Search for functional NF-kB binding sites via meta-analysis of NGS experiments in human cell lines
The NF-kB family of transcription factors plays the critical role in inflammation, immunity, cell proliferation, differentiation and metastasis. NF-kB dimers recognize 9-11 nucleotide sequences called kB sites. There are about 13600 human genes with kB sites in promoter region; however not all of them are transcriptionally active. Thus search for functional kB sites could assist in understanding the basic principles that underlie NF-kB regulation.
The combination of chromatin immunoprecipitation (ChIP) and next-generation sequencing (NGS), namely ChIP-seq, has become a powerful technique to capture potential genomic binding sites of transcription factors, histone modifications and chromatin accessible regions. Using ChIP-seq datasets deposited in the public databases, such as GEO and ENCODE we revealed physical binding sites of p65 (RelA) NF-kB subunit in various human cell lines: MCF-7, HUVEC, HeLa, SGBS and A549. For this purpose, we compared datasets for untreated cells and TNF-alpha-stimulated cells which contain activated p65 (RelA) protein.
The effect of binding of NF-kB with predicted kB-sites on gene transcription in cell lines listed above was confirmed by RNA-seq data. The analysis of accessibility of genomic regions to NF-kB binding was carried out using epigenetic ChIP-seq data. Regulatory elements of the genes were distinguished by the histone modifications (H3K4me1, H3K4me3, H3K27ac and H3K36me3). The analysis revealed a consistent pattern of the regulation of NF-kB- dependent transcription, the correlations of histone modifications and localization and consensus sequence of kB-sites. Regulatory elements (promoters and enhancers) were identified genes via the chromatin context information. Obtained data can be used for experimental validation of NF-kb -dependent regulation mechanisms by the binding kB-sites in the regulatory regions.