Developmentally Regulated Enhancers and Chromatin Architecture in Human Neurogenesis

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PROJECT SUMMARY/ABSTRACT Mammalian development relies on spatiotemporally orchestrated transcriptional programs that define cell fates. While the mechanisms that govern such transcriptional states remain unclear, clues are emerging from the study of enhancers within the non-coding genome and their long-range interactions with gene loci otherwise distant on a linear scale. Such contacts are facilitated by the three-dimensional (3D) organization of chromatin, a nuclear property whose dynamic regulation allows for cell fate decisions during embryonic development. SOX2 is a transcription factor (TF) critical to the self-renewal and pluripotency of embryonic stem cells (ESCs). In early embryonic development, SOX2 expression persists in neural progenitors, where it is also crucial to their self- renewal and multipotency, but it is not transcribed in mesendoderm. In human ESCs (hESCs), pluripotency- associated TFs, such as OCT4, form heteromultimers with SOX2 and are thought to drive SOX2 transcription by binding the SOX2 promoter. However, the mechanism whereby SOX2 transcription selectively persists in neuroectoderm in the absence of pluripotency TFs remains unknown. Our group, which is interested in human neural stem cell (NSC) biology in health and disease, recently discovered that, in human NSCs, transcription of SOX2 is regulated by a novel enhancer located 600 kb away from the gene locus via 3D chromatin looping. Importantly, this putative enhancer is repressed in hESCs and mesendodermal progenitors where the contact between the distal enhancer and promoter is lost. These findings lead us to hypothesize that, during differentiation of hESCs to early neural precursors, SOX2 transcription becomes de novo dependent on a developmentally regulated distant enhancer, which exerts its effects on the SOX2 locus via dynamically configured chromatin looping dependent on the chromatin organizer CTCF. In support of this hypothesis, we found that CRISPR excision of critical CTCF DNA binding motifs in the SOX2 genomic neighborhood disrupt this 3D chromatin loop to impair neuralization of hESCs and bias hESC differentiation to endodermal fates in teratoma assays. The proposed research plan will answer the following questions: 1) What are the effects of experimentally induced enhancer silencing and activation in human neurogenesis? 2) How does perturbation of the 3D chromatin folding affect neural development and how does it modulate enhancer activity? 3) Does SOX2 act as a conditional initiator of enhancer activation depending on protein-protein interactions with developmentally regulated TFs? Our studies will shed light on previously unrecognized but critical long-range interactions between a gene essential to neural development, SOX2, and a distant enhancer, which are mediated by dynamic reorganization of 3D genome architecture. Elucidating the underlying mechanisms will allow us to apply these biological concepts toward understanding and treating neurodevelopmental disorders.