HOXC6 Is Transcriptionally Regulated via Coordination of MLL Histone Methylase and Estrogen Receptor in an Estrogen Environment

Homeobox (HOX)-containing gene HOXC6 is a critical player in mammary gland development and milk production, and is overexpressed in breast and prostate cancers. We demonstrated that HOXC6 is transcriptionally regulated by estrogen (E2). HOXC6 promoter contains two putative estrogen response elements (EREs), termed as ERE11/2 and ERE21/2. Promoter analysis using luciferase-based reporter assay demonstrated that both EREs are responsive to E2, with ERE11/2 being more responsive than ERE21/2. Estrogen receptors (ERs) ERα and ERβ bind to these EREs in an E2-dependent manner, and antisense-mediated knockdown of ERs suppressed the E2-dependent activation of HOXC6 expression. Similarly, knockdown of histone methylases MLL2 and MLL3 decreased the E2-mediated activation of HOXC6. However, depletion of MLL1 or MLL4 showed no significant effect. MLL2 and MLL3 were bound to the HOXC6 EREs in an E2-dependent manner. In contrast, MLL1 and MLL4 that were bound to the HOXC6 promoter in the absence of E2 decreased upon exposure to E2. MLL2 and MLL3 play key roles in histone H3 lysine-4 trimethylation and in the recruitment of general transcription factors and RNA polymerase II in the HOXC6 promoter during E2-dependent transactivation. Nuclear receptor corepressors N-CoR and SAFB1 were bound in the HOXC6 promoter in the absence of E2, and that binding was decreased upon E2 treatment, indicating their critical roles in suppressing HOXC6 gene expression under nonactivated conditions. Knockdown of either ERα or ERβ abolished E2-dependent recruitment of MLL2 and MLL3 into the HOXC6 promoter, demonstrating key roles of ERs in the recruitment of these mixed lineage leukemias into the HOXC6 promoter. Overall, our studies demonstrated that HOXC6 is an E2-responsive gene, and that histone methylases MLL2 and MLL3, in coordination with ERα and ERβ, transcriptionally regulate HOXC6 in an E2-dependent manner.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (93 K)Download as PowerPoint slideResearch Highlights► HOXC6 is transcriptionally regulated by E2. ► Histone methylases MLL2 and MLL3, along with ERs, play critical roles in the E2-mediated activation of HOXC6. ►MLL1 and MLL4, as well as nuclear receptor corepressors (N-CoR and SAFB1), are bound to the HOXC6 promoter in the absence of E2 and are replaced with MLL2 and MLL3 during E2-mediated gene activation.