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E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints
Ren, Bing; Cam, Hieu; Takahashi, Yasuhiko; Volkert, Thomas; Terragni, Jolyon; Young, Richard A; Dynlacht, Brian David
The E2F transcription factor family is known to play a key role in the timely expression of genes required for cell cycle progression and proliferation, but only a few E2F target genes have been identified. We explored the possibility that E2F regulators play a broader role by identifying additional genes bound by E2F in living human cells. A protocol was developed to identify genomic binding sites for DNA-binding factors in mammalian cells that combines immunoprecipitation of cross-linked protein-DNA complexes with DNA microarray analysis. Among approximately 1200 genes expressed during cell cycle entry, we found that the promoters of 127 were bound by the E2F4 transcription factor in primary fibroblasts. A subset of these targets was also bound by E2F1. Most previously identified target genes known to have roles in DNA replication and cell cycle control and represented on the microarray were confirmed by this analysis. We also identified a remarkable cadre of genes with no previous connection to E2F regulation, including genes that encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint. Our data indicate that E2F directly links cell cycle progression with the coordinate regulation of genes essential for both the synthesis of DNA as well as its surveillance
PMCID:155321
PMID: 11799067
ISSN: 0890-9369
CID: 33142
Active repression and E2F inhibition by pRB are biochemically distinguishable
Ross JF; Naar A; Cam H; Gregory R; Dynlacht BD
To understand mechanistically how pRB represses transcription, we used a reconstituted transcription assay and compared pRB activity on naked versus chromatin templates. Surprisingly, when pRB was directly recruited to a naked template, no transcriptional repression was observed. However, we observed active repression when the same promoter was assembled into chromatin. Histone deacetylases do not appear to play a role in this observed repression. Further experiments showed repression could occur after preinitiation complex assembly, in contrast with pRB inhibition of E2F, suggesting discrete mechanisms by which pRB directly inhibits an activator such as E2F or actively represses proximally bound transcription factors
PMCID:312635
PMID: 11230147
ISSN: 0890-9369
CID: 33143
Transcriptional regulation of cell cycle progression
Chapter by: Dynlacht BD
in: Transcription factors by Locker J [Eds]
Oxford : BIOS, Academic Press, 2001
pp. ?-?
ISBN: 1859961827
CID: 2840
Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression
Takahashi Y; Rayman JB; Dynlacht BD
The E2F transcription factor plays a pivotal role in the timely activation of gene expression during mammalian cell cycle progression, whereas pRB and related proteins control cell growth in part through the ability to block the action of E2F. To identify physiologically important E2F-responsive promoters and to study their occupancy and histone acetylation state in vivo, we have taken advantage of a cross-linking approach in synchronized, living cells. We find that the pattern of E2F and pRB-related polypeptides recruited to these promoters changes in a strikingly dynamic fashion as cells progress from quiescence into G(1) and S phase: Repression of each promoter in quiescent cells is associated with recruitment of E2F-4 and p130 and low levels of histone acetylation, but by late G(1), these proteins are replaced largely by E2F-1 and E2F-3, in concert with acetylation of histones H3 and H4 and gene activation. These findings suggest that repression and activation of E2F-responsive genes may occur through distinct E2F heterodimers that direct the sequential recruitment of enzymes able to deacetylate and then acetylate core histones
PMCID:316494
PMID: 10766737
ISSN: 0890-9369
CID: 33144
Human PC4 is a substrate-specific inhibitor of RNA polymerase II phosphorylation
Schang LM; Hwang GJ; Dynlacht BD; Speicher DW; Bantly A; Schaffer PA; Shilatifard A; Ge H; Shiekhattar R
The activity of cyclin-dependent protein kinases (cdks) is physiologically regulated by phosphorylation, association with the specific cyclin subunits, and repression by specific cdk inhibitors. All three physiological regulatory mechanisms are specific for one or more cdks, but none is known to be substrate specific. In contrast, synthetic cdk peptide inhibitors that specifically inhibit cdk phosphorylation of only some substrates, 'aptamers,' have been described. Here, we show that PC4, a naturally occurring transcriptional coactivator, competitively inhibits cdk-1, -2, and -7-mediated phosphorylation of the largest subunit of RNA polymerase II (RNAPII), but it does not inhibit phosphorylation of other substrates of the same kinases. Interestingly, the phosphorylated form of PC4 is devoid of kinase inhibitory activity. We also show that wild-type PC4 but not the kinase inhibitory-deficient mutant of PC4 represses transcription in vivo. Our results point to a novel role for PC4 as a specific inhibitor of RNAPII phosphorylation
PMID: 10692395
ISSN: 0021-9258
CID: 33145
Mechanism of transcriptional repression of E2F by the retinoblastoma tumor suppressor protein
Ross JF; Liu X; Dynlacht BD
The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes
PMID: 10078202
ISSN: 1097-2765
CID: 33146
Activity and nature of p21(WAF1) complexes during the cell cycle
Cai K; Dynlacht BD
Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ('free' p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21
PMCID:22818
PMID: 9770473
ISSN: 0027-8424
CID: 33147
Dual cyclin-binding domains are required for p107 to function as a kinase inhibitor
Castano E; Kleyner Y; Dynlacht BD
The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks
PMCID:109123
PMID: 9710622
ISSN: 0270-7306
CID: 33148
Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry
Zhao J; Dynlacht B; Imai T; Hori T; Harlow E
To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry
PMCID:316526
PMID: 9472014
ISSN: 0890-9369
CID: 33170
Regulation of transcription by proteins that control the cell cycle
Dynlacht BD
In eukaryotes, progression of a cell through the cell cycle is partly controlled at the level of transcriptional regulation. Yeast and mammalian cells use similar mechanisms to achieve this regulation. Although gaps still remain, progress has been made recently in connecting the links between the cell's cycle and its transcriptional machinery
PMID: 9296491
ISSN: 0028-0836
CID: 33149