Research Spotlight

Abstract

Earlier, we showed that jumonji domain containing protein 6 (JMJD6) interacted with HOTAIR promoter (-123 to -103 bp, termed JMJD6 interaction region [JIR]) and for maximal induction, an additional (-216 to -123 bp) region was required. In silico prediction and ENCODE data from MCF7 cells showed Y-box interacting protein 1 (YBX1) peaks in this region (YIR). Publicly available mass spectrometry data of proteins following JMJD6 immunoprecipitation identified YBX1 as an interacting partner. In this study, we validate JMJD6-YBX1 interaction in breast cancer cell lines using co-immunoprecipitation assays with recombinant, endogenous and in vitro synthesized proteins. Domain mapping using deletion constructs revealed that the A/P domain of YBX1 interacted with the JMJC domain of JMJD6. These proteins also positively regulated each other's expression in breast cancer cell lines. Further, YBX1 augmented luciferase activity of HOTAIR promoter constructs, pHP216 and pHP123, in MCF7, Vec and JMJD6 overexpressing cells. siRNA-mediated depletion, mutation of YIR region or knocking out YBX1 (YKO cells) diminished luciferase activity. ChIP and ChIP-re-ChIP assays verified co-occupancy of both proteins in the HOTAIR promoter region. Electrophoretic mobility shift assays confirmed complex formation with YIR and JIR probes. Mutation of the YIR region and YKO resulted in loss of complex formation with both probes. Taken together, these data imply that YBX1 is crucial for physically recruiting JMJD6 to the HOTAIR promoter. Their interaction and positive feed-forward loop, perpetuated by JMJD6 and YBX1 inter-regulation, culminates in HOTAIR induction, which in turn is known to drive tumour progression.