Multiple E2F-Induced MicroRNAs Prevent Replicative Stress in Response to Mitogenic Signaling

Abstract

Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G₁/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G₁/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G₁/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Lucía Barrado and Susana Temiño for technical assistance, Jon Fernández and Olatz Zenarruzabeitia for help with generation of E2F3-knockdown fibroblasts, J. Méndez for providing MCM5 antibodies, K. Helin for kindly providing E2F-inducible cells and vectors, and Ignacio Pérez de Castro and Oscar Fernández-Capetillo for valuable help and discussions.

U.L. is a recipient of a Basque Government fellowship for graduate studies. This work was funded by grants from the Association International for Cancer Research (AICR 08-0188 to M.M.), Fundación Mutua Madrileña Automovilista and Fundación Ramón Areces (to M.M.), Basque Government Department of Industry (Etortek-IE06-178 to A.M.Z.), and the MICINN (SAF2006-09437 and SAF2009-11426 to J.F.-P., SAF2005-07930-C02-01 and SAF2009-12037 to A.M.Z., and SAF2006-05186 and SAF2009-07973 to M.M.). The CBM/UAM group is supported by the CIBERER network of the MICINN. The Cell Division and Cancer Group of the CNIO is supported by the OncoCycle Programme (grant S-BIO-0283-2006) from the Comunidad de Madrid. A.M.Z. and M.M. are supported by the OncoBIO Consolider-Ingenio 2010 Programme (grant CSD2007-00017) from the MICINN, Madrid, Spain.