E2f3b plays an essential role in myogenic differentiation through isoform-specific gene regulation

Abstract

Current models posit that E2F transcription factors can be divided into members that either activate or repress transcription, in part through collaboration with the retinoblastoma (pRb) tumor suppressor family. The E2f3 locus encodes E2f3a and E2f3b proteins, and available data suggest that they regulate cell cycle-dependent gene expression through opposing transcriptional activating and repressing activities in growing and quiescent cells, respectively. However, the role, if any, of E2F proteins, and in particular E2f3, in myogenic differentiation is not well understood. Here, we dissect the contributions of E2f3 isoforms and other activating and repressing E2Fs to cell cycle exit and differentiation by performing genome-wide identification of isoform-specific targets. We show that E2f3a and E2f3b target genes are involved in cell growth, lipid metabolism, and differentiation in an isoformspecific manner. Remarkably, using gene silencing, we show that E2f3b, but not E2f3a or other E2F family members, is required for myogenic differentiation, and that this requirement for E2f3b does not depend on pRb. Our functional studies indicate that E2f3b specifically attenuates expression of genes required to promote differentiation. These data suggest how diverse E2F isoforms encoded by a single locus can play opposing roles in cell cycle exit and differentiation.[Keywords: E2F; E2f3b; cell cycle; ChIP-on-chip; myogenic differentiation] Supplemental material is available at http://www.genesdev.org.

Tissue-specific targeting of cell fate regulatory genes by E2f factors

“…We found a high enrichment for both E2f3 and E2f4 targets in well-characterized E2f-regulated functions, including the DNA damage response, cell cycle regulation, chromatin organization, gene expression and cell death 23,24,[26][27][28][29][30][31] (Figure 2a, Table 1). Previously established E2f target genes identified in NPCs, most common to both E2f3&4, include Pcna, Ccne, Top2a, Cdc2a, Cdc25a, Mcm4, Rad51, Brca1/2, Ezh2 and pRb and E2f family members 24,26,[32][33][34] (complete lists in Supplementary Tables S1 and S2). In agreement with pRb/E2f family loss-offunction studies, we also observed a strong enrichment in processes related to differentiation and development, including those specific to the nervous system ( Figure 2a, Table 1).…”

“…We determined the genomic binding sites of E2f3 and E2f4 in NPCs derived from telencephalic tissue at embryonic day 14.5 (E14.5), a peak stage of cortical neurogenesis. As E2f binding sites are typically found within close proximity of a transcriptional start site (TSS) 8,17,20,24,26 we focused our analysis on promoter regions by coupling chromatin immunoprecipitation (ChIP) using antibodies towards E2f3 and E2f4 (Figure 1a and b) with proximal promoter DNA microarrays (details in Supplementary Text S1). We have validated the specificity of these antibodies previously 8 and here (Supplementary Figure S1 and S2).…”

“…Based on their binding profiles in muscle cells, 24 we hypothesized that E2f3a&b may bind unique target genes in NPCs. Thus, we performed ChIP-chip for E2f3 on chromatin from E2f3a − / − and E2f3b − / − NPCs.…”

“…24 In this analysis, we compared only those peaks that were discovered using the same antibody (sc-878) in each cell type and that fell within genomic regions that were surveyed in both experiments. We found a surprisingly low degree of overlap, with just 30% of MB genes also identifying as targets in NPCs (Figure 6a).…”

“…Genome-wide targets have been reported for E2f4 (and E2f1) in a number of immortalized and transformed cell lines, 20,22,23 and for E2f3 in immortal mouse myoblasts, myotubes and embryonic fibroblasts. 24,25 E2f targets have, however, not been identified on a genome-wide level in any neural cells, nor in any primary cell types.…”