(a) The COMPASS family of H3K4 methylases in Drosophila (three members; left) and mammals (six members; right). Subunits common to all COMPASS family members in eukaryotes fromyeast to human are shown in green; complex-specific subunits are shown in blue, orange and purple. Magenta indicates Host Cell Factor (HCF1), a substochiometric component found in some branches of the COMPASS family. (b) Mll2 and Mll3 mRNA levels after RNAi-mediated knockdown in mouse embryonic stem cells with shRNAs targeting GFP (shGFP), Mll3 (shMll3) or Mll2 (shMll2). Expression determined by quantitative reverse-transcription PCR is shown relative to β-actin (Actb). Bars indicate range of values for two technical replicates, representative of three biological-replicate experiments. (c) ChIP-seq track file examples of H3K4me3 at mouse Hox gene clusters. Red and black bars above the tracks indicate bivalent and nonbivalent regions, respectively. C.p.m., counts per million mapped reads. H3K27me3 data from Mikkelsen et al.4 are shown for comparison (purple). (d) ChIP-seq track examples of bivalent and nonbivalent chromatin in control and Mll2-shRNA–treated cells. Bivalently marked genes such as Prr18, Brachyury (T ), Vgll4 and Syn2 are shown with red bars above the tracks as in (c).
(a) H3K4me3 occupancy change in Mll2- and Mll3-depleted mouse embryonic stem cells for all H3K4me3-enriched genes. Left, ChIP-seq enrichment profiles for ±5 kb around the TSS of all H3K4me3-enriched genes. Bivalent genes are shown as a separate group from the H3K4me3-only modified genes. Right, H3K4me3 occupancy log2 fold change after depletion of Mll2 (shMll2/shGFP) or Mll3 (shMll3/shGFP), measured ±5 kb around the TSS (indicated by arrowheads). (b) Percentage of bivalent and nonbivalent genes with H3K4me3 occupancy loss. The percentages are shown at four levels: total H3K4me3-enriched genes, genes with >50% decrease of H3K4me3, genes with >75% decrease and genes with >87.5% decrease. Numbers shown are the total number of genes for each level. H3K27me3 data in a and b are from Wamstad et al.20. (c) Average gene occupancy plots of H3K4me3 in shGFP (blue), shMll2 (red) and shMll3 (green) knockdown embryonic stem cells. Top and bottom panels show nonbivalent and bivalent genes, respectively. Plots are averaged from both H3K4me3 ChIP-seq biological replicates. (d) Gene ontology (GO) analysis of genes with H3K4me3 loss after Mll2 knockdown. Benjamini-corrected P values as reported by DAVID21, 22 are shown.
(a) Expression analysis of mouse V6.5 embryonic stem cells infected with GFP-shRNA (shGFP) or Mll2-shRNA (shMll2) lentivirus and induced to differentiate with 2 μM retinoic acid for 6 h (RA 6) or 12 h (RA 12). Heat maps of expression and H3K4me3 occupancy are shown. Genes with expression increased by 6 h and 12 h of RA treatment are sorted by the wild-type occupancy of H3K4me3 and separated as bivalent or not bivalent. Left, expression heat map in control and Mll2-shRNA–treated cells. r.p.k.m., reads per kilobase per million mapped reads. Middle, log2 fold changes for expression after Mll2 knockdown. Induction defects would appear as an increase in green in the 6 and 12 h time points. Right, log2 fold change of H3K4me3 occupancy. (b) Scatter plots of log2 fold changes in expression after RA induction at 6 h (top) or 12 h (bottom). Each dot represents a gene that was induced by 6 h. Correlation coefficients are calculated in the log2 scale. (c) Gene expression track examples before and after RA induction. (d) Induction kinetics at one and three hours of RA treatment. Bars represent the range of values for two technical replicates, representative of three biological replicate experiments. A second Mll2-shRNA with an alternate sequence is denoted shMll2_2.
The Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells
Deqing Hu, Alexander S Garruss, Xin Gao, Marc A Morgan, Malcolm Cook, Edwin R Smith & Ali Shilatifard
Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalentlymarked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation.