Chromatin Mechanisms of Neuronal Maturation

神经元分化需要基因表达程序的精确编排。时间上的控制 转录对出生后大脑成熟的有丝分裂后神经元特别重要，因为这些细胞 需要表达在大脑的特定关键时期提炼神经元功能和电路的基因产物 发展。已知染色质结构的表观遗传调节在建立细胞类型中起作用 在决定细胞命运的早期阶段，基因表达的特定程序，但染色质 在分化的末期调节基因表达的机制仍未完全阐明 明白了。这项提议的目标是确定协调诱导的染色质机制 中枢神经系统成熟神经元中的基因。为了发现神经元分化的这些染色质机制， 为了确定这些机制是如何在神经元成熟的多个阶段进行调节的，我们 已经确定了染色质的可及性、增强子的激活和分化过程中的基因表达 出生后小鼠小脑颗粒神经元(CGN)的活体观察我们观察到数以千计的 调节元件显示染色质可及性随着CGN的分化而变化，我们已经验证了 许多这些差异可及的区域作为神经元发育阶段特异性增强子发挥作用 基因。此外，我们的数据确定了一组特定的基因在成熟的CGN中选择性表达 早期与抑制性组蛋白H3赖氨酸27三甲基化(H3K27me3)相关的小脑 CGN分化的有丝分裂后阶段随着CGN的成熟而失去这一标志。与Active的角色一致 在组蛋白去甲基化的诱导下，我们发现这些基因中至少有一部分显示出 在缺乏H3K27去甲基酶KDM6B的CGN中，H3K27me3表达增加，mRNA表达降低。这个 这一建议的首要假设是，H3K27me3动力学的分子调节因子在 有丝分裂后神经元在神经元成熟的计时过程中起着关键作用。为了解决这一假设，我们提出了 1)研究H3K27在有丝分裂后神经元中的动态去甲基化 在体内成熟，2)确定H3K27去甲基酶KDM6B在神经元成熟中的作用，以及3) 确定成熟神经元转录去抑制的增强子机制。这些研究将结合在一起 确定在成熟的CGN中介导H3K27me3基因特异性丢失的分子机制，以及 他们将直接测试H3K27me3沉默基因的发育去抑制的重要性 功能神经元的成熟。

Neuronal differentiation requires the precise orchestration of gene expression programs. Temporal control of transcription is particularly important in maturing postmitotic neurons of the postnatal brain because these cells need to express gene products that refine neuronal function and circuitry during specific critical periods of brain development. Epigenetic regulation of chromatin structure is known to play a role in establishing cell-type specific programs of gene expression during early stages of cell fate determination, but the chromatin mechanisms that regulate gene expression during terminal phases of differentiation remain to be fully understood. The goal of this proposal is to identify chromatin mechanisms that coordinate the induction of genes in maturing neurons of the CNS. To discover these chromatin mechanisms of neuronal differentiation, and to determine how these mechanisms are regulated across multiple stages of neuronal maturation, we have characterized chromatin accessibility, enhancer activation, and gene expression in differentiating cerebellar granule neurons (CGNs) of the postnatal mouse in vivo. We have observed that thousands of regulatory elements show chromatin accessibility changes as CGNs differentiate, and we have verified that many of these differentially accessible regions function as developmental stage-specific enhancers of neuronal genes. Furthermore our data identify a specific group of the genes selectively expressed in mature CGNs of the cerebellum that are associated with repressive histone H3 lysine 27 trimethylation (H3K27me3) at early postmitotic stages of CGN differentiation yet lose this mark as CGNs mature. Consistent with a role for active histone demethylation in the induction of these genes, we find that at least a subset of these genes show increased H3K27me3 and reduced mRNA expression in CGNs lacking the H3K27 demethylase Kdm6b. The overarching hypothesis of this proposal is that the molecular regulators of H3K27me3 dynamics in postmitotic neurons play a key role in timing neuronal maturation. To address this hypothesis we propose the following Specific Aims: 1) To characterize the dynamic demethylation of H3K27 in postmitotic neurons maturing in vivo, 2) To determine the role of the H3K27 demethylase Kdm6b in neuronal maturation, and 3) To identify enhancer mechanisms of transcriptional derepression in maturing neurons. Together these studies will identify the molecular mechanisms that mediate the gene-specific loss of H3K27me3 in maturing CGNs, and they will directly test the importance of the developmental derepression of H3K27me3-silenced genes for functional neuronal maturation.