The COMPASS protein family catalyzes histone H3 Lys 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells. We found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, DNA demethylation on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4 trimethylation underlies transcriptional regulation on MLL2/COMPASS targets.
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RNA-seq, ChIP–seq and bisulfite sequencing raw data are available in the Gene Expression Omnibus database under accession GSE129037. Additional data supporting the findings of this study are available from the corresponding author upon request. Source data for Figs. 2 and 6 and Extended Data Figs. 2, 3 and 5 (full scans of the immunoblots) and Figs. 5 and 7 (statistical source data) are provided with the paper.
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We thank the Shilatifard laboratory members for helpful suggestions and discussions. K.A.H. and B.D.S. are supported by NIH K08HL128867. C.C.S. is supported, in part, by the NIH Predoctoral to Postdoctoral Transition Award F99CA234945. K.C. is supported, in part, by the NIH Pathway to Independence Award K99HD094906. A.P. is supported by the NIH Pathway to Independence Award K99CA234434. E.R.S. is supported by NIH R50CA211428. We thank N. J. Ethen for the graphical representation of the model (Fig. 8). Studies in the Shilatifard laboratory related to COMPASS are supported by the NCI Outstanding Investigator Award R35CA197569.
The authors declare no competing interests.
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a–h, Interleaved scatter plot of indicated genes’ normalized RNA-seq counts in the indicated conditions for the genes Magohb (a), Zdhhc2 (b), Zdhhc2(c), Sh3bgrl2 (d), Actr10 (e), Dnmt1 (f), Dnmt3a (g) and Crabp1 (h) (n = 2). CPM, counts per million.
a, FACS analysis of mCherry-Magohb MLL2KO mESCs transformed with an empty vector or a MLL2 expressing vector. The experiment was repeated three times independently with similar results. b, Western blot for FLAG-MAGOHB expression in WT cells expressing a shRNA control (shNT) or a shRNA targeting MLL2 (shMLL2). The experiment was repeated two times independently with similar results. c, Western blot for CAS9, FLAG-MAGOHB and TUBULIN (loading control) protein levels in WT, Magohb KI WT cells (WT-MagohbKI) and Magohb KI MLL2KO cells (MLL2KO-MagohbKI). The experiment was repeated two times independently with similar results. d, Top panel: mCherry high cells were isolated via two successive rounds of FACS in MLL2KO mESCs. Bottom panel: mCherry low cells were isolated via two successive rounds of FACS in WT mESCs. The experiment was repeated three times independently with similar results. Uncropped gels are available as source data. Source data
a, CRISPR screen results representing the number of enriched sgRNA in the ‘Sorted cells’ compared to the ‘Total population’ per gene for WT screen. The majority of the genes fall into the category ‘0 sgRNA enriched out of 4’. b, Expression levels of Magohb as assessed by reverse transcription quantitative PCR in MLL2KO mESCs expressing a control sgRNA (Control) or sgRNAs targeting various genes (n = 2). c, Western blot of MAGOHB and HSP90 (loading control) protein levels. Two different exposures are shown. The experiment was repeated two times independently with similar results. d, Quantification of two independent replicates of Extended Data Fig. 2c western blots. Uncropped gels are available as source data. Source data
a, Boxplot analysis of cluster1 gene expression in 1) MLL2KOshNT vs. WTshNT mESCs and 2) MLL2KOshCXXC1 vs. WTshNT mESCs. P values were computed using Wilcoxon test (two-sided), n = 2,021. The boxplots indicate the median (middle line), the third and first quartiles (box) and the first and fourth quartiles (error bars). b, Donut chart of cluster 1 distribution of genes based on their level of rescue in MLL2KO shCXXC1 cells compared to WT cells. ‘Full rescue’ genes were characterized by a log2FC ≥ 0 comparing MLL2KOshCXXC1 to WT mESCs, ‘Partial rescue’ genes were characterized by a log2FC > 0 comparing MLL2KOshCXXC1 and MLL2KO mESCs and ‘No rescue’ were characterized by a log2FC ≤ 0 comparing MLL2KOshCXXC1 and MLL2KO mESCs. c, RNA-seq tracks for Zdhhc2 in WT, MLL2KOshNT, MLL2KOshCXXC1, MLL2ΔSETshNT, MLL2ΔSETshCXXC1 and TMutant. The experiment was repeated two times independently with similar results. d, Boxplot analysis of cluster1 gene expression in 1) MLL2ΔSETshNT vs. WTshNT mESCs and 2) MLL2ΔSETshCXXC1 vs. WTshNT mESCs, 3) TMutantshNT vs. WTshNT mESCs, 4) TMutantshCXXC1 vs. WTshNT mESCs and 5) MLL2KOshCXXC1 vsTMutantshNT mESCs (n = 2).
a, Western blot of DNMT1, DNMT3A and HSP90 (loading control) protein levels in WTshNT, MLL2KOshNT and MLL2KOshCXXC1 mESCs. The experiment was repeated two times independently with similar results. b, RNA-seq and SET1A ChIP-seq tracks for Dnmt3a. The experiment was repeated two times independently with similar results. c, Western blot of DNMT1 and HSP90 (loading control) protein levels in WTsgNT, MLL2KOsgNT and MLL2KOsgDnmt1. The experiment was repeated two times independently with similar results. d, Heatmaps show the corresponding log2 fold changes in gene expression in 1) MLL2KO vs. WT mESCs, 2) MLL2KO sgDnmt1 vs. MLL2KOsgNT mESCs and 3) MLL2KO treated for 4 days with 100 nM 5dAza vs. MLL2KO mESCs. e, Box-and-Whisker plot quantifying changes in CpG methylation around TSS (±3 kb) of cluster2 clusters identified in Fig. 3a (N = 3 biological replicates, n = 13420). The boxplots indicate the median (middle line), the third and first quartiles (box) and the first and fourth quartiles (error bars). f, Flow cytometry on mCherry-Magohb KI WT or MLL2KO mESCs expressing a dead Tet1 catalytic domain (dTet1) or an active Tet1 catalytic domain (Tet1) targeted to a control region 5 kb upstream Magohb or Magohb promoter. The experiment was repeated two times independently with similar results. Uncropped gels are available as source data. Source data
a, Heatmaps show the corresponding log2 fold changes in gene expression in 1) MLL2KO vs. WT mESCs, 2) MLL2KOSUZ12KO#1 vs. MLL2KO mESCs, 3) MLL2KOSUZ12KO#2 vs. MLL2KO mESCs, 4) MLL2KOSUZ12KO#3 vs. MLL2KO mESCs and 5) MLL2KOSUZ12KO#4 vs. MLL2KO mESCs. The experiment was repeated two times independently with similar results. b, ChIP-seq tracks of H3K27me3 occupancy at the Snx24 locus in WT, MLL2KO and MLL2KO 100 nM 5dAza for 4 days treated mESCs. The experiment was repeated two times independently with similar results.
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Douillet, D., Sze, C.C., Ryan, C. et al. Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb and DNA methylation. Nat Genet 52, 615–625 (2020). https://doi.org/10.1038/s41588-020-0618-1
Nature Reviews Genetics (2020)