PARP-1 binds E2F-1 independently of its DNA binding and catalytic domains, and acts as a novel coactivator of E2F-1-mediated transcription during re-entry of quiescent cells into S phase.
The transcription factor E2F-1 is implicated in the activation of S-phase genes as well as induction of apoptosis, and is regulated by interactions with Rb and by cell cycle-dependent alterations in E2F-1 abundance. We earlier demonstrated a pivotal role for poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of E2F-1 expression and promoter activity during S-phase re-entry when quiescent cells re-enter the cell cycle. We now investigate the putative mechanism(s) by which PARP-1 may upregulate E2F-1 promoter activity during S-phase re-entry. DNase-1 footprint assays with purified PARP-1 showed that PARP-1 did not directly bind the E2F-1 promoter in a sequence-specific manner. In contrast to p53, a positive acceptor in poly(ADP-ribosyl)ation reactions, E2F-1 was not poly(ADP-ribosyl)ated by wild-type PARP-1 in vitro, indicating that PARP-1 does not exert a dual effect on E2F-1 transcriptional activation. Protein-binding reactions and coimmunoprecipitation experiments with purified PARP-1 and E2F-1, however, revealed that PARP-1 binds to E2F-1 in vitro. More significantly, physical association of PARP-1 and E2F-1 in vivo also occurred in wild-type fibroblasts 5 h after re-entry into S phase, coincident with the increase in E2F-1 promoter activity and expression of E2F-1-responsive S-phase genes cyclin A and c-Myc. Mapping of the interaction domains revealed that full-length PARP-1 as well as PARP-1 mutants lacking either the catalytic active site or the DNA-binding domain equally bind E2F-1, whereas a PARP-1 mutant lacking the automodification domain does not, suggesting that the protein interaction site is located in this central domain. Finally, gel shift analysis with end-blocked E2F-1 promoter sequence probes verified that the binding of PARP-1 to E2F-1 enhances binding to the E2F-1 promoter, indicating that PARP-1 acts as a positive cofactor of E2F-1-mediated transcription.
Simbulan-Rosenthal
Cynthia M
CM
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA. simbulac@georgetown.edu
Rosenthal
Dean S
DS
Luo
RuiBai
R
Samara
Raed
R
Espinoza
Luis A
LA
Hassa
Paul O
PO
Hottiger
Michael O
MO
Smulson
Mark E
ME
D000818:Animals; D001665:Binding Sites; D018797:Cell Cycle Proteins; D019925:Cyclin A / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016259:Genes, myc / Q000502:physiology; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011233:Precipitin Tests; D016196:S Phase / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D014157:Transcription Factors; D011065:Poly(ADP-ribose) Polymerases