Inactivation of Rb and E2f8 Synergizes To Trigger Stressed DNA Replication during Erythroid Terminal Differentiation

Abstract

Rb is critical for promoting cell cycle exit in cells undergoing terminal differentiation. Here we show that during erythroid terminal differentiation, Rb plays a previously unappreciated and unorthodox role in promoting DNA replication and cell cycle progression. Specifically, inactivation of Rb in erythroid cells led to stressed DNA replication, increased DNA damage, and impaired cell cycle progression, culminating in defective terminal differentiation and anemia. Importantly, all of these defects associated with Rb loss were exacerbated by the concomitant inactivation of E2f8. Gene expression profiling and chromatin immunoprecipitation (ChIP) revealed that Rb and E2F8 cosuppressed a large array of E2F target genes that are critical for DNA replication and cell cycle progression. Remarkably, inactivation of E2f2 rescued the erythropoietic defects resulting from Rb and E2f8 deficiencies. Interestingly, real-time quantitative PCR (qPCR) on E2F2 ChIPs indicated that inactivation of Rb and E2f8 synergizes to increase E2F2 binding to its target gene promoters. Taken together, we propose that Rb and E2F8 collaborate to promote DNA replication and erythroid terminal differentiation by preventing E2F2-mediated aberrant transcriptional activation through the ability of Rb to bind and sequester E2F2 and the ability of E2F8 to compete with E2F2 for E2f-binding sites on target gene promoters.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01651-13.

ACKNOWLEDGMENTS

We are grateful to Amgen for providing erythropoietin and Harvey Lodish and Joe Shuga for their help with the in vitro erythroid differentiation assay. We also thank Gustavo Leone and Alain de Bruin for providing the E2f8^loxP/loxP mice, Ursula Klingmueller and James D. Engel for providing the EpoR-GFPCre mice, Kathleen McGrath and Scott Peslak for the ImageStream procedure, Utz Herbig for immunofluorescence staining, and Nury Yim, Dan Li, Lester Foldi, and Mai Abd Al Qader for technical assistance.

This study was supported by grants from the Leukemia Research Foundation, the New York Community Trust, and the Foundation of the University of Medicine and Dentistry of New Jersey.