Regulation of neurogenin-dependent gene expression by geminin. Xenopus laevis

Transcriptional targets of neurogenin (Ngnr1) were identified by over-expression of an inducible form of neurogenin in Xenopus ectodermal explants. The effects of co-expressing the nucleoprotein geminin on Ngnr1-dependent target gene transactivation were defined. Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized both neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, geminin antagonized the ability of neural bHLH transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. Geminin is highly expressed in undifferentiated neuronal precursor cells but is downregulated prior to differentiation. Therefore, these data support a model whereby geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis. Overall design: A dexamethasone-inducible (GR ligand binding domain fused) form of Xenopus neurogenin-related 1, NgnrGR, was over-expressed in Xenopus embryonic ectodermal explants, in the presence or absence of over-expressed geminin. Induction of Ngnr1 activity was used to define direct targets as previously described (EMBO J. 26(24): 5093-5108). The ability of geminin to suppress Ngnr1-dependent transactivation of its target gene programs was determined.通过在非洲爪蟾（Xenopus）外胚层外植体（ectodermal explants）中过表达诱导型神经素（neurogenin），鉴定了神经素相关1（neurogenin-related 1, Ngnr1）的转录靶标（transcriptional targets），并明确了共表达核蛋白Geminin（nucleoprotein geminin）对Ngnr1依赖型靶基因转录激活（transactivation）的调控作用。调控谱系受限祖细胞（lineage-restricted progenitors）向终末分化细胞（terminally differentiated cells）的转化，是神经系统发育的核心环节之一。本研究以非洲爪蟾原发神经发生（primary neurogenesis）过程及体外培养的哺乳动物神经元分化体系为模型，从机制层面探究了Geminin在神经发生调控中的功能。后续实验分别采用了体外培养的胚胎干细胞（embryonic stem cells）、胚胎癌细胞（embryonal carcinoma cells）来源的神经细胞，以及小鼠前脑（forebrain）来源的神经干细胞（neural stem cells）。在所有实验体系中，Geminin均可拮抗神经源性碱性螺旋-环-螺旋转录因子（neural bHLH transcription factors）激活促神经发生转录程序的能力。进一步研究发现，Geminin可在促神经发生转录因子编码基因位点上诱导形成二价染色质状态（bivalent chromatin state），该状态同时兼具组蛋白激活修饰与抑制修饰的特征。这种表观遗传状态（epigenetic state）可抑制未分化干细胞及神经元前体细胞（neuronal precursor cells）向神经元谱系定型相关基因的表达。Geminin在未分化神经元前体细胞中高表达，但在细胞分化前会被下调。因此，本研究数据支持如下模型：Geminin通过调控促神经发生转录因子编码基因的表观遗传状态与表达水平，维持神经元前体细胞状态。后续其他发育信号可进一步调控这些细胞向终末神经元或神经胶质细胞分化，在哺乳动物神经发生过程中完成细胞命运转换。实验整体设计：将非洲爪蟾神经素相关1（Ngnr1）的地塞米松诱导型（dexamethasone-inducible）形式——融合糖皮质激素受体（glucocorticoid receptor, GR）配体结合域的NgnrGR，在是否共过表达Geminin的条件下，转入非洲爪蟾胚胎外胚层外植体中。如先前研究所述（EMBO J. 26(24): 5093-5108），通过诱导Ngnr1活性以鉴定其直接转录靶标。本研究同时检测了Geminin抑制Ngnr1依赖型靶基因程序转录激活的能力。