Abnormal neuronal development can lead to a wide array of mental disorders. This is thought to be caused, amongst others, by aberrant gene regulation during early neural development. While we know the role of several key regulators, no study has thoroughly investigated the regulatory landscape during early time points of this process. The primary goal of this project is to comprehensively map and investigate the function of gene regulatory elements associated with early neural differentiation and understand their role in neural disease. Holding a dual appointment at leading labs in Berkeley and UCSF, creates a unique opportunity to tackle these questions in an interdisciplinary approach that combines experimental and computational methods. I will build on my statistical genetics and functional genomics training and add to them novel skills that I will develop in the training phase of this proposal, including experimental design of CRISPR/Cas9 genome editing assays, novel computational modeling and fundamentals of the etiology of neurological disorders. I will create a model system that extensively characterizes early neural development from undifferentiated ESCs (0 hour) and through six different early time points of neural induction (3, 6, 12, 24, 48, and 72 hours). First, I will leverage our recently assembled genomic (RNA/ChIP/ATAC - seq) and functional (Massive Parallel Reporter Assay - MPRA) profiling of differentiating hESC to identify responsive genomic regions, the transcription factors (TFs) that bind them, and their relationship with gene expression (Aim K1); Subsequently, I will explore regions and factors that can trigger neural differentiation from hESC without environmental signal by (i) designing a single- cell RNA profiling of pooled overexpression for top TF candidates and (ii) targeting selected regulatory elements using CRISPR/Cas9 based activation. This analysis will elucidate determinants of neural induction (Aim K2). In the independent phase of this project, I will focus on selected regions to perform a ?perturbation? MPRA by systematically targeting the binding sites of selected TFs in those regions, leading to a refined regulatory model, which will provide more insight about functional binding (Aim R1); Finally, I will map variants that are associated with neural phenotype and reside in candidate enhancer regions, to perform a comparative reference/alternative MPRA that will specifically highlight variants that alter the regulatory potential of a region and indicate their functionality in early neural development (Aim R2). Overall, this framework will identify key enhancers linked to neural differentiation, and provide hypotheses about the mechanism by which TFs interact in those regions to control transcription and which variants have a potential effect on neural phenotype during these early stages. Furthermore, this will allow to showcase the ability of genomic methods to force differentiation from stem cells without the use of any extrinsic factors which can potentially contribute to neurological disorder therapy. Through this proposed training and research, I will gain the necessary skills to achieve my ultimate career goal of leading a successful and independent research laboratory.
Over 18% of U.S. disability-adjusted life years (DALYs) are attributed to neuropsychiatric disorders and genetic aberrations associated with such disorders, in particular those involved in early neuronal development, often reside in uncharacterized noncoding genomic regions. To identify genetic factors that lead to neurological disorders, I plan to combine genomic and functional assays with regulatory logic modeling, to comprehensively map transcriptional and epigenetic changes that are associated with pluripotency exit and neural induction. These experiments will significantly increase our understanding of early neural development in a systematic and unbiased manner and contribute to neurological disorder therapy.
