Role of the Rett Syndrome-causing gene MeCP2 in 3D chromosomal organization and rescue of cellular disease phenotypes

Rett 综合征致病基因 MeCP2 在 3D 染色体组织和细胞疾病表型拯救中的作用

• 批准号: 10065818
• 负责人: X. Shawn Liu
• 金额: $ 24.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065818
• 关键词: 3-Dimensional; Academic Medical Centers; Affect; Alleles; Binding; Bioinformatics; Biological Assay; Brain; CCCTC-binding factor; CRISPR/Cas technology; Cell Nucleus; Cells; Cerebrum; ChIP-seq; Chromatin; Chromatin Interaction Analysis by Paired-End Tag Sequencing; Chromatin Loop; Chromosome Structures; Co-Immunoprecipitations; Collaborations; Cytosine; DNA Methylation; DNA Sequence Alteration; DNA-Binding Proteins; DNMT3a; Data; Data Set; Defect; Development; Disease; Electrophysiology (science); Enhancers; Epigenetic Process; Expression Profiling; Female; Fostering; Frameshift Mutation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Guide RNA; Health; Heterochromatin; Histone Acetylation; Histone-Lysine N-Methyltransferase; Histones; Human; Impairment; Institutes; Intellectual functioning disability; Investigation; Knock-out; Knowledge; Label; Laboratories; Laboratory Research; Link; Liquid substance; Maps; Mediating; Mental Retardation; Methyl-CpG-Binding Protein 2; Microcephaly; Mission; Modification; Molecular; Morphology; Mus; Mutation; National Institute of Mental Health; Neighborhoods; Neurobiology; Neurodevelopmental Disorder; Neurons; Neurotransmitters; Nuclear Proteins; Nucleosomes; Organoids; Pathway interactions; Patients; Phase; Phenotype; Play; Property; Proteins; Protocols documentation; Psyche structure; Public Health; RNA analysis; Regulation; Reporter; Research; Research Personnel; Rett Syndrome; Role; Series; Subfamily lentivirinae; Symptoms; Syndrome; System; Testing; Therapeutic; Transcriptional Regulation; Transgenes; United States National Institutes of Health; Western Blotting; X Chromosome; X Inactivation; autism spectrum disorder; brain cell; brain size; cohesin; demethylation; disability; disease phenotype; electrical property; experimental study; high resolution imaging; human embryonic stem cell; improved; induced pluripotent stem cell; innovation; insight; loss of function mutation; male; mammalian genome; mature animal; mutant; nervous system disorder; neuronal cell body; neurophysiology; novel; novel therapeutic intervention; postnatal; precursor cell; promoter; restoration; tool; transcriptome sequencing

