The MLL3/MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers

Abstract

Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (complex of proteins associated with Set1) family from Saccharomyces cerevisiae to humans, and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which is enriched at transcription start sites in all eukaryotes. Our recent studies of Drosophila melanogaster demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 (GeneID 58508) and MLL4 (GeneID 8085), are most closely related to Drosophila Trr. Here, we use chromatin immunoprecipitation-sequencing (ChIP-seq) of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and find that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, we demonstrate that MLL3 and MLL4 are major regulators of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, we find a redundant role between Mll3 (GeneID 231051) and Mll4 (GeneID 381022) in enhancer H3K4 monomethylation in mouse embryonic fibroblast (MEF) cells. These findings suggest that mammalian MLL3 and MLL4 function in the regulation of enhancer activity and that mutations of MLL3 and MLL4 that are found in cancers could exert their properties through malfunction of these Trr/MLL3/MLL4-specific (Trrific) enhancers.

Fig 1

MLL3/MLL4 are required for bulk levels of H3K4me1 in HCT116 cells. (A) The COMPASS family of H3K4 methylases in Drosophila and mammals. There are three COMPASS family members in Drosophila (dCOMPASS) (14) (left panels). Mammals have six COMPASS members, two members for each Drosophila version (middle and right panels) (hCOMPASS, human COMPASS). The subunits common to all COMPASS family members are highlighted in gray. Complex-specific components are represented in blue, orange, and green for the Set1, Trx, and Trr subfamilies, respectively. Hcf1 (pink) is reported to be in Set1 and Trithorax subfamily complexes but not in the Trr family (40). (B) Schematic representation of the MLL4 alleles in parental HCT116 cells (MLL4^WT) and MLL4 disrupted HCT116 cells (MLL4^Δset) as generated previously (22). A DNA fragment harboring a stop codon was inserted upstream of the SET domain-coding exon of the MLL4 alleles (22). Parental HCT116 cells have a homozygous frameshift mutation before the SET domain of MLL3 (parental HCT116 cells were denoted as MLL3^Δset in subsequent figures), so the MLL4^Δset HCT116 cells lack methyltransferase activity for both MLL3 and MLL4 (designated MLL3^Δset/4^Δset in subsequent figures). (C) Deficiency of MLL4 results in a large reduction of H3K4me1 levels in HCT116 cells. Whole-cell lysates isolated from parental HCT116 cells and MLL3^Δset/4^Δset cells were analyzed by Western blotting using the indicated antibodies. MLL3^Δset/4^Δset cells show bulk reduction of H3K4me1 levels without a detectable effect on other histone modification levels globally. (D) Ectopic expression of MLL4 in MLL3^Δset/4^Δset cells restores the H3K4me1 levels similar to those in the parental HCT116 cells. vec, vector. (E) Mll4 mRNA levels after lentivirus-mediated knockdown in mouse embryonic fibroblasts (MEFs). Mll4 expression was determined with quantitative reverse transcription-PCR and is normalized to β-actin (Actb). (F) Mll3 functions redundantly with Mll4 in H3K4 monomethylation in MEFs. Mll4 was knocked down in wild-type and Mll3 knockout (KO) MEFs, respectively, and whole-cell lysates were analyzed by Western blotting with the indicated antibodies. Note that Mll4 knockdown reduces H3K4me1 level in Mll3 knockout MEFs, while its reduction in WT cells demonstrates no detectable effect on H3K4me1 levels.

