PROJECT SUMMARY/ABSTRACT Loss-of-function and gain-of-function mutations in the X-chromosome gene MECP2 are both associated with severe syndromic neurodevelopmental disorders, indicating that the developing brain is highly sensitive to MECP2 dosage. However, partial reduction in MECP2 function is associated with a milder form of intellectual disability and autism that manifests more strongly in males than females. Similarly, male mice with modest changes in MECP2 expression levels display social and behavioral defects without other severe neurological symptoms. This raises the possibility that human mutations that modestly alter MECP2 expression levels—such as non-coding mutations in MECP2 cis-regulatory regions (CREs)—could cause autism-like phenotypes in males. The broad objective of this proposal is to improve the functional map of MECP2 CREs towards determining whether mutations that impact CRE activity contribute to male-biased autism. Previous studies identified two distal MECP2 CREs (one enhancer and one repressor), as well as a CRE proximal to the MECP2 promoter. However, there are two additional regions of open chromatin within or flanking MECP2 that have not been investigated. I will test these candidate CREs for MECP2 regulatory activity (Aim 1) by deleting each region in human neurons, then evaluating MECP2 RNA and protein levels. I will then determine if these neurons display MECP2-related molecular phenotypes by comparing their transcriptome to the established dysregulated transcriptomic profiles in MECP2 disease neurons. In addition to expanding the catalog of MECP2 CREs, I will functionally dissect the three known MECP2 CREs to identify key transcription factor binding sites (TFBS) within each CRE (Aim 2). Each CRE is hundreds of nucleotides long, making it challenging to know which TFBSs are most responsible for CRE activity and where CRE-disrupting mutations might lie. I will shuffle each predicted TFBS within each CRE and screen them for an impact on CRE activity using a massively parallel reporter assay, then validate candidate TFBSs using a traditional luciferase reporter assay. Cumulatively, these two Aims will expand and refine the map of MECP2 CREs. This work will provide the groundwork for identifying and interpreting variants in these CREs that could alter MECP2 expression and therefore may contribute to male-biased neurodevelopmental disorders such as autism. This research will be augmented and strengthened by the exceptional training provided by the Sponsor, Dr. Huda Zoghbi, and the greater academic environment of Baylor College of Medicine and Texas Medical Center. This proposal offers the chance to develop new technical skills with in vitro neuronal models, molecular biology techniques, and bioinformatics, as well as the opportunity to hone my scientific reasoning, writing, and presentation skills. Successfully completing these research and training plans will ideally position me to transition into a career as an ...