Massive GGAAs in genomic repetitive sequences serve as a nuclear reservoir of NF-κB

Nuclear factor kB（NF-kB） is a DNA-binding transcription factor. Characterizing its genomic binding sites is crucial for understanding its gene regulatory function and mechanism in cells. This study characterized the binding sites of NF-kB ReIA/p65 in the tumor neurosis factor-a（TNFa） stimulated HeLa cells by a precise chromatin immunoprecipitation-sequencing（ChIP-seq）. The results revealed that NF-kB binds nontraditional motifs（nt-motifs） containing conserved GGAA quadruplet. Moreover, nt-motifs mainly distribute in the peaks nearby centromeres that contain a larger number of repetitive elements such as satellite, simple repeats and short interspersed nuclear elements（SINEs）. This intracellular binding pattern was then confirmed by the in vitro detection, indicating that NF-kB dimers can bind the nontraditional kB（nt-KB） sites with low affinity. However, this binding hardly activates transcription.This study thus deduced that NF-kB binding nt-motifs may realize functions other than gene regulation as NF-kB binding traditional motifs（t-motifs）. To testify the deduction, many ChIP-seq data of other cell lines were then analyzed. The results indicate that NF-kB binding nt-motifs is also widely present in other cells. The ChIP-seq data analysis also revealed that nt-motifs more widely distribute in the peaks with low-fold enrichment. Importantly, it was also found that NF-kB binding nt-motifs is mainly present in the resting cells, whereas NF-kB binding t-motifs is mainly present in the stimulated cells. Aston？ishingly, no known function was enriched by the gene annotation of nt-motif peaks. Based on these results, this study proposed that the nt-KB sites that extensively distribute in larger numbers of repeat elements function as a nuclear reservoir of NF-kB. The nuclear NF-kB proteins stored at nt-KB sites in the resting cells may be recruited to the t-KB sites for regulating its target genes upon stimulation.