Coordinate actions between methyl-CpG binding proteins in neuronal development

 描述(由申请人提供)：甲基-CpG结合蛋白(MBPS)是表观遗传调控的中心角色，通过将DNA甲基化转化为表型变化。MeCP2基因突变会导致Rett综合征和某些自闭症谱系障碍。在自闭症患者和智力障碍患者的子集中发现了MBD1的变化。我们已经证明，MBD1基因缺失的小鼠表现出学习障碍和自闭症样行为缺陷。因此，MeCP2和MBD1都是自闭症相关基因(SFARI)。我们已经证明，MeCP2和MBD1在未成熟的干细胞和出生后大脑中的新神经元中都有表达，并调节神经发生和神经元的成熟。我们发现，这种调控至少部分是通过控制非编码microRNA的表达来实现的。虽然许多研究都集中在MeCP2靶标的识别上，但特定神经细胞类型的MeCP2靶标仍然很大程度上未知。此外，几乎没有采取任何行动来确定目标。 大脑中的MBD1。文献表明，MBD1和MeCP2可能有不同的靶点，但两者都表现出相似的异染色质结合，并至少共享几个非编码基因靶点。这两种蛋白在神经元发育过程中的功能关系还没有被探索过。我们观察到，同时缺乏MBD1和MeCP2的小鼠在出生后第21天比单一突变小鼠死亡更早，这与出生后突触发生和神经元成熟期以及神经元DNA甲基化的动态变化相吻合。因此，在此期间同时缺乏MBD1和MeCP2是极其有害的，但原因尚不清楚。此外，尽管这两种蛋白质之间存在明显的功能补偿，但问题仍然存在，为什么MBD1不能完全补偿MeCP2的缺失，以及哪些基因组结合靶点可能是这种补偿缺失的原因。直接比较全基因组的MBD1和MeCP2靶标将为理解MBD对神经发育和疾病的调控提供有价值的信息。PI和合作者非常适合填补这些知识空白。在这个探索性的R21方案中，我们的目标是利用张博士创建的MeCP2-FLAG系和我们新创建的MBD1-FLAG小鼠系来直接比较MBD1和MeCP2在大脑发育中的靶点。我们的目的是验证这一假设，即MBD1和MeCP2具有共同和独特的靶点，这些靶点是它们在出生后发育过程中特定功能的基础。我们将首先确定这些蛋白质的基因组结合图谱。然后我们将确定基因组结合的特异性是否对基因表达和神经元成熟有影响。拟议的研究是建立在私人投资和合作者的免费专业知识的基础上的。本研究的结果将为了解MBP的生物学功能及其在基因调控中的意义提供必要的信息。由于这两种MBP对自闭症和神经发育障碍都很重要，这项研究将揭示与发育障碍有关的新基因和途径。

 DESCRIPTION (provided by applicant): Methyl-CpG binding proteins (MBPs) are central players of epigenetic regulation by translating DNA methylation into phenotypic changes. Mutations in MeCP2 result in Rett Syndrome and certain autism- spectrum disorders. Alterations in MBD1 are found in a subset of autistic patients and intellectual disabilities. We have demonstrated that MBD1 null mice display learning impairment and autism-like behavioral deficits. Thus, both MeCP2 and MBD1 are autism-associated genes (SFARI). We have shown that both MeCP2 and MBD1 are expressed in immature stem cells and new neurons in the postnatal brains and regulate neurogenesis and neuronal maturation. We found that such regulations are at least in part through controlling the expression of noncoding microRNAs. Although many studies have focused on MeCP2 target identification, MeCP2 targets in specific neural cell types remain largely unknown. In addition, little has been done to identify the targets of MBD1 in the brain. Literatures have suggested that MBD1 and MeCP2 may have different sets of targets, however both protein exhibit similar heterochromatin binding and share at least several noncoding gene targets. Functional relationship between these two proteins during neuronal development has not been explored. We observed that mice lacking both MBD1 and MeCP2 die much earlier than single mutant mice, at postnatal day 21, which coincides with the postnatal synaptogenesis and neuronal maturation period as well as the dynamic changes in DNA methylation in neurons. Therefore a lack of both MBD1 and MeCP2 during this period is extremely detrimental but the reason is unknown. In addition, despite the apparent functional compensations between these two proteins, questions remained why MBD1 cannot fully compensate for MeCP2 deficiency and which genomic binding targets might be responsible for this lack of compensation. A direct comparison of genome-wide MBD1 and MeCP2 targets will yield valuable information for understanding MBD regulation of neurodevelopment and disorders. The PI and collaborators are uniquely suited to fill these knowledge gaps. In this exploratory R21 proposal, we aim to take advantage of a MeCP2-FLAG line created by collaborator Dr Chang and our newly created MBD1-FLAG mouse line to directly compare the targets between MBD1 and MeCP2 in developing brain. We aim to test the hypothesis that MBD1 and MeCP2 have both common and unique targets which underlie their specific functions during postnatal development. We will first identify genomic binding profiles of these proteins. We will then determine whether the genomic binding specificities have impact on gene expression and neuronal maturation. The proposed study is built upon the complimentary expertise of the PI and collaborators. The results of this study will provide much needed understanding in biological functions of MBP and its significance in gene regulation. Since both MBPs are important for autism and neurodevelopmental disorders, the study will unveil the novel genes and pathways involved in developmental disorders.