Differential and overlapping targets of the transcriptional regulators NRF1, NRF2, and NRF3 in human cells.

The nuclear factor (erythroid 2)-like (NRF) transcription factors are a subset of cap'n'collar transcriptional regulators. They consist of three members, NRF1, NRF2, and NRF3, that regulate the expression of genes containing antioxidant-response elements (AREs) in their promoter regions. Although all NRF members regulate ARE-containing genes, each is associated with distinct roles. A comprehensive study of differential and overlapping DNA-binding and transcriptional activities of the NRFs has not yet been conducted. Here, we performed chromatin immunoprecipitation (ChIP)-exo sequencing, an approach that combines ChIP with exonuclease treatment to pinpoint regulatory elements in DNA with high precision, in conjunction with RNA-sequencing to define the transcriptional targets of each NRF member. Our approach, done in three U2OS cell lines, identified 31 genes that were regulated by all three NRF members, 27 that were regulated similarly by all three, and four genes that were differentially regulated by at least one NRF member. We also found genes that were up- or down-regulated by only one NRF member, with 84, 84, and 22 genes that were regulated by NRF1, NRF2, and NRF3, respectively. Analysis of the ARE motifs identified in ChIP peaks revealed that NRF2 prefers binding to AREs flanked by GC-rich regions and that NRF1 prefers AT-rich flanking regions. Thus, sequence preference, likely in combination with upstream signaling events, determines NRF member activation under specific cellular contexts. Our analysis provides a comprehensive description of differential and overlapping gene regulation by the transcriptional regulators NRF1, NRF2, and NRF3.