Chromatin Alterations in Rett Syndrome

描述(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(MECP2)的基因的有害突变有关，但尚不清楚MECP2缺乏如何导致神经元疾病。MeCP2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，这些组蛋白与包裹在组蛋白周围的DNA一起构成染色质的基本结构单位，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)验证组蛋白超乙酰化有助于Rett综合征表型的假设(特定目标1)。我们将监测野生型和突变型小鼠大脑皮层发育过程中亲离子谷氨酸受体亚单位基因调控序列的H3和H4共价修饰的变化，以及印迹基因Necdin和Xist基因的富含甲基CpG的调控序列(特定目标2)。我们将在原代皮质培养中检测药物诱导的组蛋白乙酰化变化是否会影响神经元的生长和存活(特定目标3)。最后，我们将研究被诊断为Rett综合征并确认MECP2突变的受试者死后大脑皮层的组蛋白甲基化，并与被诊断为自闭症谱系障碍的受试者和非神经对照组进行比较。基于初步数据，我们的中心假设是：1)抑制组蛋白脱乙酰酶(HDAC)的药物治疗将加速MECP2缺陷小鼠的疾病发展；2)HDAC抑制剂损害培养的MECP2缺陷神经元的生长和存活；3)组蛋白甲基化在RERT受试者的大脑皮层中失调；4)MECP2突变小鼠大脑中发育调节的组蛋白乙酰化和甲基化改变。我们的实验包括体内和体外对HDAC抑制剂和激活剂药物的研究，使用条件突变小鼠，在不同发育阶段通过cre/IoxP介导的大脑皮质切除MeCP2，并辅之以收集人类死后组织。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，以及通过定点修饰定义的针对H3和H4表位的抗体。预计这些方法将提供关于皮质神经元组蛋白修饰的发育调控的清晰和全面的图景，包括MECP2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.