K. Yoshimura Y. Mol. Hum. Reprod. 2007; 13: 141-148Crossref PubMed Scopus (25) Google Scholar). Evidence indicates that BET family proteins are multifunctional and regulate cell growth and transcription (3Ladurner A.G. Inouye C. Jain R. Tjian R. Mol. Cell. 2003; 11: 365-376Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 4Matangkasombut O. Buratowski S. Mol. Cell. 2003; 11: 353-363Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Denis G.V. Vaziri C. Guo N. Faller D.V. Cell Growth Differ. 2000; 11: 417-424PubMed Google Scholar, 10Crowley T.E. Kaine E.M. Yoshida M. Nandi A. Wolgemuth D.J. Mol. Endocrinol. 2002; 16: 1727-1737Crossref PubMed Scopus (43) Google Scholar). In line with this evidence, there are reports indicating that Brd4 is involved in cell growth regulation; Brd4-/- embryos fail to grow and die early at around the time of implantation (11Houzelstein D. Bullock S.L. Lynch D.E. Grigorieva E.F. Wilson V.A. Beddington R.S. Mol. Cell. Biol. 2002; 22: 3794-3802Crossref PubMed Scopus (228) Google Scholar). Similarly, Brd4-/- embryonic stem cells do not grow in culture (12Nishiyama A. Dey A. Miyazaki J. Ozato K. Mol. Biol. Cell. 2006; 17: 814-823Crossref PubMed Scopus (54) Google Scholar). Moreover, in some malignant cells, Brd4 is fused to the NUT gene, and the fusion protein exhibits a growth regulatory activity (13French C.A. Kutok J.L. Faquin W.C. Toretsky J.A. Antonescu C.R. Griffin C.A. Nose V. Vargas S.O. Moschovi M. Tzortzatou-Stathopoulou F. Miyoshi I. Perez-Atayde A.R. Aster J.C. Fletcher J.A. J. Clin. Oncol. 2004; 22: 4135-4139Crossref PubMed Scopus (309) Google Scholar, 14Haruki N. Kawaguchi K.S. Eichenberger S. Massion P.P. Gonzalez A. Gazdar A.F. Minna J.D. Carbone D.P. Dang T.P. J. Med. Genet. 2005; 42: 558-564Crossref PubMed Scopus (36) Google Scholar). In addition, overexpression of Brd4 in cultured cells is shown to alter their growth properties, in part due to the interaction of Brd4 with growth regulatory proteins such as RFC140 or Sipa1 (15Maruyama T. Farina A. Dey A. Cheong J. Bermudez V.P. Tamura T. Sciortino S. Shuman J. Hurwitz J. Ozato K. Mol. Cell. Biol. 2002; 22: 6509-6520Crossref PubMed Scopus (123) Google Scholar, 16Farina A. Hattori M. Qin J. Nakatani Y. Minato N. Ozato K. Mol. Cell. Biol. 2004; 24: 9059-9069Crossref PubMed Scopus (59) Google Scholar). The reports that Brd4 facilitates partition of Papillomavirus genomes during mitosis also support the idea that Brd4 plays a role in cell division (17You J. Croyle J.L. Nishimura A. Ozato K. Howley P.M. Cell. 2004; 117: 349-360Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 18Baxter M.K. McPhillips M.G. Ozato K. McBride A.A. J. Virol. 2005; 79: 4806-4818Crossref PubMed Scopus (103) Google Scholar). So far, however, the mechanism(s) by which Brd4 participates in cell growth regulation has remained elusive. With respect to transcriptional regulation, the yeast BET family protein Bdf1 is shown to be broadly involved in gene transcription (3Ladurner A.G. Inouye C. Jain R. Tjian R. Mol. Cell. 2003; 11: 365-376Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 4Matangkasombut O. Buratowski S. Mol. Cell. 2003; 11: 353-363Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Bdf1 is incorporated into the general transcription factor complex and regulates many yeast genes (4Matangkasombut O. Buratowski S. Mol. Cell. 2003; 11: 353-363Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). It also inhibits Sir2-mediated gene silencing (3Ladurner A.G. Inouye C. Jain R. Tjian R. Mol. Cell. 2003; 11: 365-376Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The mammalian Brd2 is also reported to interact with general transcription factors and takes part in transcription (10Crowley T.E. Kaine E.M. Yoshida M. Nandi A. Wolgemuth D.J. Mol. Endocrinol. 