Chromatin Alterations in Rett Syndrome

描述（由申请人提供）：Rett综合征是一种儿童早期神经系统疾病。它与编码甲基-CpG结合蛋白2（MECP 2）的基因的有害突变有关，但仍不清楚MECP 2缺陷如何导致神经元疾病。MECP 2被认为调节核心组蛋白的乙酰化、甲基化和其他翻译后修饰，核心组蛋白与包裹在它们周围的DNA一起构成染色质的基本结构单元，从而调节基因表达、DNA修复和染色体分离。 我们的中心目标是1）检验组蛋白过度乙酰化导致Rett综合征表型的假设（特异性目的1）。我们将监测在发育中的野生型和突变小鼠大脑皮层发育调节的变化H3和H4共价修饰的离子型谷氨酸受体亚基基因的调控序列的网站，在β-珠蛋白基因座和在甲基-CpG丰富的印记基因Necdin和Xist基因的调控序列（具体目标2）。我们将在原代皮层培养中研究药物诱导的组蛋白乙酰化变化是否影响神经元的生长和存活（具体目标3）。最后，我们将研究诊断为Rett综合征并确认MECP 2突变的受试者死后大脑皮层中的组蛋白甲基化，并与诊断为自闭症谱系障碍的受试者和非神经系统对照进行比较。基于初步数据，我们的中心假设是：1）用抑制组蛋白去乙酰化酶（HDAC）的药物治疗将加速MECP 2缺陷小鼠的疾病发展; 2）HDAC抑制剂损害培养的MECP 2缺陷神经元的生长和存活; 3）Rett受试者大脑皮层中组蛋白甲基化失调; 4）在MECP 2突变小鼠的脑中，在限定的基因组区域处发育调节的组蛋白乙酰化和甲基化被改变。 我们的实验包括HDAC抑制剂和激活剂药物的体内和体外研究，使用条件突变小鼠，在不同的发育阶段在大脑皮层中进行cre/IoxP介导的Mecp 2切除，并补充收集人死后组织。我们的实验将依赖于染色质免疫沉淀分析，免疫印迹和免疫组织化学与抗体对H3和H4表位定义的位点特异性修饰。预计这些方法将提供关于皮质神经元中组蛋白修饰的发育调节的清晰和全面的图片，包括MECP 2缺陷脑中的潜在变化。

DESCRIPTION (provided by applicant): Rett syndrome is a neurological disease of early childhood. It is associated with deleterious mutations of the gene encoding methyI-CpG-binding protein 2 (MECP2) but it remains unclear how MECP2-deficiency results in neuronal disease. MECP2 is thought to regulate acetylation, methylation and other post-translational modifications of the core histones, that together with DNA wrapped around them comprise the fundamental structural unit of chromatin and thus regulate gene expression, DNA repair and chromosome segregation. Our central goals are 1) to test the hypothesis that histone hyperacetylation contributes to the Rett syndrome phenotype (Specific Aim 1). We will monitor in developing cerebral cortex of wildtype and mutant mice developmentally regulated changes in H3 and H4 covalent modifications at the site of regulatory sequences of ionotropic glutamate receptor subunit genes, at the beta-globin locus and at methyI-CpG-rich regulatory sequences of the imprinted gene Necdin and of the Xist gene (Specific Aim 2). We will examine in primary cortical cultures if drug-induced changes in histone acetylation affect neuronal growth and survival (Specific Aim 3). Finally, we will study histone methylation in postmortem cerebral cortex of subjects diagnosed with Rett syndrome and confirmed MECP2 mutations, in comparison to subjects diagnosed with autism spectrum disorder and to non-neurological controls. Based on preliminary data, our central hypotheses are: 1) Treatment with drugs that inhibit histone deacetylases (HDACs) will accelerate development of disease in MECP2-deficient mice; 2) HDAC inhibitors impair growth and survival of cultured MECP2-deficient neurons; 3) Histone methylation is dysregulated in cerebral cortex of Rett subjects; 4) developmentally regulated histone acetylation and methylation at defined genomic regions is altered in brain of MECP2 mutant mice. Our experiments include in vivo and vitro studies with HDAC inhibitor and activator drugs, using conditional mutant mice with cre/IoxP mediated Mecp2 excision in cerebral cortex at different stages of development, complemented by a collection of human postmortem tissue. Our experiments will rely on chromatin immunoprecipitation assays, immunoblotting and immunohistochemistry with antibodies against H3 and H4 epitopes defined by site-specific modifications. It is expected that these approaches will provide a clear and comprehensive picture on the developmental regulation of histone modifications in cortical neurons, including potential changes in MECP2-deficient brain.