Investigation of Transcription Factor Binding Sequences and Target Genes using Protein Binding Microarrays
This thesis 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 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 HNF4α DNA binding interactions using PBMs. We investigate binding specificities for various isoforms and species of HNF4α. We then use PBMs to rank ~ 4,000 HNF4α binding sequences in order of binding affinity. Using this training set we identify/predict novel HNF4α binding sequences and rules, and from these rules we generate a model for HNF4α 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 HNF4α direct target genes.
In Chapter 3, we identify HNF4α association with Alu repeats, a novel finding. We investigate HNF4α binding to Alu sequences in in vitro and in vivo in the promoters of HNF4alpha-regulated genes, and thus reveal a novel association between HNF4α 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 HNF4αand TCF-1 and those bind both HNF4α and COUP-TF2. This finding suggests competition between these TFs on the promoters of their target genes. Additionally, we investigate the effect of coregulator PGCα and the effect of the endogenous ligand linoleic acid HNF4α DNA binding.
This study significantly advances our knowledge of binding sequences, binding motifs, target genes, and transcriptional regulation for several transcription factors, HNF4α, COUP-TF2 and TCF-1. It also sheds light on evolution of HNF4α binding sequences through Alu repetitive elements. It suggests a strong framework for comprehensive investigation of transcriptional regulation in mammalian systems for other transcription factors.