| Determining the genetic underpinnings of normal developmental and abnormal disease processes is central to the field of human genetics. Whereas the completion of the Human Genome Project yielded a genetic parts list, recent work has emphasized understanding how those parts are deployed--how those genes are turned on at the right time and right place. In this thesis, I discuss two major projects designed to explicate the regulation of TBX5, a gene critical to heart development and implicated in severe morphological heart disorders. Birthed from the shortcomings of genome-wide association and exome sequencing studies, the first project assesses the contribution of genetic variation in non-coding, regulatory elements (enhancers) to congenital heart disease (CHD). We first identified 3 enhancers that contribute to TBX5 expression, and subsequently discovered a mutation in one of these elements that destroys its cardiac enhancer activity in mouse. Importantly, we found this variant nucleotide present in the Brazilian population at large. Based on its frequency therein, this variant could contribute to as many cases of CHD as HOS, effectively doubling, as a conservative estimate, the number of cases of CHD attributable to TBX5 variation. The second project also focuses on the regulatory dynamics of heart development, but at the epigenetic level. We hypothesize that the regionalized expression of some genes in the heart is the result of distinct patterns of histone modifications, which potentiate or prohibit their expression. Using ChIP-seq for chromatin marks of activation (H3K4me1) and repression (H3K27me3), in combination with transcriptome profiling via RNA-seq, we found a significant set of genes that demonstrate a coordinated epigenetic program that reinforces regionalized expression. TBX5 fits this pattern, with regionalized cardiac and limb expression correlated with coordinated epigenetic marking. Furthermore, we demonstrate that the trans factors necessary to activate TBX5 are present throughout the heart, implying some other mechanism—which we believe to be epigenetic closure—restricts expression to certain domains. In summary, my work has furthered our understanding of the interactions between cis-regulatory elements and epigenetic marks in determining the spatial, temporal and quantitative expression patterns of genes, as well as illustrated how genetic variation within these cis-regulatory sequences likely underlie the etiology of a significant fraction of human diseases, including congenital malformations. |
