Identification and characterization of binding sites for NF-kB transcription factors in T cells

Transcription of genes is controlled by trans-acting DNA binding transcription factors interacting with cis-regulatory elements containing their cognate binding sites. The interaction between transcription factors and their binding sites has long been studied to understand transcriptional regulatory networks. Initial studies were performed through in vitro experiments, which have provided insights into transcription factor-DNA interactions and allowed the identification of regulatory elements for transcription factors of interest. However, these studies provided little information regarding proteinDNA interactions occurring in living cells. Thus, chromatin immunoprecipitation (ChIP) was developed to allow the detection of in vivo protein-DNA interactions in living cells. More recently, protein-DNA interactions have been studied using ChIP combined with microarray on a genome-wide scale. NF-kB family of transcription factors play an important role in the regulation of many genes that are involved in a large number of cellular processes such as immune and inflammatory responses, cell development, cell survival, and cell proliferation. The main aim of the research reported here was to identify and characterize transcription factor binding sites for the NF-KB transcription factor family members, cReI and RelA on a genome-wide scale. Firstly, ChIP assays were developed and used to investigate the kinetics of c-Rel and RelA binding at the known inducible interleukin-Z (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) proximal promoter regions in EL-4 and primary CD4+ T cells. This study showed that the two transcription factors have distinct kinetics of binding in activated T cells, suggesting that they may play distinct roles in the control of gene expression in T cells. Secondly, ChIP was expanded to a genome-wide scale by combining with DNA microarray (ChIP-on-chip), which identified the c-Rel and RelA binding regions across the genome. These binding regions were located not only upstream of the transcription start site (TSS) of genes but also within genes. c-Rel and RelA bound to an overlapping set of regions and while the RelA binding regions were enriched for NF-kB consensus sequences, the c-Rel regions were not. ChIP-on-chip combined with expression profiling data revealed that genes located near c-Rel binding regions were more likely to be responsive to T cell activation and/or c-Rel overexpression compared with the entire gene set on the expression array. However, only a relatively small number of c-Relbinding genes were inducible. Validation studies showed that about 31% of the c-ReI binding regions tested bound c-Rel in ChIP-PCR assays and the expression of a small number of these genes was decreased in the absence of c-Rel. Finally, c-Rel binding regions were enriched for IRF transcription factor binding regions and IRF-1 also bound to these regions. Taken together, these studies demonstrated that two NF:kB proteins have distinct binding profiles in activated T cells and identified binding regions of these transcription factors across the genome. c-Rel target genes were identified and a link between c-Rel and IRF binding regions was elucidated