The Rb/E2F pathway has long been appreciated for its role in regulating cell cycle progression. Emerging evidence indicates that it also influences physiological events beyond regulation of the cell cycle. We have previously described a requirement for Rb/E2F mediating neuronal migration; however, the molecular mechanisms remain unknown, making this an ideal system to identify Rb/E2F-mediated atypical gene regulation in vivo. Here, we report that Rb regulates the expression of neogenin, a gene encoding a receptor involved in cell migration and axon guidance. Rb is capable of repressing E2F-mediated neogenin expression while E2F3 occupies a region containing E2F consensus sites on the neogenin promoter in native chromatin. Absence of Rb results in aberrant neuronal migration and adhesion in response to netrin-1, a known ligand for neogenin. Increased expression of neogenin through ex vivo electroporation results in impaired neuronal migration similar to that detected in forebrain-specific Rb deficiency. These findings show direct regulation of neogenin by the Rb/E2F pathway and demonstrate that regulation of neogenin expression is required for neural precursor migration. These studies identify a novel mechanism through which Rb regulates transcription of a gene beyond the classical E2F targets to regulate events distinct from cell cycle progression.The Rb pathway is best characterized for its role in regulating cell cycle progression through E2F-mediated transcriptional regulation of classical cell cycle machinery target genes. Recently, however, accumulating in vivo and in vitro evidence is emerging to suggest that Rb and E2F are capable of regulating expression of atypical target genes with functions other than cell cycle regulation in cell-type-specific manners (reviewed in reference 35). In vivo, several studies have emerged that implicate Rb and E2F interaction in novel processes beyond well-characterized roles in cell cycle regulation (10; for a review, see reference 6). In the nervous system, in particular, we have recently shown that an Rb-E2F3 interaction mediates migration of a subpopulation of GABAergic interneurons (34). In the same study, we also observed deregulation of a number of genes with known roles in neuronal migration in cell populations lacking Rb, suggesting a role for E2F3 in regulating transcription of novel targets (34). A second cell cycle-independent role for E2F3a in regulating Rb-mediated interneuron differentiation was also reported in the retina (9). Thus far, in vivo studies have failed to identify the mechanism through which these cell cycle-independent processes occur.In parallel, in vitro several microarray studies examining changes in gene expression in response to various models of deregulated E2F expression have each identified groups of overlapping novel target genes with well-characterized roles in differentiation, development, and migration (5,15,25,31,39,41,60). More recently, chromatin immunoprecipitation (ChIP)-on-chip studies have identified putative E2F binding site...

Cell cycle proteins are important regulators of diverse cell fate decisions, and in this capacity have pivotal roles in neurogenesis and brain development. The mechanisms by which cell cycle regulation is integrated with cell fate control in the brain and other tissues are poorly understood, and an outstanding question is whether the cell cycle machinery regulates fate decisions directly or instead as a secondary consequence of proliferative control. Identification of the genes targeted by E2 promoter binding factor (E2f) transcription factors, effectors of the pRb/E2f cell cycle pathway, will provide essential insights into these mechanisms. We identified the promoter regions bound by three neurogenic E2f factors in neural precursor cells in a genome-wide manner. Through bioinformatic analyses and integration of published genomic data sets we uncovered hundreds of transcriptionally active E2f-bound promoters corresponding to genes that control cell fate processes, including key transcriptional regulators and members of the Notch, fibroblast growth factor, Wnt and Tgf-β signaling pathways. We also demonstrate a striking enrichment of the CCCTC binding factor transcription factor (Ctcf) at E2f3-bound nervous system-related genes, suggesting a potential regulatory co-factor for E2f3 in controlling differentiation. Finally, we provide the first demonstration of extensive tissue specificity among E2f target genes in mammalian cells, whereby E2f3 promoter binding is well conserved between neural and muscle precursors at genes associated with cell cycle processes, but is tissue-specific at differentiation-associated genes. Our findings implicate the cell cycle pathway as a widespread regulator of cell fate genes, and suggest that E2f3 proteins control cell typespecific differentiation programs by regulating unique sets of target genes. This work significantly enhances our understanding of how the cell cycle machinery impacts cell fate and differentiation, and will importantly drive further discovery regarding the mechanisms of cell fate control and transcriptional regulation in the brain, as well as in other tissues.