2002; 16: 1727-1737Crossref PubMed Scopus (43) Google Scholar). We have previously found that Brd4 complexes with the kinase active form of P-TEFb and enhances human immunodeficiency virus-1 long terminal repeat transcription (19Jang M.K. Mochizuki K. Zhou M. Jeong H.S. Brady J.N. Ozato K. Mol. Cell. 2005; 19: 523-534Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 20Yang Z. Yik J.H. Chen R. He N. Jang M.K. Ozato K. Zhou Q. Mol. Cell. 2005; 19: 535-545Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar). P-TEFb, composed of cyclin T and Cdk9, phosphorylates the C-terminal domain of RNA polymerase II (pol II) 9The abbreviations used are: RNA pol IIRNA polymerase IIshRNAsmall hairpin RNAChIPchromatin immunoprecipitationKDknockdownMEFmouse embryonic fibroblastsqRT-PCRquantitative reverse transcription PCRRbretinoblastomaCtrlcontrol. and contributes to transcriptional elongation (21Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar, 22Chao S.H. Price D.H. J. Biol. Chem. 2001; 276: 31793-31799Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Additionally, Brd4 interacts with components of the Mediator complexes that associates with pol II preinitiation complexes, further suggesting a role in transcription (19Jang M.K. Mochizuki K. Zhou M. Jeong H.S. Brady J.N. Ozato K. Mol. Cell. 2005; 19: 523-534Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar, 20Yang Z. Yik J.H. Chen R. He N. Jang M.K. Ozato K. Zhou Q. Mol. Cell. 2005; 19: 535-545Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 23Wu S.Y. Chiang C.M. J. Biol. Chem. 2007; 282: 13141-13145Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). Recently, Brd4 is shown to repress human Papillomavirus gene transcription by interacting with the viral transactivator E2 and blocking the recruitment of TFIID and pol II to the viral promoters (24Wu S.Y. Lee A.Y. Hou S.Y. Kemper J.K. Erdjument-Bromage H. Tempst P. Chiang C.M. Genes Dev. 2006; 20: 2383-2396Crossref PubMed Scopus (168) Google Scholar). Despite the evidence for regulating viral gene transcription, little is known about the role of Brd4 in the transcriptional regulation of cellular genes. Moreover, it has been unclear as to whether cell growth regulation by Brd4 is in any way coupled with its potential role in transcription. RNA polymerase II small hairpin RNA chromatin immunoprecipitation knockdown mouse embryonic fibroblasts quantitative reverse transcription PCR retinoblastoma control. To address the role of Brd4 in cell growth and transcription, we employed a shRNA approach and knocked down Brd4 expression in NIH3T3 cells and primary mouse embryonic fibroblasts (MEFs), non-transformed cells with a normal growth property. Our data indicate that Brd4 has a vital role in promoting cell cycle progression from G0 to G1 and entry into S in these cells. In microarray analysis, a large number of genes showed a marked increase in transcript expression during G0-G1 progression in control cells. However, this extensive G1 gene up-regulation was largely absent in Brd4 knockdown (KD) cells. Chromatin immunoprecipitation (ChIP) analysis showed that Brd4 was recruited to a number of G1 gene promoters, coinciding with up-regulation of G1 gene expression in control cells, but not in Brd4 KD cells. Furthermore, RNA pol II and Cdk9 were recruited to these G1 genes in a Brd4-dependent manner, suggesting that Brd4 facilitates productive transcription of multiple G1 genes. These results provide a mechanistic basis by which Brd4 regulates cell growth. Cell Culture, Brd4 shRNA, and Rescue Vectors—NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium with 10% donor serum. For synchronization, cells were incubated in 0. 