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

修改后的项目摘要/摘要部分 作为美国国立卫生研究院K99/R00独立奖获得者，我将搬到哥伦比亚大学医学中心，开始我的研究实验室，担任R00阶段的首席研究员。Rett综合征(RTT)是一种X连锁的出生后进行性神经发育障碍，与严重的精神残疾和孤独症样综合征有关。这种疾病是由X染色体上DNA结合蛋白MeCP2(甲基CpG结合蛋白2)功能丧失突变引起的，是女性智力障碍的第二大常见原因。MeCP2的缺失会导致数千个基因的表达变化，损害大多数脑细胞和神经回路，并扰乱所有神经递质系统。然而，MeCP2如何作为基因活性的全球抑制因子和基因表达的激活剂，在该领域仍然是一个悬而未决的问题。小头畸形(脑体积缩小)已被记录为RTT的标志，对hESC/IPSC来源的RTT神经元的分析也显示胞体大小减小。我们对人类RTT突变神经元的初步研究显示了一组细胞表型，包括胞体大小减小、电学特性受损和染色体结构缺陷。因此，我们假设MeCP2参与了3D染色体图谱的组织，有助于基因表达的调节和随后的神经生物学。我们证明了MeCP2蛋白在异染色区形成动态的液状凝聚体，并在细胞核中集中了异染因子hp1α，但不是活跃转录的成分。MeCP2的这种凝聚特性有助于3D基因组的区隔和转录机制的调节(目标1，K99期)。然后我们发现MeCP2蛋白的固有无序区-2(IDR-2)参与了异染色质凝集物的形成。缺乏IDR-2的常见RTT突变体MeCP2-R168X不能形成异染色质凝集物来浓缩异染色质因子，导致转录调控缺陷，提供了MeCP2介导的三维染色体组织(Aim 2，K99期)的分子机制。在恢复MeCP2表达后，RTT成年动物的RTT样症状在小鼠中的发展可以逆转。由于大多数女性RTT患者仍携带MeCP2的野生型等位基因，受到随机X染色体失活(XCI)的影响，如果能重新激活非激活X染色体(XI)中的MeCP2野生型等位基因，将对治疗有好处。我们通过将催化失活的Cas9与Tet1/DNMT3A融合，开发了一种DNA甲基化编辑工具。最近，我们扩展了这个工具箱，以操纵其他表观遗传修饰，包括组蛋白乙酰化和DNA环化。我们将使用这些工具通过重新激活XI(Aim 3，R00期)上的野生型MECP2等位基因来逆转RTT表型。这个项目将填补我们对MeCP2在3D染色体结构组织中的功能的认识空白，并测试逆转RTT表型的新的治疗方法。

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.