Investigation of transcription factor binding sequences and target genes using protein binding microarrays

This dissertation describes the investigation of binding rules and DNA binding sequences for several transcription factors (TFs). We develop Protein Binding Microarrays (PBMs) to study the interactions between TFs and DNA in vitro and we use a support vector machine (SVM) algorithm to capture these interactions in silico. We then apply this methodology to study the binding of TFs to promoters and repetitive sequences in a genomewide fashion.In Chapter 2, we thoroughly investigate HNF4alpha/DNA binding interactions using PBMs. We investigate binding specificities for various isoforms and species of HNF4alpha. We then use PBMs to rank &sim 4,000 HNF4alpha binding sequences in order of binding affinity. Using this training set we identify/predict novel HNF4alpha binding sequences and rules, and from these rules we generate a model for HNF4alpha binding. We then use this large dataset, in combination with ChIP-on-chip and RNAi followed by an expression profiling to identify hundreds of novel HNF4alpha direct target genes.In Chapter 3, we identify HNF4alpha association with Alu repeats, a novel finding. We investigate HNF4alpha binding to Alu sequences in in vitro and in vivo in the promoters of HNF4alpha-regulated genes, and thus reveal a novel association between HNF4alpha and Alu repeats.Finally in Chapter 4, we leverage the PBM technology to investigate the binding properties of transcription factors COUP-TF2 and TCF-1. We identify many sequences that bind both HNF4alpha and TCF-1 and those bind both HNF4alpha and COUP-TF2. This finding suggests competition between these TFs on the promoters of their target genes. Additionally, we investigate the effect of coregulator PGCalpha and the effect of the endogenous ligand, linoleic acid,on HNF4alpha DNA binding.This study significantly advances our knowledge of binding sequences, binding motifs, target genes, and transcriptional regulation for several transcription factors, HNF4alpha, COUP-TF2 and TCF-1. It also sheds light on evolution of HNF4alpha binding sequences through Alu repetitive elements. Finally, it provides a powerful framework for the comprehensive investigation of transcriptional regulation in mammalian systems for other transcription factors.