Genome-wide analysis of proteins that bind to DNA and regulate gene expression, with particular emphasis on imprinted genes

Regulation of gene expression is a complicated process, subject to different mechanisms operating at different levels. At the genomewide level, work in this thesis describes the use of chromatin immunoprecipitation and next-generation sequencing to interrogate the coincident and allele-specific binding of the proteins CTCF, cohesin, ATRX and MeCP2. Identifying regions co-binding these proteins helps generate a model to understand how these proteins influence gene expression, particularly at imprinted loci. Using these tools we are able to understand more about the proteins that participate in the genomic landscape around imprinted loci and drive this unusual mode of gene regulation. These loci act as models for studying DNA binding proteins and their roles in transcription. At the level of the individual locus, mechanisms of gene regulation were investigated using imprinted retrogenes as a model. Retrogenes are transcriptionally active intronless genes located within an intron of a ‘host’ gene. The role of epigenetic factors influencing gene transcription were investigated at the H13/Mcts2 locus. Expression of an intronic retrogene can cause premature termination of a ‘host’ transcript. Our hypothesis is that this premature termination can be caused by transcription of the retrogene interfering with host gene transcription. We have designed a construct based on this locus, which allows us to regulate the expression through the retrogene to study this in more detail. These studies provide a mechanistic component to our whole genome analyses.