5% donor serum for 60 h and then released in the complete media. MEFs were prepared from embryos of day 13 post-coitus and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Brd4 shRNA and control shRNA were cloned into the pSUPER retro vector (Oligoengine). The Brd4 shRNA sequence was from +604 to +623 relative to the transcription start site, GATCCCCGCATCAACTTCTCCGCAGATTCAAGAGATCTGCGGAGAAGTTGATGCTTTTTGGAAA. The control siRNA was a shuffled sequence of the above, GATCCCCATGCACGTGCACATATCCCTTCAAGAGAGGGATATGTGCACGTGCATTTTTTGGAAA. BOSC23 packaging cells were transfected with the above vectors, and viral supernatants were harvested 2 days later. NIH3T3 and MEFs were transduced with viral supernatants by spinoculation (2800 rpm, 28 °C, 2 h) in the presence of 4 μg/ml Polybrene (Sigma-Aldrich) and selected by puromycin (5 μg/ml) for 2 days. A retroviral rescue vector expressing a Brd4 mutant resistant to Brd4 shRNA inhibition was constructed from the pMSCVneo vector expressing wild type Brd4 (15Maruyama T. Farina A. Dey A. Cheong J. Bermudez V.P. Tamura T. Sciortino S. Shuman J. Hurwitz J. Ozato K. Mol. Cell. Biol. 2002; 22: 6509-6520Crossref PubMed Scopus (123) Google Scholar) using the oligomer GCTTCTACATCACCGCAGA that changed the Brd4 nucleotide sequence without changing the amino acid sequence (mutations are underlined). NIH3T3 cells were first transduced with the rescue vector (or empty vector) and selected by G418 for 4 days followed by the second transduction with the pSUPERretro vectors containing Brd4 shRNA as above. Microarray Analysis and Quantitative Real Time PCR (qRT-PCR)—Total RNA was extracted from cells using TRIzol (Invitrogen) and purified by the RNeasy kit (Qiagen), and cDNA was prepared using SuperScript II RNase H-Reverse (Invitrogen). The NIA 15K mouse cDNA microarray system was used (25Tanaka T.S. Jaradat S.A. Lim M.K. Kargul G.J. Wang X. Grahovac M.J. Pantano S. Sano Y. Piao Y. Nagaraja R. Doi H. Wood III, W.H. Becker K.G. Ko M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9127-9132Crossref PubMed Scopus (364) Google Scholar) (http://lgsun.grc.nia.nih.gov/cDNA/15k.html). cDNA from cells expressing control or Brd4 shRNA and those from reference RNA were labeled with Cy3 and Cy5, respectively, by reverse transcription using the MICROMAX direct cDNA microarray system (PerkinElmer Life Sciences). Twenty-five μg of labeled probes were added to the slides and incubated overnight at 65 °C. Slides were washed in 0.5× SSC (1× SSC 0.15 m NaCl and 0.015 m sodium citrate), 0.01% SDS and 0.06× SSC, 0.01% SDS and 0.06× SSC. Data were analyzed using the ScanArray Express (PerkinElmer Life Sciences). Spots showing signals lower than the average background of the entire Chip were eliminated. Normalization of Cy3 to Cy5 signals was carried out by the global median normalization. To minimize sample to sample variability, hybridization was carried out with duplicate slides, and spots with signals that lie outside the best-fit line were eliminated (26Kadota K. Miki R. Bono H. Shimizu K. Okazaki Y. Hayashizaki Y. Physiol. Genomics. 2001; 4: 183-188Crossref PubMed Scopus (54) Google Scholar). To reduce biological variability, three independently prepared RNA samples were analyzed, and the spots showing high variability were removed. Microarray signals were represented by a log2 ratio relative to signals by reference RNA. The principal component analysis and hierarchical clustering analysis were performed by the "Cluster" program (27Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13268) Google Scholar, 28Landgrebe J. Wurst W. Welzl G. Genome Biology. 2002; 3 (RESEARCH0019)Crossref PubMed Google Scholar). For qRT-PCR, amplification of sample cDNA was monitored with the SYBR green in a kit along with the ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. Transcript levels were normalized by 18 S rRNA levels. Primers used for qRT-PCR are in shown in supplemental Table S1. Flow Cytometry and Immunoblot—To monitor cell cycle profiles, cells were stained with propidium iodide and analyzed on FACSCalibur interfaced with the Cell Quest software (BD Biosciences) as described in Nishiyama et al. (12Nishiyama A. Dey A. Miyazaki J. Ozato K. Mol. Biol. Cell. 2006; 17: 814-823Crossref PubMed Scopus (54) Google Scholar). To detect apoptosis, cells were stained with annexin V-fluorescein isothiocyanate (BD Biosciences Pharmingen) according to the manufacturer's instructions. Total cell proteins were extracted by radioimmune precipitation assay buffer, and 40 μg of proteins were resolved on a 4-20% gradient SDS-polyacrylamide gel electrophoresis and immunoblotted with antibodies for cyclin D1, TATA box-binding protein (TBP) (Santa Cruz), cyclin D2, cyclin E, and phosphorylated Rb (Cell Signaling) by the standard procedures. Antibody for unphosphorylated Rb was from Pharmingen. Antibodies for Brd2 and Brd4 were described (5Dey A. Chitsaz F. Abbasi A. Misteli T. