NRF2/long noncoding RNA ROR signaling regulates mammary stem cell expansion and protects against estrogen genotoxicity.

Long noncoding RNAs (lncRNAs) have emerged as key regulators of gene expression in embryonic stem cell (ESC) self-renewal and differentiation. In ESCs, lncRNAs are regulated at the genetic level via transcription factor binding to lncRNA gene promoters. Here we demonstrate that the key cytoprotective transcription factor NRF2 controls lncRNA expression in mammary stem cells. By profiling lncRNAs in wild-type and NRF2 knockdown mammary stem cells, we demonstrate that the lncRNA ROR, a regulator of embryonic stem cell pluripotency, is overexpressed upon NRF2 knockdown. We performed promoter analyses and examined predicted NRF2 binding elements in the ROR promoter using luciferase reporter constructs of a ROR promoter deletion series. Our studies revealed that NRF2 binds to two specific NRF2 response elements flanking the ROR promoter and that these two NRF2 response elements are equally important to suppress ROR transcription. In addition, we identified associated H3K27me3 chromatin modification and EZH2 binding at the ROR promoter that was dependent on NRF2 binding. We observed that NRF2 knockdown or ROR overexpression leads to increased stem cell self-renewal in mammary stem cells. Furthermore, we demonstrate Nrf2 regulation of the mammary stem cell population in vivo. These observations provide further evidence for the critical role of NRF2 in maintaining normal stem cell subpopulations in mammary epithelium.