SIGNAL TRANSDUCTION PATHWAYS REGULATING NEURON DIFFERENTIATION

拟议研究的长期目标是阐明控制遗传的表观遗传机制 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， Chd 4酶在小鼠小脑颗粒神经元连接中的作用。令人惊讶的是， 条件性Chd 4基因敲除小鼠的小脑显示Chd 4触发组蛋白变体H2A.z的沉积 在体内的神经元活性依赖基因的启动子，从而触发其关闭。纯化 来自同步发育的颗粒神经元的核糖体相关mRNA显示，条件性敲除 Chd 4的表达削弱了神经元在体内进行树突修剪时活性依赖基因的关闭。 因此，Chd 4依赖的活性基因关闭驱动体内颗粒神经元树突修剪。我们 这些发现定义了一种表观遗传机制，该机制关闭了活性依赖性转录，从而调节 大脑中的树突图案。这些发现还提出了有关监管的基本问题， Chd 4控制基因表达和大脑中神经元连接的机制。ATP酶Chd 4 代表核小体重塑和脱乙酰酶（NuRD）复合物的核心亚基。我们将阐明 蛋白质Mbd 3的作用，这是组装NuRD复合物所必需的，在Chd 4诱导的H2A.z中。 依赖性关闭的活性基因和颗粒神经元树突图案在小鼠大脑中的体内。我们将 还确定Chd 4是否像染色质重塑酶p400一样，直接将H2A.z掺入 核小体，并评估p400在Chd 4/H2A.z表观遗传途径中的作用。最后，我们将描述 Chd 4/H2A.z表观遗传通路在以下背景下颗粒神经元反应中的生物学作用： 小脑回路这项拟议中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Chd 4在内的表观遗传调节因子的突变导致 神经发育障碍的认知，包括自闭症和智力残疾，我们的研究也将摆脱 这些主要的脑部疾病的致病机制。

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.