Fig 2

MLL4 is preferentially associated with enhancer regions in HCT116 cells. (A) An MLL4 antibody was tested in immunoprecipitation (IP) to study the disruption pattern for MLL4. Note that the MLL4 antibody is capable of immunoprecipitating the core COMPASS subunit RBBP5 in the parental HCT116 cells but not in the MLL3^Δset/4^Δset cells. For a control, we observed that antibodies to MLL (MLL1) are capable of immunoprecipitating RBBP5 in both the parental HCT116 cells and the MLL3^Δset/4^Δset cells. The MLL3 gene is disrupted in HCT116 cells, and accordingly, antibodies to MLL3 can only bring down the background levels of RBBP5. (B) Since our studies in panel A confirmed the specificity of MLL4 antibodies, we tested MLL4 distribution pattern in HCT116 cells using these antibodies. (Left) Pie chart showing the percentage of MLL4 peaks that overlap the transcription start site (TSS), within a gene (Intragenic), or upstream or downstream of the nearest gene. (Right) Positions of MLL4 non-TSS peak summit to the TSS of nearest genes. (C) A representative genomic snapshot of MLL4 peaks showing that MLL4 colocalizes with p300 and H3K4me1 at enhancer regions (red box) or overlaps with H3K4me3 in promoter regions (green box). Histone H3K27ac ChIP-seq data in HCT116 cells are from Frietze et al. (34). (D) Enrichment of binding profiles for MLL4, p300, H3K4me1, H3K4me3, and H3K27ac are shown within 50 kb around the TSS of the MLL4 nearest gene. (E) KEGG pathway analysis of the nearest genes to MLL4 peaks.

Fig 3

Histone H3K4me1 is diminished at enhancer regions in the absence of MLL4. (A) Genome browser track examples for H3K4me1, H3K4me2, and H3K4me3 occupancy in the parental HCT116 and MLL3^Δset/4^Δset cells. Published ChIP-seq data for H3K27ac in HCT116 cells (34) are used to help identify enhancer regions. Chr., chromosome. (B) Coverage profiles of H3K4me1, H3K4me2, and H3K4me3 in parental HCT116 and MLL3^Δset/4^Δset cells. A total of 27,341 putative enhancers and 13,095 TSS with H3K4me1 enrichment in parental HCT116 cells are shown. A region within 5 kb around the center of each putative enhancer (top panel) or transcription start site (TSS) (bottom panel) is displayed. Regions are sorted from highest to lowest H3K4me1 occupancy in parental HCT116 cells. (C) Quantitation of the H3K4 methylation states for the same 5-kb window around putative enhancers from panel B. (D) Quantitation of the H3K4 methylation states for the same 5-kb window around the TSS from panel B.

Fig 4

Existence of MLL3/MLL4-independent enhancers in HCT116 cells. (A) Coverage profiles showing a large percentage of putative enhancers exhibit a significant loss in H3K4me1 (P < 1e−3) in the absence of MLL3/MLL4 (we call these enhancers Trr/MLL3/MLL4-specific [Trrific] enhancers), while a minority of putative enhancers is independent of MLL3/MLL4 (we call these enhancers Trr/MLL3/MLL4-independent enhancers). (B) Gene ontology (GO) analysis of genes closest to Trrific enhancers and Trr/MLL3/MLL4-independent enhancers. Benjamini-corrected P values are shown.

Fig 5

Mll4 occupies enhancers in mouse embryonic stem cells. (A) Pie charts showing the genomic distribution of Mll4 peaks. (Left) Percentages of Mll4 peaks that overlap transcription start sites (TSS), reside within gene bodies, or are upstream or downstream of the nearest genes are indicated. (Right) Positions of Mll4 non-TSS peak summit to the TSS of the nearest genes. (B) Genome browser track examples showing Mll4 binding to regions surrounding Nanog and Lefty1 loci in mouse embryonic stem cells. Previously described enhancer regions are shown outlined in a red box. Numbers on the y axis are in counts per million. (C) Enrichment profiles showing a global view of Mll4 binding sites to their 5,522 nearest genes. Since our antibodies were generated toward human MLL4, its strength in mouse cells is lower than its strength in human cells. The high-confidence peaks for Mll4, H3K4me1, p300, H3K27ac, and H3K4me3 are shown for regions 50 kb upstream and downstream of the TSS for the genes nearest Mll4 in mouse ES cells. (D) GO term enrichment analysis for the genes nearest Mll4 peaks identifies several biological processes associated with development.

Fig 6

A model for differential genomic localization of histone H3K4me3 and H3K4me1 and implementation by distinct branches of the COMPASS family. The MLL3/4 branches of the COMPASS family are major histone H3K4 monomethylases (H3K4me1) at enhancers. In contrast, SET1A/B-COMPASS branches function as H3K4 trimethylases (H3K4me3) at promoters.