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8758-8763Crossref PubMed Scopus (502) Google Scholar). Acid extraction of total histones and immunoblot analysis were described (12Nishiyama A. Dey A. Miyazaki J. Ozato K. Mol. Biol. Cell. 2006; 17: 814-823Crossref PubMed Scopus (54) Google Scholar). ChIP Assay—Chromatin immunoprecipitation was performed using indicated antibodies essentially as described (29Dahl J.A. Collas P. Front. Biosci. 2007; 12: 4925-4931Crossref PubMed Scopus (40) Google Scholar). Briefly, control and Brd4 KD NIH3T3 cells (1 × 106) were cross-linked with 1% paraformaldehyde at 37 °C for 10 min and lysed in buffer containing 50 mm Tris HCl, pH 8.0, 10 mm EDTA, 1% SDS, and a protease inhibitor cocktail (Roche Applied Science). Chromatin was sheared by sonication to generate 200-1000-bp DNA fragments and diluted by 10-fold in radioimmune precipitation assay buffer (10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 0.5 mm EGTA, 1% Triton X, and 0.1% SDS, 0.1% sodium deoxycholate, 140 mm NaCl). Rabbit antibodies for Brd4 (prepared in this laboratory) (5Dey A. Chitsaz F. Abbasi A. Misteli T. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8758-8763Crossref PubMed Scopus (502) Google Scholar) or Cdk9 (Santa Cruz) or RNA pol II (Covance) or preimmune or normal rabbit IgG were incubated with protein A-coupled paramagnetic beads (Dynalbeads, Invitrogen). Chromatin preparations were then incubated with antibody-precoated magnetic beads above for 2 h to overnight at 4 °C. Immune complexes were washed in radioimmune precipitation assay buffer three times. Antibody bound chromatin was reverse-cross-linked, and DNA was purified by phenol-chloroform and ethanol. DNA was then subjected to quantitative PCR using the primer set compiled in supplemental Table S1. The percentage input was calculated as (2(CT input - CT IP sample)) × 100/CT input. (CT, cycle threshold, IP, immunoprecipitation.) Brd4 shRNA Inhibits G1 Progression in NIH3T3 Cells and MEFs— To study the role of Brd4 in cell growth and transcription, we took a shRNA approach to inhibit the expression of Brd4 in fibroblasts. This approach was a practical alternative to studying Brd4-/- cells, which do not grow in culture (11Houzelstein D. Bullock S.L. Lynch D.E. Grigorieva E.F. Wilson V.A. Beddington R.S. Mol. Cell. Biol. 2002; 22: 3794-3802Crossref PubMed Scopus (228) Google Scholar, 12Nishiyama A. Dey A. Miyazaki J. Ozato K. Mol. Biol. Cell. 2006; 17: 814-823Crossref PubMed Scopus (54) Google Scholar). NIH3T3 cells were transduced with a retroviral vector for Brd4 shRNA or control shRNA. Immunoblot analysis presented in Fig. 1A showed that introduction of the Brd4 shRNA stably knocked down Brd4 protein expression by ∼ 90% as compared with the introduction of control shRNA, which did not affect Brd4 levels. Brd4 expression in Brd4 KD cells remained low at least for 14 days after vector transduction without altering expression of a related protein, Brd2, as well as TBP and other general transcription factors (see below). Several additional control shRNAs including Brd2 shRNA did not change Brd4 expression, supporting specificity of Brd4 shRNA (data not shown). It should be noted here that our shRNA targeted both the long and short forms of Brd4, the latter suggested to exist in some cells (11Houzelstein D. Bullock S.L. Lynch D.E. Grigorieva E.F. Wilson V.A. Beddington R.S. Mol. Cell. Biol. 2002; 22: 3794-3802Crossref PubMed Scopus (228) Google Scholar). So far, however, we have detected only a single Brd4 mRNA (6.5 kilobases) and protein (∼200 kDa) in all cells examined, including NIH3T3 and MEFs (2Dey A. Ellenberg J. Farina A. Coleman A.E. Maruyama T. Sciortino S. Lippincott-Schwartz J. Ozato K. Mol. Cell. Biol. 2000; 20: 6537-6549Crossref PubMed Scopus (236) Google Scholar, 12Nishiyama A. Dey A. Miyazaki J. Ozato K. Mol. Biol. Cell. 2006; 17: 814-823Crossref PubMed Scopus (54) Google Scholar), suggesting that the short Brd4 isoform occurs rarely, if at all. Brd4 KD cells grew more slowly than cells with control shRNA as tested over a 12-day period after shRNA vector introduction. As shown in Fig. 1B, total cell yields were more than 10-fold lower in Brd4 KD cells compared with control shRNA (hereafter control) cells on both days 8 and 12. To assess the basis of this growth inhibition, cells were synchronized to G0 by 60 h of serum starvation and released into complete media to allow progression to G1, and cell cycle profiles were monitored by flow cytometry. As seen in Fig. 1C, control cells, upon exiting G0, proceeded through G1 and reached S at 16-20 h. By 24 h, control cells moved further to G2, and some cells entered G1 again. In contrast, Brd4 KD cells remained at G1 during this time and did not reach S even at 24 h, indicating solid G1 arrest (see supplemental Fig. S1 for the percentage of cells in G1, S, and G2/M). Confirming the failed S phase entry, Brd4 KD cells did not incorporate [H3]thymidine during this period as presented in Fig. 1D.G1 arrest observed in Brd4 KD cells did not appear to be a simple delay in S phase entry in that a certain fraction of cells underwent apoptosis, as evidenced by increased annexin V uptake by Brd4 KD cells at 24 h (Fig. 1E). Staining with 7-amino-actinomycin D, another apoptosis detecting agent, also supported increased cell death in Brd4 KD cells (data not shown). Although NIH3T3 cells are non-transformed cells that exhibit a normal growth property, they are tissue culture-adapted cells. To ascertain if Brd4 knockdown exerts similar growth inhibitory effect on fresh fibroblasts, we tested primary MEFs transduced with Brd4 shRNA vector. Immunoblot data in Fig. 2A confirmed that Brd4 shRNA, but not control shRNA, knocked down Brd4 expression in MEFs without affecting TFIIB and α-tubulin expression. Furthermore, we found that upon G0 synchronization and release, Brd4 KD MEFs were arrested at G1, whereas the majority of control MEFs proceeded to S phase by 16 h (Fig. 2B, supplemental Fig. S1C). These results show that a reduction in Brd4 expression causes G1 arrest in fibroblasts. Immunoblot analysis was next performed for NIH3T3 cells with Brd4 shRNA to examine the expression of Cyclins involved in G1 progression, namely cyclin D1, D2, E1, and E2, known to play a role in G1/S progression (30Sherr C.J. Roberts J.M. Genes Dev. 2004; 18: 2699-2711Crossref PubMed Scopus (913) Google Scholar, 31Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (963) Google Scholar, 32Massague J. Nature. 2004; 432: 298-306Crossref PubMed Scopus (985) Google Scholar). As seen in Fig. 2C, their expression was markedly increased during G1 in control cells. However, in Brd4 KD cells they showed a meager increase during the same period. Levels of Rb, cyclin C, and cyclin D3 were similar in control and Brd4 KD cells throughout the period. However, progressive Rb phosphorylation, evident in control cells (see pRb with arrowheads in Fig. 2C), was not observed in Brd4 KD cells, in line with G1 arrest and apoptosis (33Harbour J.W. Dean D.C. Nat. Cell Biol. 2000; 2: 65-67Crossref PubMed Scopus (422) Google Scholar). Similarly, in Brd4 KD MEFs, cyclin D1 expression was reduced relative to control cells (Fig. 2A). These results indicate that Brd4 is required for G1 cyclin expression, and Rb phosphorylation is critical for G1 progression. Microarray Analysis Reveals Deficiency in G1 Gene Expression in Brd4 KD Cells—To study the role of Brd4 in genome wide gene expression, microarray analysis was performed with synchronized control and Brd4 KD cells. We analyzed cells at G0 and early G1 rather than those at later stages. We felt that gene expression at early stages would reflect the effect of Brd4 knockdown on gene expression more directly than at later stages or in unsynchronized cells. Given that cell cycle status becomes different in control and Brd4 KD cells toward S phase (Fig. 1C), gene expression at later stages may be influenced by secondary effects such as differences in cell cycle stages more extensively than at an earlier stage. Furthermore, a preliminary microarray analysis with unsynchronized cells did not show a consistent, large scale difference in control and Brd4 KD cells, presumably due to mixed cell populations (data not shown). RNA samples from control and Brd4 KD cells at 0, 4, and 8 h after release were analyzed using the NIA 15 K mouse cDNA array (25Tanaka T.S. Jaradat S.A. Lim M.K. Kargul G.J. Wang X. Grahovac M.J. Pantano S. Sano Y. Piao Y. Nagaraja R. Doi H. Wood III, W.H. Becker K.G. Ko M.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9127-9132Crossref PubMed Scopus (364) Google Scholar). After normalization by the reference RNA and removal of spots giving high variability in three independent sets of synchronized samples, 8,000-10,000 genes could be compared between control and Brd4 KD cells at all three time points. Signals higher or lower than >0.8-fold by log2 were regarded as significant and analyzed further. Control and Brd4 KD cells exhibited a large difference in gene expression profiles at 8 h, whereas differences were less at 0 and 4 h. This was verified by the lower coefficients of correlation at 8 h relative to 0 and 4 h in scatter plot analysis (Fig. 3A). Similarly, expression profiles within control or KD cells differed most at 8 h in the principal component analysis that allows classifying expressed genes into groups of similar biological status and phenotype (Fig. 3B) (28Landgrebe J. Wurst W. Welzl G. Genome Biology. 2002; 3 (RESEARCH0019)Crossref PubMed Google Scholar). Thus, many genes were up-regulated at 8 h in control cells, in line with the induction of many genes during G1 (30Sherr C.J. Roberts J.M. Genes Dev. 2004; 18: 2699-2711Crossref PubMed Scopus (913) Google Scholar, 32Massague J. Nature. 2004; 432: 298-306Crossref PubMed Scopus (985) Google Scholar). In Brd4 KD cells, however, this large scale up-regulation was largely absent (Fig. 3C, supplemental Fig. S2); as many as 1108 genes (11%) were underexpressed in Brd4 KD cells at 8 h relative to control cells. At 0 and 4 h, about 200 genes showed underexpression in Brd4 KD cells relative to control cells. Contrary to the large number of genes underexpressed in Brd4 KD cells, only ∼100 genes were scored overexpressed in KD cells relative to control cells at all three time points. This score is likely to be an overestimate, because in the subsequent qRT-PCR analysis many of these genes did not reveal reduced expression in Brd4 KD cells (see the legend to Fig. 3C). This may be due to the conservative normalization we adopted for our microarray analysis. Collectively, these results indicate that Brd4 KD cells fail to up-regulate expression of many G1 genes, consistent with the idea that Brd4 has a role in the expression of multiple G1 genes. Our data also suggest that Brd4 represses relatively few genes during G0/G1 progression. Genes underexpressed in Brd4 KD cells during G1 were of diverse functions and included those important for G1/S progression, such as Ccnd1 and Ccnd2 (cyclin D1 and cyclin D2), as illustrated in hierarchical clustering analysis in supplemental Fig. S2. In addition, Orcl2 (Orc2) and Mcm2, components of the origin recognition complex, Dnttip, Dhfr, Top2a, Pcna, critical for DNA replication, were underexpressed in Brd4 KD cells (34DePamphilis M.L. Cell Cycle. 2005; 4: 70-79Crossref PubMed Scopus (96) Google Scholar). Furthermore, genes involved in chromatin regulation during G1/S transition, such as Chaf1a (known as CAF-1), Hmgb1, and And-1 (35Hoek M. Stillman B. Proc