The Function And Molecular Mechanisms Of JMJD6 In Breast Cancer And Its Drug Screening

Regulation of enhancer activity is important to controlling gene expression programs,but the mechanisms in mediating enhancer activation remain poorly understood.Here,we report that recruitment of the JmjC domain-containing protein 6(JMJD6)to estrogen receptor alpha(ER?)bound active enhancers is required for RNA polymerase II recruitment and enhancer RNA production on enhancers,resulting in transcriptional pause release of cognate estrogen target genes.JMJD6 was found to interact with MED 12 in the Mediator complex to regulate its recruitment.Unexpectedly,JMJD6 is necessary for MED 12 to interact with CARM1,which methylates MED 12 at multiple arginine sites and regulates its chromatin binding.JMJD6 is required for estrogen/ER?-induced breast cancer cell growth and tumorigenesis,which is dependent on its enzymatic activity.However,due to the absence of any selective inhibitors,the mechanisms of the demethylase activity of JMJD6 in regulating cellular responses remains poorly understood.In this study,following our structure-based virtual screening protocol,several small molecule inhibitors capable of inhibiting JMJD6 demethylase activity were discovered.Among them WL12 exhibited direct binding interactions to JMJD6.The IC50 of WL12 targeting JMJD6 was measured by in-vitro demethylation assay.Moreover,WL12 exhibited selectivity over other JmjC domaincontaining proteins,such as KDM3A,KDM4C,KDM5C and KDM7B.WL12 treatment led to a significant increase of HSP70R469me1,which was presumably through inhibiting JMJD6.WL12 was shown to be able to suppress JMJD6-dependent cancer cell proliferation,including cervical and liver cancer cells.Importantly,this compound exhibited selectivity over normal cells.Our results have uncovered a critical regulator of estrogen/ER?-induced enhancer coding gene activation and breast cancer cell potency,providing a potential therapeutic target of cancers.WL12 is potent and JMJD6-selective inhibitor that can be used to physiologically and pathologically investigate the roles of JMJD6 demethylation.