Investigations into the function and regulation of the C-terminal binding protein (CTBP-1) in C. elegans

Abstract

C-terminal Binding Protein (CtBP) is a transcriptional co-repressor which plays roles in development and apoptosis. There are two highly related CtBP genes in vertebrates (CtBPl and CtBP2). Knock-out of both CtBPl and CtBP2 results in lethality early in embryogenesis. Recently, we cloned a single CtBP in Caenorhabditis elegans (C. elegans). Like its mammalian counterparts, the C. elegans CtBP, called CTBP-1, functions as a transcriptional co-repressor and docks onto transcription factors containing an amino acid motif of the form PXDLS. The CtBP family of proteins are mechanistically well characterised, however, there is limited information on their in vivo functions. In this thesis, we explored how CTBP-1 functions using C. elegans as a model. First, we found that CTBP-1 is predominantly expressed in the nervous system. With mutant analysis we identified a role for CTBP-1 in specification of a subset of cholinergic neurons, and our data suggests that this may occur through interaction with neuron-specific transcription factor ZAG-1. To identify genes regulated by CTBP-1 for correct specification of neurons, cell-specific microarray experiments were employed where a comparison of gene expression profiles of all neurons from ctbp-l(ok498) mutants and wild type animals identified putative CTBP-1 targets, some of which display neuronal roles. In addition to targets of CTBP-1 regulation, regulators of CTBP-1 were investigated through a mutagenesis screen which led to the isolation of mutants with changes in CTBP-1 expression. This approach revealed a new regulator of CTBP-1 which was identified by next-generation sequencing. We confirmed the identification of this gene by successfully rescuing a CTBP-1 over-expression phenotype in epidermal cells caused by a nonsense mutation in this novel regulator. Bioinformatic analysis has shown that this novel regulator displays homology to human Ino80d, a subunit of an ATP-dependent chromatin remodelling complex called Ino80. Taken together, these results indicate a role for CTBP-1 in the nervous system and propose a possible mechanism by which CTBP-1 expression is regulated. Given the evolutionary conservation of CtBP from nematodes to mammals, our results may ultimately facilitate the understanding of CtBP function in neuronal regulation in higher organisms including humans, and identify novel components of the gene regulatory networks in which CtBP is involved.