SIGNAL TRANSDUCTION PATHWAYS REGULATING NEURON DIFFERENTIATION

这项拟议研究的长期目标是阐明控制 大脑中的神经元连接。我们最近发现了染色质重塑的重要作用 CHD4酶在小鼠小脑颗粒神经元连接中的作用。令人惊讶的是，全基因组分析 条件性CHD4基因敲除小鼠的小脑显示CHD4触发组蛋白变异体H_2A.z的沉积 在体内神经元活动依赖基因的启动子，从而触发它们的关闭。提纯 来自同步发育的颗粒神经元的核糖体相关mRNAs表明条件性基因敲除 在活体中，当神经元经历树突修剪时，CHD4的作用损害了活性依赖基因的关闭。 因此，依赖于CHD4的活性基因的关闭在体内驱动颗粒神经元树突的修剪。我们的 研究结果定义了一种表观遗传机制，该机制关闭了依赖活性的转录，从而调节了 大脑中的树枝状图案。这些发现还提出了有关监管和监管的根本问题 CHD4的机制--控制大脑中的基因表达和神经元连接。ATPase CHD4 代表核小体重塑和脱乙酰酶(NuRD)复合体的核心亚单位。我们会澄清 在CHD4诱导的H_2A.z中，组装NuRD复合体所需的蛋白质MBD3的作用。 活体小鼠脑内活性基因的依赖关闭和颗粒神经元树突的形成。我们会 还要确定CHD4是否像染色质重塑酶P400一样，直接将H2A.z结合到 并评估P400在CHD4/H_2A.z表观遗传途径中的作用。最后，我们将描述 CHD4/H2A.z表观遗传通路在颗粒神经元反应中的生物学作用 小脑回路。拟议的研究将促进我们对表观遗传机制的理解 控制大脑中的神经元连接。因为包括CHD4在内的表观遗传调控因子的突变会导致 认知的神经发育障碍，包括自闭症和智力残疾，我们的研究也将放弃 阐明这些主要脑部疾病背后的致病机制。

The long-term goals of the proposed research are to elucidate the epigenetic mechanisms that control neuronal connectivity in the brain. We have recently discovered essential roles for the chromatin remodeling enzyme Chd4 in granule neuron connectivity in the mouse cerebellum. Strikingly, genome-wide analyses of the cerebellum in conditional Chd4 knockout mice reveal that Chd4 triggers deposition of the histone variant H2A.z at promoters of neuronal activity-dependent genes in vivo, thereby triggering their shutoff. Purification of ribosome-associated mRNAs from synchronously developing granule neurons shows that conditional knockout of Chd4 impairs shutoff of activity-dependent genes when neurons undergo dendrite pruning in vivo. Accordingly, Chd4-dependent shutoff of activity genes drives granule neuron dendrite pruning in vivo. Our findings define an epigenetic mechanism that shuts off activity-dependent transcription and thereby regulates dendrite patterning in the brain. These findings also raise fundamental questions on the regulation and mechanisms of Chd4-control of gene expression and neuronal connectivity in the brain. The ATPase Chd4 represents the core subunit of the nucleosome remodeling and deacetylase (NuRD) complex. We will elucidate the role of the protein Mbd3, which is required for the assembly of the NuRD complex, in Chd4-induced H2A.z- dependent shutoff of activity genes and granule neuron dendrite patterning in the mouse brain in vivo. We will also determine whether Chd4, like the chromatin remodeling enzyme p400, directly incorporates H2A.z into nucleosomes, and assess the role of p400 in the Chd4/H2A.z epigenetic pathway. Finally, we will characterize the biological role of the Chd4/H2A.z epigenetic pathway in granule neuron responses in the context of cerebellar circuitry. The proposed research will advance our understanding of the epigenetic mechanisms that control neuronal connectivity in the brain. Because mutations of epigenetic regulators including Chd4 cause neurodevelopmental disorders of cognition including autism and intellectual disability, our studies will also shed light on pathogenic mechanism underlying these major disorders of the brain.