Preliminary Studies On The Molecular Mechanism Underlying The Transcriptional Activation Function Of C-terminal Binding Protein

C-terminal Binding Proteins(CtBPs)are best known as evolutionarily conserved transcriptional corepressors.CtBP interacts with many DNA-binding transcription factors as well as chromatin remodelers,such as histone methyltransferases and histone deacetylases via its PXDLS motif,which offers a molecular explaination on how CtBP exerts its repression function.Elegant examples are illustrated in Droshophila,where CtBP works as a major co-factor for transcriptional repressors,such as Knirps,Snail and Hairy,in early embryonic development in Drosophila.One important advance towards understanding CtBP function is that CtBP can not only repress,but also activate gene transcription in a context-dependent manner.For example,it has been shown that CtBP directly activates and represses Wnt target genes.We found recently in our lab that CtBP genetically antagonizes the homeotic transformation caused by loss of PcG gene function,indicating transcriptional activation function of CtBP in the epigenetic regulation of gene expression.However,the mechanism underlies the CtBP mediated transcriptional activation is not clear.In the present study,we aimed to explore whether CtBP might interact with the proteins with activation function by means of biochemical approaches.In addition,by fly genetics,we intended to further study the target genes likely to be activated by CtBP from the previous studies.The following are main results of this thesis,1.CtBP interacts with transcriptional activatorsWe first investigated the interaction of CtBP with Fs(1)h,a trxG protein homologous to mammalian BRD4,well known to be involved in transcriptional activation.In light of a conserved variant PXDLS-like motif found in Fs(1)h and its orthologous mammalian counterparts,we used GST pull down assay to demonstrate that CtBP interact directly with Fs(1)h-PMDLG protein fragment in vitro,which indicates the direct association between CtBP and Fs(1)h.Furthermore,we performed unbiased screen for CtBP-interacting proteins using tandem mass spectrometry.We found several more proteins involved in transcriptional activation.These results indicate that CtBP may exert its activation role by recruiting proteins with transcriptional activation function.2.CtBP regulates the transcription activation of Brk genes with a higher sensitivtiy than Hox genesIn order to directly confirm the transcription activation function of CtBP,we studied the target genes activated by CtBP in Drosophila.Previous studies have shown that CtBP can inhibit the Dpp signaling pathway,while Brk is also the negative regulatory factor of this pathway.We ask if CtBP affect the Dpp signaling pathway by influencing the expression level of Brk genes? We found that ectopic CtBP indeed led to increased brk expression.In addition,maternally knocked down CtBP during embryonic stage also resulted lowered the expression of Brk.These results indicate that CtBP universally activates Brk.Previous lab work found that CtBP is crucial for Hox genes expression when PcG was knocked down,we therefore detected whether CtBP could activate these Hox genes in embryos.However,neither overexpression nor maternally knocked down of CtBP in embryos affect the Ubx or Abd-B pattern.These observation might reflect that the CtBP activation are with varied sensitivity under different contexts.In summary,we show that CtBP interacts with proteins with activation function,which offer a possible explaination of CtBP activation and suggest that such an activation role may be direct.The observation that CtBP activates Dpp signaling target gene more sensitively than Hox genes provides bases for further exploration of the complexities of the transcriptional activation function of CtBP.