JMJD6 and YBX1 physically interact and regulate HOTAIR proximal promoter

Abstract

In a previous study, we showed JMJD6 interacted with HOTAIR promoter (-123 to -103 bp upstream of TSS, JIR) and augmented its transcription. Maximal JMJD6 mediated induction required (-216 to -123 bp) region. In silico prediction and ENCODE data suggested that YBX1 could be that potential candidate and in this study the region is designated as YBX1 interacting region (YIR). In breast cancer cell lines, we show that JMJD6 and YBX1 regulate each others expression and physically interact with each other when recombinantly expressed, as endogenous proteins and when synthesized in vitro. Domain mapping indicated that A/P domain of YBX1 interacted with JMJC domain of JMJD6. Luciferase activity of HOTAIR promoter constructs, pHP216 and pHP123, increased in presence of YBX1 in MCF7, Vec and JMJD6 overexpressing JOE cells but was lost in the presence of JMJD6 and YBX1 siRNAs. Interestingly, activity of pHP123 that lacks YIR also decreased upon YBX1 knock out (YKO). Next, by individual JMJD6, YBX1 and ChIP-re-ChIP assays we demonstrate that both proteins co-occupy this promoter region. Further, electrophoretic mobility shift assays showed that YIR probes retarded two complexes, which lost intensity in YKO cells. Interestingly, JIR-protein complex disappeared in YKO cells. Together these data imply that YBX1 not only enhanced promoter activity but may also be involved in JMJD6 recruitment. Taken together, our data proposes that the interaction and positive feed forward loop perpetuated by JMJD6 and YBX1 may culminate in HOTAIR induction, which in turn is known to drive tumor progression.