The transcriptional regulator, REST (RE1-Silencing Transcription Factor; also known as Neuron-Restrictive Silencer Factor, NRSF), has been shown to repress neuronal-specific genes in cells outside the nervous system through binding to a DNA element, the RE1 (Repressor Element 1; also known as Neuron-Restrictive Silencer Element, NRSE). Although a variety of genes have previously been identified as REST targets, the extent of the gene network regulated by REST is not known. Furthermore, the contribution of REST to the transcription of its target genes has been somewhat controversial. For example, studies have shown that perturbation of REST function has little or no effect on its targets, in vivo, while other studies maintain its role as a master regulator of the neuronal phenotype.

The overall goal of my thesis work has been to provide a deeper understanding of how REST determines cell phenotype through identification and functional analysis of its target gene repertoire. Exposing the network of REST regulated genes is crucial to understanding how changes in its regulation could potentially lead to disease states including epithelial cancers and paraneoplastic diseases.

To interrogate genomes for transcription factor binding sites and identify the repertoire of occupied genes, in vivo, a novel technique was developed. This technique, SACO (Serial Analysis of Chromatin Occupancy) combines chromatin immunoprecipitation (ChIP) analysis and SAGE (Serial Analysis of Gene Expression) to allow for high-throughput sequencing of transcription factor-bound chromatin. The SACO technique was applied to a nonneuronal mouse kidney cell line for the identification of REST binding sites, in vivo. The analyses performed on the REST-SACO library resulted in a number of new findings including (1) novel variations of the REST binding site (2) extension of the diverse gene network potentially regulated by REST (3) expansion of target genes shown to be differentially regulated by REST (4) REST regulation of brain-specific microRNAs and (5) degenerate REST binding sites that co-localize with canonical sites to potentially aid in the recruitment of REST to its targets. Collectively, these studies have expanded our knowledge of the gene network regulated by REST while providing insight into how REST differentially regulates its target gene repertoire.