Modified Project Summary/Abstract Section As an NIH Pathway to Independence K99/R00 awardee, I am moving to Columbia University Medical Center to start my research laboratory as a principle investigator for the R00 phase. Rett syndrome (RTT) is an X-linked postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like syndromes. The disease is caused by loss-of-function mutations of the DNA binding protein MeCP2 (Methyl CpG-binding Protein 2) in the X chromosome and represents the second most common cause of intellectual disability in females. Loss of MeCP2 leads to expression changes in thousands of genes, compromises the majority of brain cells and circuits, and dysregulates all neurotransmitter systems. However, how MeCP2 can act as a global repressor of gene activity as well as an activator for gene expression remains an open question in the field. Microcephaly (the reduction in brain size) has been documented as a hallmark of RTT, and analysis of hESC/iPSC-derived RTT neurons showed a reduced soma size as well. Our preliminary studies on human RTT mutant neurons showed a panel of cellular phenotypes including reduced soma size, impaired electrical properties, and defects in chromosomal structures. Therefore, we hypothesized that MeCP2 is involved in the organization of 3D chromosomal landscape contributing to the regulation of gene expression and subsequent neurobiology. We demonstrated that MeCP2 proteins form dynamic liquid-like condensates at the heterochromatic regions and concentrate heterochromatic factor HP1α but not components of active transcription in the nucleus. This condensate property of MeCp2 contributes to the compartmentation of 3D genome and the regulation of transcription machinery (Aim 1, K99 phase). Then we found that the intrinsically disordered region-2 (IDR-2) of MeCP2 protein mediates the formation of heterochromatin condensate. A common RTT mutant MeCP2-R168X lacking IDR-2 fails to form heterochromatin condensates to concentrate the heterochromatic factor and causes defects in the transcription regulation, providing a molecular mechanism of MeCP2-mediated 3D chromosomal organization (Aim 2, K99 phase). Development of RTT-like symptoms in mice can be reversed in RTT adult animals following the restoration of MeCP2 expression. As most female RTT patients still carry a wild type allele of MeCP2 subject to the random X-chromosome inactivation (XCI), it will be of therapeutic benefit if the wild type allele of MeCp2 in the inactive X chromosome (Xi) can be reactivated. We developed a DNA methylation editing tool by fusion of a catalytically inactive Cas9 with Tet1/Dnmt3a. Recently we expanded this toolbox to manipulate other epigenetic modifications including histone acetylation and DNA looping. We will use these tools to reverse the RTT phenotypes via reactivation of the wild type MECP2 allele on the Xi (Aim 3, R00 phase).This project will fill the gaps in our knowledge of MeCP2 function in the organization of 3D chromosomal structure and test the novel therapeutic approach to reverse RTT phenotypes.

修改的项目摘要/摘要部分 作为通往独立K99/R00获奖者的NIH途径，我搬到哥伦比亚大学医学中心，开始研究我的研究实验室，作为R00阶段的主要研究者。 RETT综合征（RTT）是一种与严重的精神残疾和自闭症综合症相关的X连锁后进行性神经发育障碍。该疾病是由X染色体中DNA结合蛋白MECP2（甲基CpG结合蛋白2）的功能丧失突变引起的，这代表了女性智障的第二大常见原因。 MECP2的丧失会导致数千种基因的表达变化，损害大多数脑细胞和电路，并失调所有神经递质系统。但是，MECP2如何充当基因活性的全局复制品以及基因表达的激活因子仍然是该领域的一个空缺问题。小头畸形（脑大小的减小）已被记录为RTT的标志，对hESC/IPSC衍生的RTT神经元的分析也显示出降低的SOMA大小。我们对人RTT突变神经元的初步研究显示了一系列细胞表型，包括降低的体体大小，电性能受损以及染色体结构中的缺陷。因此，我们假设MECP2参与了3D染色体景观的组织，这有助于调节基因表达和随后的神经生物学。我们证明了MECP2蛋白在异质区域形成动态液体样冷凝物，并浓缩异质因子HP1α，而不是细胞核中活性转录的成分。 MECP2的这种冷凝物特性有助于3​​D基因组的隔室和转录机械的调节（AIM 1，K99相）。然后，我们发现MECP2蛋白的内在无序区域2（IDR-2）介导了异染色质冷凝物的形成。缺乏IDR-2的常见RTT突变体MECP2-R168X未能形成异染色质冷凝物来浓缩异质因子并导致转录调节中的缺陷，从而提供了MECP2介导的3D染色体组织的分子机制（AIM 2，K99相）。恢复MECP2表达后，RTT成年动物的RTT样症状的发展可以逆转。由于大多数女性RTT患者仍然携带MECP2的野生等位基因，但如果MECP2的野生类型等位基因在不活动的X染色体（XI）中可以进行耐受。我们通过将催化性无活性Cas9与TET1/DNMT3A融合开发了DNA甲基化编辑工具。最近，我们扩展了此工具箱，以操纵其他表观遗传修饰，包括组蛋白乙酰化和DNA循环。我们将使用这些工具通过在XI上的野生型MECP2等位基因重新激活RTT表型（AIM 3，R00相）。该项目将填补我们对3D染色体结构组织中MECP2功能知识的空白，并测试新型热方法以逆转RTT RTT现象。