Distribution of NF-κB-binding sites across human chromosome 22

Abstract

We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-B family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity, and inflammation. The binding sites of the NF-B family member p65 were determined by using chromatin immunoprecipitation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5 end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-B binding was also found at genes whose expression was regulated by tumor necrosis factor ␣, a known inducer of NF-B-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor ␣. Many of these latter genes are either known to be activated by NF-B under other conditions or are consistent with NF-B's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-B binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.U nderstanding the targets regulated by transcription factors and where they bind relative to these targets in an unbiased fashion in mammalian cells is highly desirable. We and others have developed a procedure for mapping in vivo targets of transcription factors by chromatin immunoprecipitation (ChIP) with antibodies to a transcription factor of interest to isolate protein-bound DNA, followed by probing a microarray containing genomic DNA sequences with the immunoprecipitated DNA (ChIP chip) (1-3). This approach was first used successfully in yeast and has more recently been used in a limited fashion to identify transcription factor binding sites in mammalian cells (4-6). However, a large-scale, unbiased global analysis of the distribution of mammalian transcription factor binding sites along large genomic regions has not been previously explored.In this study we employ a microarray containing the entire nonrepetitive sequence of chromosome 22 to determine the chromosome-wide binding profile for the transcription factor NF-B. The NF-B͞Rel family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including immune and inflammatory responses (7,8), and the activation pathway has been studied extensively over the last two decades (9, 10). Numerous NF-B target genes have also been identified; however, it remains unclear how many of these are direct targets of the transcription factor (11).There are five mammalian NF-B family members (p50, p52, RelA͞p65, RelB, and c-rel), all of which function as homo-or heterodimers. The different dimers exhibit varying binding aff...

Transcriptional regulation via the NF-κB signaling module

“…Most kB sites appear to be 10 bp in length with the consensus sequence 5 0 -GGGRN W YYCC-3 0 (where R denotes a purine base, N denotes any base, W denotes an adenine or thymine and Y denotes a pyrimidine base) (Sen and Baltimore, 1986;Chen and Ghosh, 1999). However, this degenerate kB DNA consensus sequence may still be more restrictive than what may be revealed by systematic in vitro (Linnell et al, 2004) and in vivo (Martone et al, 2003;Schreiber et al, 2006) studies. (However, in vivo studies do not distinguish between direct DNA-binding events and protein-protein interaction-mediated promoter association by NF-kB).…”

“…Since then, high throughput technologies have revealed that the vast majority of the inflammatory gene expression program is NF-kBdependent (Hoffmann and Baltimore, 2006), and that DNA-bound NF-kB can be detected at many chromosomal locations (Martone et al, 2003;Schreiber et al, 2006). Given NF-kB's critical involvement in so many physiological responses, a major challenge of current research is to elucidate how NF-kB signaling in response to diverse stimuli can mediate distinct cellular responses in particular physiological contexts.…”

Crosstalk via the NF-κB signaling system

“…Interestingly, a recent study, revealed that in dendritic cells TLR4 induced expression of inflammatory or T-cell stimulatory genes require distinct RelA or cRel subunits (Wang et al, 2007), suggesting that there is a regulatory code for canonical NF-κB dimers that remains to be elucidated. Given the abundance of NF-κB (more than 100,000 molecules per cell), it may not be surprising that genome-wide chromatin immunoprecipitation (ChIP) analysis revealed a large number of unexpected binding sites of unknown functional relevance (Martone et al, 2003;Schreiber et al, 2006). Therefore, such chromatin location studies must be coupled to functional analysis using genetic perturbations (knockouts and knock-in mutants) to elucidate the transcriptional regulatory code of NF-κB.…”

A plethora of sites

“…Several groups recently adapted this technology to identify transcription factor binding sites in human cells for selected regions of the human genome 3-5 . Two teams, one involving a collaboration between Affymetrix and Harvard 6 and the other at Yale 7,8 , have now extended this technology to map binding sites along entire human chromosomes.Martone et al 7 and Euskirchen et al 8 mapped the binding sites of NF-κB (p65) and CREB, respectively, along human chromosome 22, and Cawley et al 6 mapped the binding sites of Sp1, c-Myc and p53 on chromosomes 21 and 22. In each case, genomic tiling arrays were used.…”

“…Several groups recently adapted this technology to identify transcription factor binding sites in human cells for selected regions of the human genome [3][4][5] . Two teams, one involving a collaboration between Affymetrix and Harvard 6 and the other at Yale 7,8 , have now extended this technology to map binding sites along entire human chromosomes.…”