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. During the K99 phase of the award, I created a model system that extensively characterizes neural induction (Aim K1). Using neural induction from human pluripotent stem cells (hPSCs) as a paradigm, we interrogated these dynamics by performing RNA-seq, ChIP-seq and ATAC-seq at seven time points during early neural differentiation. We found that changes in DNA accessibility precede H3K27ac, which is followed by gene expression changes. Using massively parallel reporter assays (MPRAs) to test the activity of 2,464 candidate regulatory sequences at all seven time points, we show that many of these sequences have temporal activity patterns that correlate with their respective cell-endogenous gene expression and chromatin changes. A prioritization method incorporating all genomic and MPRA data further identified key transcription factors (TFs) involved in driving neural fate. These results provide a comprehensive resource of genes and regulatory elements that orchestrate neural induction and illuminate temporal frameworks during differentiation (Cell Stem Cell 2019). I extended this work to identify functional TF binding in early neural differentiation (Aim K2). To that end, I designed a MPRA experiment to test the effect of perturbing hundreds of TF binding sites across hundreds of regulatory regions relevant to neural differentiation. These perturbed regions along with wild type regions and control sequences were tested in similar temporal points. Computational analysis highlights: Temporal TF binding effects; Different types of effects; Interactions between TFs (Manuscript in preparation). In the R00 phase of the award, 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 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.
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.
