EXPLORING THE CONSEQUENCES OF REGULATORY VARIATION ON THE GENESIS AND FUNCTION OF MELANOCYTES
Baker, Maggie Bryce
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Our understanding of genetic disease is increasingly dependent on understanding the effects of transcriptional regulation. While we have a strong command on predicting the effects of variation in coding sequence, our ability to predict the impact of variation in non-coding genomic space, specifically enhancers, is limited. Therefore, our aim for this study was to study enhancers using pigmentproducing melanocyte cells as a model with the ultimate goal of a better understanding of the mechanics of enhancer function in a native chromatin context. In vitro and in vivo reporter assays are cost- and time-efficient tools with which to test the activity of putative regulatory sequences, but remove them from their endogenous context, rendering them a sub-optimal approach to enhancer discovery and functional validation. Recently there has been a surge of research demonstrating the importance of chromatin conformation in regulating appropriate transcriptional control. 4C-seq is an emerging technology which allows identification of all sequences in close physical proximity to a given locus, in this case, the promoter of Sox10, a melanocyte-critical gene. Because enhancers act by physically looping into proximity of their cognate promoter, we expected to identify previously validated enhancers at that locus as well as new sequences that interact with the promoter. We identified three distinct regions of interaction containing all previously identified melanocyte enhancers. We propose a stem-loop like structure with multiple interacting domains contacting the Sox10 promoter within a chromatin substructure. Our results are supported by previous functional work and a deletion mutant mouse line missing non-coding sequence in one of the interaction domains we identified. The mice display reduced Sox10 expression and present with multiple pigment-related phenotypes. The ultimate goal of future work is to employ CRISPR/Cas9 technology to examine the effects of various disruptions, deletions, and combinations of deletions on gene transcription. It is expected that disrupted TF binding will also impact the physical relationship between an enhancer and its cognate promoter. What is less predictable is what impact such a change in interaction stability/frequency will have 2 on other interactions that may exist in the collective chromatin structure promoting transcription. It may be that removal or disruption of an enhancer sequence impacts only its own relationship with the promoter, but it is also possible that stability of chromatin looping may be more broadly affected. Having described the catalog of interactions at Sox10, we will be able to selectively evaluate the impact of enhancer mutation on chromatin structure and gene regulation, both globally and at individual loci.