The Atoh1 targetome in murine postnatal cerebellum

A relatively small number of proneural transcription factors specify a multitude of neural progenitor populations in the developing mammalian brain. Despite their importance, little is known about their targets, cellular processes they regulate, or what genomic sites they occupy in vivo. We used an integrated experimental and computational approach, combining RNA-seq, Histone-seq, and Atoh1 ChIP-seq in cerebellar tissue, to identify over 600 targets of Atoh1, an important developmental transcription factor. We validated 10% of these targets and found that Atoh1 directly regulates genes involved not only in early proliferation but also later differentiation, migration, cell adhesion, metabolism, and cytoskeletal organization by recognizing a novel 10 nucleotide Atoh1 E-Box associated motif (AtEAM). This Atoh1 "targetome" is not only a resource for studies aimed at understanding the molecular mechanisms involved in cerebellar development, but our integrated approach provides a framework for future in vivo studies of transcription factors and their targetomes. Examination of RNA-seq WT and null expression data (2 reps each), 2 different histone modifications (2 rep each) and IgG control, and Atoh1 DNA-binding (2 reps each) and control in the developing cerebellum在发育的哺乳动物脑中，少量神经前体转录因子（proneural transcription factors）即可特化出大量神经祖细胞群体。尽管这类转录因子发挥着关键作用，但目前学界对其靶基因、所调控的细胞生物学过程，以及它们在体内结合的基因组位点仍知之甚少。我们采用实验与计算相结合的整合研究策略，在小脑组织中联合运用RNA测序（RNA-seq）、组蛋白测序（Histone-seq）以及Atoh1染色质免疫共沉淀测序（ChIP-seq），成功鉴定出重要发育转录因子Atoh1的600余个靶基因。我们验证了其中10%的靶基因，发现Atoh1可通过识别一种全新的10核苷酸Atoh1 E盒相关基序（AtEAM），直接调控参与早期增殖、晚期分化、迁移、细胞黏附、代谢及细胞骨架组织的诸多基因。该Atoh1“靶基因组（targetome）”不仅为解析小脑发育的分子机制提供了重要研究资源，我们所采用的整合研究策略也为未来开展转录因子及其靶基因组的体内研究提供了通用框架。本研究针对发育中小脑开展了多组检测，涵盖RNA-seq野生型（WT）与敲除型（null）表达数据（各设2次生物学重复）、2种不同组蛋白修饰的检测数据（各2次重复）及IgG对照数据，以及Atoh1 DNA结合检测数据（2次重复）与对照数据