Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals1,2,3. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.
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Herz, H.M. et al. Enhancer-associated H3K4 monomethylation by Trithorax-related, the Drosophila homolog of mammalian Mll3/Mll4. Genes Dev. 26, 2604–2620 (2012).
Hu, D. et al. The MLL3/MLL4 branches of the COMPASS family function as major histone H3K4 monomethylases at enhancers. Mol. Cell. Biol. 33, 4745–4754 (2013).
Shilatifard, A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annu. Rev. Biochem. 81, 65–95 (2012).
Banerji, J., Rusconi, S. & Schaffner, W. Expression of a β-globin gene is enhanced by remote SV40 DNA sequences. Cell 27, 299–308 (1981).
Fukaya, T., Lim, B. & Levine, M. Enhancer control of transcriptional bursting. Cell 166, 358–368 (2016).
Levine, M., Cattoglio, C. & Tjian, R. Looping back to leap forward: transcription enters a new era. Cell 157, 13–25 (2014).
Smith, E. & Shilatifard, A. Enhancer biology and enhanceropathies. Nat. Struct. Mol. Biol. 21, 210–219 (2014).
Dorsett, D. Distant liaisons: long-range enhancer–promoter interactions in Drosophila. Curr. Opin. Genet. Dev. 9, 505–514 (1999).
Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).
Heintzman, N.D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).
Lee, J.E. et al. H3K4 mono- and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation. eLife 2, e01503 (2013).
Piunti, A. & Shilatifard, A. Epigenetic balance of gene expression by Polycomb and COMPASS families. Science 352, aad9780 (2016).
Lawrence, M.S. et al. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature 505, 495–501 (2014).
Sedkov, Y. et al. Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein. Mech. Dev. 82, 171–179 (1999).
Takahashi, Y.H. et al. Regulation of H3K4 trimethylation via Cps40 (Spp1) of COMPASS is monoubiquitination independent: implication for a Phe/Tyr switch by the catalytic domain of Set1. Mol. Cell. Biol. 29, 3478–3486 (2009).
Collins, R.E. et al. In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J. Biol. Chem. 280, 5563–5570 (2005).
Kanda, H., Nguyen, A., Chen, L., Okano, H. & Hariharan, I.K. The Drosophila ortholog of MLL3 and MLL4, trithorax related, functions as a negative regulator of tissue growth. Mol. Cell. Biol. 33, 1702–1710 (2013).
Eissenberg, J.C. et al. The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase. Nat. Struct. Mol. Biol. 14, 344–346 (2007).
Gildea, J.J., Lopez, R. & Shearn, A. A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156, 645–663 (2000).
Kagey, M.H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).
Michaelis, C., Ciosk, R. & Nasmyth, K. Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35–45 (1997).
Phillips-Cremins, J.E. et al. Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153, 1281–1295 (2013).
Rollins, R.A., Korom, M., Aulner, N., Martens, A. & Dorsett, D. Drosophila nipped-B protein supports sister chromatid cohesion and opposes the stromalin/Scc3 cohesion factor to facilitate long-range activation of the cut gene. Mol. Cell. Biol. 24, 3100–3111 (2004).
Rollins, R.A., Morcillo, P. & Dorsett, D. Nipped-B, a Drosophila homologue of chromosomal adherins, participates in activation by remote enhancers in the cut and Ultrabithorax genes. Genetics 152, 577–593 (1999).
Gause, M., Misulovin, Z., Bilyeu, A. & Dorsett, D. Dosage-sensitive regulation of cohesin chromosome binding and dynamics by Nipped-B, Pds5, and Wapl. Mol. Cell. Biol. 30, 4940–4951 (2010).
Mohan, M. et al. The COMPASS family of H3K4 methylases in Drosophila. Mol. Cell. Biol. 31, 4310–4318 (2011).
Jang, Y., Wang, C., Zhuang, L., Liu, C. & Ge, K. H3K4 methyltransferase activity is required for MLL4 protein stability. J. Mol. Biol. 429, 2046–2054 (2017).
Dorighi, K.M. et al. Mll3 and Mll4 facilitate enhancer RNA synthesis and transcription from promoters independently of H3K4 monomethylation. Mol. Cell 66, 568–576 (2017).
Wang, C. et al. Enhancer priming by H3K4 methyltransferase MLL4 controls cell fate transition. Proc. Natl. Acad. Sci. USA 113, 11871–11876 (2016).
Papp, B. & Müller, J. Histone trimethylation and the maintenance of transcriptional ON and OFF states by trxG and PcG proteins. Genes Dev. 20, 2041–2054 (2006).
Rickels, R. et al. An evolutionary conserved epigenetic mark of Polycomb response elements implemented by Trx/MLL/COMPASS. Mol. Cell 63, 318–328 (2016).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Lawrence, M. et al. Software for computing and annotating genomic ranges. PLOS Comput. Biol. 9, e1003118 (2013).
Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Shen, L., Shao, N., Liu, X. & Nestler, E. ngs.plot: quick mining and visualization of next-generation sequencing data by integrating genomic databases. BMC Genomics 15, 284 (2014).
Saldanha, A.J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).
We thank members of the Shilatifard laboratory for their critical feedback during preparation of the manuscript and L. Shilatifard for editorial assistance. R.R. is supported by the NIH/NCI under the Ruth L. Kirschstein National Research Service Award (F31CA213928). A.P. is an EMBO postdoctoral fellow (ALTF 372-2015), and his work in the Shilatifard laboratory is supported by AIRC and Marie Curie Actions–People–COFUND. H.-M.H. is supported by the NIH (R00CA181506). E.R.S. is supported by the NIH (R50CA211428). C.C.S. is supported in part by an NIH/NCI training grant (T32CA09560). Studies on Drosophila Trr and the mammalian MLL3 and MLL4 COMPASS-like family in the Shilatifard laboratory are supported in part by a generous Outstanding Investigator Award from the National Institute of Health to A.S. (R35CA197569).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Chromatograms confirming the correct mutations incorporated into trr genomic DNA and expressed as mRNA; relevant codons are highlighted in green. The catalytically inactive (C2398A) mutant is a TGC-to-GCC conversion, and the catalytically hyperactive (Y2383F) mutant is a TAC-to-TTC conversion. Note the overlapping peaks due to wild-type sequence at the endogenous trr locus (which is transcribed but truncates after 88 codons; only 3.6% of the full Trr protein). (b) The null trr allele has a point mutation (CAG>TAG) that generates a premature stop codon after the first 88 residues (Q89X). Chromatograms are shown confirming the nonsense mutation (red peak = T) present in mRNA from all three trr rescue lines (marked by a red arrowhead). Note the overlapping peaks due to lack of Q89X mutation in the three trr rescue constructs integrated into chromosome 3L.
Supplementary Figure 2 Disruption or conversion of Trr-dependent H3K4 methylation at enhancers in Drosophila.
(a) Western blots from lysates of adult fly brains confirm H3K4me1 bulk reduction in trr-C/A, and H3K4me2/3 bulk increase in trr-Y/F, similar to that shown in wing discs. (b) ChIP–seq data from adult fly brains plotted as average profiles, centered on non-TSS H3K27ac peaks; n = 1,440.(c) Box plots of Z-score averages comparing H3K4 methylation and H3K27ac at 5,367 active promoters in adult brain tissue. Plots are centered on H3K27ac peaks overlapping annotated TSSs, ±2.5 kb. P values are shown below. In comparison to the effects at enhancers, H3K4 methylation levels often show the opposite effect at TSSs. This is most likely an artifact due to redistribution of sequencing reads in the trr mutants.(d) Average meta-peak profiles for both Trr and Lpt at 6,807 genomic loci demonstrate that neither of the catalytic mutations substantially affects chromatin binding. (e) ChIP–seq track examples from wing imaginal discs demonstrate reduced H3K4me1 and H3K27ac in trr-C/A, as well as increased H3K4me3 and H3K27ac in trr-Y/F at putative enhancers, similar to adult brains. (f) Density bar plots comparing H3K4 methylation and H3K27ac Z scores at 787 active enhancers in wing imaginal discs. Plots are centered on wild-type H3K27ac (non-TSS) peaks, ±2.5 kb. Note the reductions in H3K4me1 in trr-C/A and conversion of H3K4me1 to H3K4me3 in hyperactive trr-Y/F. The Z-score scale for H3K4me1 and H3K27ac is 0 to 1.5 and for H3K4me3 is 0 to 1. Note the changes in H3K27ac that track with increased H3K4 methylation. (g) Box plots of ChIP Z-score averages, centered on wild-type H3K27ac peaks, were calculated the same as in b. Quantitative differences in H3K4me1/3 and H3K27ac are shown in wing imaginal disc enhancers (n = 787), similar to adult brain tissues. Changes in histone modification at promoters (TSSs) are negligible (N = 4077).
(a) RNA-seq analysis from adult brains displayed as MA plots comparing trr-WT with trr-C/A. Note that only four genes are differentially expressed in trr-C/A. (b) RNA-seq analysis from adult brains displayed as MA plots comparing trr-WT with trr-Y/F. Note that only two genes are differentially expressed in trr-Y/F.
Supplementary Figure 4 Trr-Y/F adults have extranumerary thoracic macrochaetae when grown at higher temperatures.
When grown at 29 °C, trr-Y/F flies exhibit extranumerary thoracic macrochaetae, as compared with either trr-WT or trr-C/A. Note that this phenotype is less penetrant in the wild-type trr background (right). Sixty flies were scored per genotype.
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Rickels, R., Herz, HM., Sze, C. et al. Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability. Nat Genet 49, 1647–1653 (2017). https://doi.org/10.1038/ng.3965
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