Elucidation of sex-biased regulatory elements through the study of chromatin interactions, gene expression, and genetic variants in a mouse liver model
Matthews, Bryan J.
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The genome is hierarchically organized into DNA-looped domains ranging from kilobase to megabase in size, largely shaped by two architectural protein factors: cohesin and CCCTC-binding factor (CTCF). DNA loops impact gene expression either directly via looping between genes and regulatory elements (i.e., enhancers) or indirectly by limiting available regulatory element interactions. In mouse liver, there are ~1,000 sex- biased genes whose activity and expression are modulated by ~5,000 mostly distal sex- biased enhancers. The impact of distal enhancer-promoter communication on sex-biased gene expression in mouse liver is explored in this thesis. First, I present a computational method to predict tissue-conserved DNA loops mediated by CTCF and cohesin using ChIP-seq and sequence information alone. Functionally, these loops indirectly guide enhancer-promoter interactions by insulating chromatin interactions, as is shown for the v Albumin gene. Next, I directly compared male and female CTCF and cohesin binding in mouse liver to identify ~1,000 sex-biased binding sites for each factor, the majority of which are distal to sex-biased genes. Chromatin interaction analysis revealed sex-biased enhancer-promoter and promoter-promoter interactions neighboring highly sex-biased genes. CTCF and cohesin contribute directly (as for A1bg and Sult3a2) and indirectly (as for C9, Nudt7, and Nox4) to chromatin interactions, and cohesin may be necessary for proper expression of distally-regulated male-biased genes. Finally, I identified a core subset of sex-biased genes and enhancers common between two genetically distant mouse strains, C57Bl/6J and CAST/EiJ, as well as genes and enhancers unique to each strain, reflecting their divergent evolution. The shared sex-biased genes are highly liver- specific in their function and expression and likely are the consequence of sex-specific selective evolutionary pressure. Strain-specific genetic variants were used to identify enhancers important for the expression of 481 sex-biased genes associated with genetic variants at 491 significantly contributing loci. A substantial fraction (36%) of all strain- specific, sex-biased enhancers overlap genetic variants within these 491 loci, evidencing the role of genetic disruption at distal sex-biased enhancers. Together, this research characterizes distal enhancer function in relation to gene expression using sex differences in mouse liver as a framework, with both DNA looping and genetic variation as useful tools to better understand genomic regulation.
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