EXPLORING THE CONSEQUENCES OF REGULATORY VARIATION ON THE GENESIS AND FUNCTION OF MELANOCYTES
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Date
2017-05-16
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Johns Hopkins University
Abstract
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
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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.
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Keywords
Sox10, melanocyte, enhancer