In mammals, hippocampal dentate gyrus granule cells (DGCs) constitute a particular neuronal population produced both during embryogenesis and adult life, and play key roles in neural plasticity and memory. However, the molecular mechanisms regulating neurogenesis in the dentate lineage throughout development and adulthood are still not well understood. The Retinoblastoma protein (RB), a transcriptional repressor primarily involved in cell cycle control and cell death, plays crucial roles during cortical development but its function in the formation and maintenance of DGCs remains unknown. Here, we show that loss of RB during embryogenesis induces massive ectopic proliferation and delayed cell cycle exit of young DGCs specifically at late developmental stages but without affecting stem cells. This phenotype was partially counterbalanced by increased cell death. Similarly, during adulthood, loss of RB causes ectopic proliferation of newborn DGCs and dramatically impairs their survival. These results demonstrate a crucial role for RB in the generation and the survival of DGCs in the embryonic and the adult brain. © 2016 Wiley Periodicals, Inc.

We have previously shown that p107, a member of the retinoblastoma (Rb) cell cycle regulatory family, has a unique function in regulating the pool of neural precursor cells. As the pool of progenitors is regulated by a limiting supply of trophic factors, we asked if the Rb/E2F pathway may control the size of the progenitor population by regulating the levels of growth factors or their receptors. Here, we demonstrate that fibroblast growth factor 2 (FGF2) is aberrantly upregulated in the brains of animals lacking Rb family proteins and that the gene encoding the FGF2 ligand is directly regulated by p107 and E2F3. Chromatin immunoprecipitation assays demonstrated that E2F3 and p107 occupy E2F consensus sites on the FGF2 promoter in the context of native chromatin. To evaluate the physiological consequence of FGF2 deregulation in both p107 and E2F3 mutants, we measured neural progenitor responsiveness to growth factors. Our results demonstrate that E2F3 and p107 are each mediators of FGF2 growth factor responsiveness in neural progenitor cells. These results support a model whereby p107 regulates the pool of FGF-responsive progenitors by directly regulating FGF2 gene expression in vivo. By identifying novel roles for p107/E2F in regulating genes outside of the classical cell cycle machinery targets, we uncover a new mechanism whereby Rb/E2F mediates proliferation through regulating growth factor responsiveness.Cell cycle genes have been found to play an important role in brain development, with numerous molecules regulating the G 1 /S transition having been shown to regulate neural precursor proliferation (reviewed in reference 38). Perhaps the most important regulators of the G 1 /S transition are the retinoblastoma protein (Rb) and its closely related family member p107. Rb is a pivotal regulator of neural precursor proliferation and the timing of cell cycle withdrawal. For example, Rb has been shown to regulate terminal mitosis of neuroblasts in the central and peripheral nervous systems and retina (7,18,34,35). Furthermore, recent evidence has emerged indicating that Rb itself is capable of regulating diverse cellular processes in the nervous system beyond proliferation. Roles for Rb have been indicated in laminar patterning of the cortex and neuronal migration (17; reviewed in reference 38). These studies highlight the importance of Rb in regulating neural cell populations. In contrast to Rb, little is known about the role of p107. While its role was originally thought to overlap with and compensate for that of Rb (29), distinct functional differences in tissues such as muscle, chondrocytes, and adipocytes, have emerged, suggesting otherwise (10, 28, 51). We have recently shown that p107 plays a unique role, one distinct from Rb, in regulating neural precursor cell numbers in the developing and adult brain (60). p107 null neural precursor cells have an enhanced capacity for self-renewal and, consistent with this, exhibit expanded populations of both precursors and progenitors. While we have previously demonstr...

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