MLL4 establishes super-enhancers and broad H3K4me3 for tumor-suppressive function

Clusters of enhancers called super-enhancers are associated with gene activation. Broad trimethyl histone H3 lysine 4 (H3K4me3) often defines actively transcribed tumor suppressor genes. However, how these epigenetic signatures are regulated for tumor suppression is poorly understood. Here, we show that brain-specific knockout of the H3K4 methyltransferase MLL4 (aka KMT2D) in mice spontaneously induces cerebellar tumors in brain while indirectly increasing expression of oncogenic programs, such as Ras activators and Notch pathway components. Mll4 loss caused widespread impairment of super-enhancers and broad H3K4me3. Notably, Mll4 loss reduced super-enhancer and broad H3K4me3 signals in tumor suppressor genes co-marked by both signatures, including Dnmt3a and Bcl6. MLL4 upregulates DNMT3A-mediated DNA methylation to downregulate expression of Ras activators and increases Bcl6 expression to suppress the Notch pathway. These findings suggest an unanticipated epigenetic tumor-suppressive mechanism in which MLL4 is required for establishing super-enhancers and broad H3K4me3 for anti-tumor programs in normal cells. Expression profiling by RNA-seq, profiling of Mll4f/f (Mll4_WT) and Mll4 BSKO (Mll4 KO) from one month and 4 months mice cerebellum, and histone modifications (H3K4me1/3, H3K27ac, and Mll4) by ChIP-seq, at four months cerebellum. ChIP assays were performed as a modification of the previously described methods (Dhar et al., 2016; Dhar et al., 2012). In brief, cerebellar (4-month-old) tissues were isolated. DNA was purified from chromatin immunoprecipitates for MLL4 or IgG using the phenol/chloroform extraction. IgG was used as a negative control for ChIP (samples not included here). Then, DNA was amplified by quantitative PCR and normalized to input. To compare H3K4me1, H3K27ac, and H3K4me3 levels between Mll4f/f and Mll4 BSKO cerebellum, ChIP with reference exogenous genome (ChIP-Rx) was carried out as previously described (Li et al., 2016; Orlando et al., 2014). This series includes H3K4me1, H3K27ac, and H3K4me3 ChIP-seq data of cerebellar neurospheres.