| Estimated to comprise ∼3.5% of the human genome, functional non-coding sequences represent a relatively unexplored source of biological information. Expected roles for these sequences include quantitative, temporal, and spatial control of gene expression. Furthermore, variation within non-coding sequences is predicted to play a significant role in human disease. However, the nature and identity of such disease-causing mutations remain largely unknown. This thesis focuses on the functional elucidation of the regulatory landscape of RET, a crucial developmental gene in which regulatory mutations are predicted to underlie susceptibility to Hirschsprung disease (HSCR; aganglionic megacolon).; First, comparative sequence analysis identified non-coding conserved sequences in a ∼220 kb interval encompassing human RET and orthologous sequence from 12 vertebrates. We demonstrate the majority of identified non-coding conserved sequences function as enhancers in vitro, largely in a cell type-dependent manner. Additionally, we show that discrete sequences within regulatory elements bind nuclear proteins.; Combined information from in vitro assays and human genetic analyses implicated an enhancer (MCS+9.7), located in RET intron 1, in HSCR susceptibility. We demonstrate that a HSCR associated variant in MCS+9.7 abrogates reporter expression in vitro. Functional analysis in mouse demonstrates that MCS+9.7 drives reporter gene expression consistent with the endogenous RET protein. Additionally, we provide evidence that SOX10 and PAX3 activate MCS+9.7.; We utilized a novel transposon-based transgenic technology in zebrafish to examine additional RET MCSs in vivo. We demonstrate that RET regulatory function is conserved from human to zebrafish, despite undetectable sequence conservation. In fact, most examined human RET MCS sequences displayed patterns of regulatory control consistent with zebrafish ret. We also examined non-coding elements conserved among teleosts, and found that most drive expression consistent with ret.; Additionally, we are assessing the necessity and disease relevance of MCS+9.7. To begin to address this issue, we generated mice transgenic for a reporter BAC encompassing Ret. We show expression of this BAC completely overlaps expression of Ret in transgenic mice and all regulatory elements necessary for appropriate Ret expression are contained within the BAC.; Systematic functional evaluation of conserved non-coding sequences, as exemplified by these studies, represent a significant step towards understanding their relevance to human genetic disease. |
