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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

Oncogene volume 22, pages 8460–8471 (2003)Cite this article

Abstract

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.

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Abbreviations

DMEM:

Dulbecco's modified Eagle's medium

FBS:

fetal bovine serum

MRC:

multiprotein DNA replication complex

PARP:

poly(ADP-ribose) polymerase

PAR:

poly(ADP-ribose)

PBS:

phosphate-buffered saline

PCNA:

proliferating-cell nuclear antigen

pol:

DNA polymerase

RFC:

replication factor C

RPA:

replication protein A

BRCT:

breast cancer susceptibility protein C-terminus

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Acknowledgements

We thank Dr ZQ Wang (IARC, Lyon, France) for the immortalized wild-type and PARP-1−/− cells, Dr Alexander Spoonde for the recombinant wild-type and mutant PARP-1. This work was supported by grants (CA25344 and PO1 CA74175) to MES from the National Cancer Institute, the US Air Force Office of Scientific Research (AFOSR-89-0053) to MES, and the US Army Medical Research and Development Command contract DAMD17-90-C-0053 to MES and DAMD17-00-C-0026 to DSR.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, 20007, DC, USA

    Cynthia M Simbulan-Rosenthal, Dean S Rosenthal, RuiBai Luo, Raed Samara, Luis A Espinoza & Mark E Smulson

  2. Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, Zurich, 8057, Switzerland

    Paul O Hassa & Michael O Hottiger

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  1. Cynthia M Simbulan-Rosenthal
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Correspondence to Cynthia M Simbulan-Rosenthal.

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Simbulan-Rosenthal, C., Rosenthal, D., Luo, R. et al. 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. Oncogene 22, 8460–8471 (2003). https://doi.org/10.1038/sj.onc.1206897

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