Genomic and regulatory network diversity revealed by REST/NRSF, maltase glucoamylase and the protocadherin gene cluster [electronic resource]

This thesis details the experimentation on three gene/gene families: the RE1-silencing transcription factor also known as neuron-restrictive silencer factor (REST/NRSF), a repressor of neuronal genes; maltase-glucoamylase (MGAM), the intestinal enzyme responsible for the breakdown of starch; and the neuronally expressed protocadherin gene family of unknown function (PCDH). The research I have completed is tied together by the fundamental question: how is diversity achieved? On the evolutionary scale, I have investigated the functional consequences of mutation and how these functional changes may have impacted the evolution of diversity. On the cellular scale, I have sought to understand how diversity is generated by attempting to define the binding networks of proteins important in the determination of cell fate in the case of REST/NRSF and in the determination of specific neuronal identities as the predicted function of the protocadherins. REST/NRSF is canonically known as a master repressor whose function is to establish and maintain the repression of neuronal genes in nonneuronal cells. While a handful of REST/NRSF target genes of neuronal function have been extensively studied, there is little consensus on the entire repertoire of REST/NRSF targets. I participated in the development of a set of software tools for transcription factors with known binding sites which allows the identification and characterization of binding sites and their potential target genes. We identified 660 putative target genes for REST/NRSF and demonstrated that this set is enriched for genes of known neuronal function and that the expression of REST/NRSF is negatively correlated with predicted target gene expression. We validated a large number of potential binding sites and experimentally determined a threshold value which enriches the set of predicted binding sites for true positive sites. Maltase glucoamylase (MGAM) is responsible for starch breakdown and is thought to be especially important in brain development when glucose demand is high. Other components of starch metabolism are known to have common variations which affect function and these high functioning variants are more common in populations with high starch diets. Research in our lab revealed a large, common deletion in the MGAM gene with a high allele frequency in Europeans, a population with comparatively increased starch intake. I mapped the endpoints of this large, common deletion and determined the likely mechanism by which the deletion occurred to be recombination between two highly similar repeats within the MGAM gene. I then demonstrated that the deletion has no gross impact on mRNA structure or expression in heterozygous form and is therefore likely to be a neutral mutation. The protocadherins are a diverse set of neuronally-expressed cell-adhesion-like molecules hypothesized to provide the molecular code necessary for the identification of individual neurons for their incorporation into appropriate neural circuits. I demonstrate that gene conversion events occur and have likely functional effects on protocadherin paralogs, a hypothesis that had only previously been supported by evolutionary evidence. Previous research suggested that endogenous cleavage products of some protocadherins localized to the nucleus and affected gene expression. I verified nuclear localization of the protocadherin paralogs previously published and demonstrated that this nuclear localization is a common feature of all protocadherin paralogs assayed. I found no evidence that the protocadherin fragment was capable of binding to DNA directly or to affect protocadherin expression, but was unable to determine whether this was indicative of a lack of these functions in vivo or a failure of my experimental system to detect them. I conclude that nuclear localization of protocadherin internal fragments is a process common to disparate members of the protocadherin cluster but the functional consequences of this nuclear localization remain unknown.

Keywords: Genetics
Advisor: Myers Richard (Richard M.) primary advisor. Thesis advisor
Barsh Gregory Stefan advisor. Thesis advisor
Brunet Anne
Stearns Tim advisor. Thesis advisor