Opposing Regulation of Sox2 by Cell-Cycle Effectors E2f3a and E2f3b in Neural Stem Cells

Abstract

The mechanisms through which cell-cycle control and cell-fate decisions are coordinated in proliferating stem cell populations are largely unknown. Here, we show that E2f3 isoforms, which control cell-cycle progression in cooperation with the retinoblastoma protein (pRb), have critical effects during developmental and adult neurogenesis. Loss of either E2f3 isoform disrupts Sox2 gene regulation and the balance between precursor maintenance and differentiation in the developing cortex. Both isoforms target the Sox2 locus to maintain baseline levels of Sox2 expression but antagonistically regulate Sox2 levels to instruct fate choices. E2f3-mediated regulation of Sox2 and precursor cell fate extends to the adult brain, where E2f3a loss results in defects in hippocampal neurogenesis and memory formation. Our results demonstrate a mechanism by which E2f3a and E2f3b differentially regulate Sox2 dosage in neural precursors, a finding that may have broad implications for the regulation of diverse stem cell populations.

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

“…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).…”

“…Importantly, we have previously demonstrated that loss of either E2f3 isoform in NPCs does not result in altered expression or a perceivable change in DNA-binding capacity of the other isoform, nor of other E2f factors. 8 Figure 2 E2f3 and E2f4 bind an overlapping set of gene promoters associated with fundamental NPC fate decisions. (a) Gene Ontology (GO) analysis of E2f3 and E2f4 target genes, expressed as the percentage of all target genes in each group (groups indicated to the right of the graph) with a particular GO annotation.…”

“…Cell cycle dynamics strongly influence neural precursor cell (NPC) maintenance and neurogenesis, [1][2][3][4] and gain-or loss-of-function studies have demonstrated key roles for cell cycle proteins, including the E2f family, in NPC fate decisions. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] E2f3 is required for proper cortical migration of neurons and to maintain the balance between NPC self-renewal, proliferation and differentiation, and its loss disrupts long-term neurogenesis and cortical function; E2f1 deficiency impairs NPC proliferation, and E2f4 deficiency leads to inhibition of NPC self-renewal and severe defects in telencephalic development. 6,[8][9][10]17 A pivotal question is whether cell fate control by the pRb/E2f pathway is largely a consequence of cell cycle regulation, or due to direct regulation of cell fate-associated genes.…”

“…We recently found that loss of E2f3a and E2f3b leads to opposing defects in NPC maintenance and differentiation. 8 The fact that this occurred without affecting cell cycle dynamics strongly suggests that fate control by E2fs is not secondary to cell cycle regulation. In addition, a number of key cell fate genes and pathways have been identified as E2f-regulated targets driving E2f-dependent fate decisions in NPCs.…”

“…In addition, a number of key cell fate genes and pathways have been identified as E2f-regulated targets driving E2f-dependent fate decisions in NPCs. These include the neurogenesis and migration genes Dlx1/Dxl2 and Neo1 (Neogenin), 18,19 the growth factor fibroblast growth factor 2 (Fgf2), 17 the pluripotency and self-renewal factor Sox2, 8 and the Notch/Hes 11 and Sonic Hedgehog pathways. 10 Together, these findings demonstrate a direct role for pRb/E2f at cell fate-associated genes, but the extent of this interaction is unknown.…”