Ascl1 Coordinately Regulates Gene Expression and the Chromatin Landscape during Neurogenesis

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe generation of neurons in the developing central nervous system requires a number of precisely orchestrated steps, whereby proliferating neural progenitors become committed to the neuronal fate, exit cell cycle, and undergo a long and complex program of migration and differentiation (). Proneural transcription factors (TFs) of the bHLH family, such as Ascl1/Mash1, are the main regulators of neurogenesis in the mammalian brain, and gain and loss-of-function analyses have shown that they are both required and sufficient to promote neurogenesis (, ). Accordingly, while genetic ablation of proneural genes in mice results in neural developmental defects associated with reduced neurogenesis, overexpression of proneural factors in neural progenitors induces a full neuronal differentiation program (, , ). In addition to its pivotal role in development, Ascl1 has been extensively used in protocols to reprogram somatic cells, including fibroblasts, astrocytes, and pericytes, into induced neurons (, , ), renewing interest in understanding the neurogenic activity of this proneural factor.Previously, we characterized the transcriptional program of Ascl1 in the ventral telencephalic region of the embryonic mouse brain by combining gene expression profiling with chromatin immunoprecipitation (ChIP), followed by hybridization to promoter oligonucleotide arrays (ChIP-chip). This work resulted in the identification of a set of Ascl1 target genes with various biological roles at distinct stages of the differentiation program, raising intriguing questions concerning the molecular basis for such temporal pattern (, ). In addition, it led to the identification of a novel function for Ascl1 in maintaining cell proliferation, mediated by the direct activation of genes that promote cell cycle progression. This resulted in a model whereby this proneural factor sequentially promotes the proliferation and differentiation of progenitor cells along the neuronal lineage, reconciling the classical view of this proneural protein as a differentiation factor with the fact that it is mostly expressed in cycling progenitors. Moreover, a recent study has shown that these two opposing activities are associated with distinct modes of Ascl1 expression, with oscillating or sustained Ascl1 promoting proliferation or differentiation, respectively ().In spite of the significant progress made on the characterization of its transcriptional targets, little is still known about how Ascl1 regulates gene expression. In particular, the relationship between Ascl1 binding, regulation of the chromatin landscape, and gene transcription is poorly understood. It was recently shown that during neuronal reprogramming, Ascl1 can access its cognate sites in nucleosomal-DNA when ectopically expressed in fibroblasts, defining it as a pioneer TF (). However, it remains to be seen whether Ascl1 works as a pioneer factor in a neurogenic context and whether binding of Ascl1 results in alterations to the chromatin landscape at its target regions, as it has been shown for some, but not all, other pioneer TFs ().Mammalian neurogenesis is not a synchronized process at a cell population level and studies to investigate the mechanistic basis of Ascl1 function at a genome-wide scale are difficult to perform in the developing embryo or in the adult brain. An alternative is the use of adherent cultures of neural stem (NS) cell lines derived from embryonic stem cells or embryonic neural precursors (, href="#bib30" rid="bib30" class=" bibr popnode">Pollard et al., 2006). These cultures provide us with reliable models to study neurogenesis in culture, without the confounding effects of cellular heterogeneity, characteristic of other cellular models such as neurospheres. In proliferating culture conditions, endogenous Ascl1 regulates a progenitor program that functions to maintain cell proliferation (href="#bib13" rid="bib13" class=" bibr popnode">Castro et al., 2011), whereas overexpression of Ascl1 leads to efficient cell cycle exit and neuronal differentiation.Here we investigate how Ascl1 activity is restricted by and impacts the chromatin landscape, when driving neuronal differentiation. We combined expression profiling with genome-wide mapping of Ascl1 binding sites (ChIP-seq) (href="#bib28" rid="bib28" class=" bibr popnode">Park, 2009), and DNase I hypersensitivity sites (DNase-seq) (href="#bib34" rid="bib34" class=" bibr popnode">Song and Crawford, 2010), in a cellular model of neurogenesis driven by overexpressed Ascl1. We identify a large number of genes directly regulated by Ascl1 and characterize widespread changes in chromatin accessibility during differentiation. Ascl1 binding correlates with activation of gene transcription, targeting not only regions of accessible but also of closed chromatin. In addition, binding of Ascl1 to DNA precedes a local increase in chromatin accessibility at the regulatory regions of its target genes, providing the first direct link between Ascl1 regulation of gene expression and local changes in chromatin landscape.