Histone H3K4 monomethylation catalyzed by Trr and mammalian COMPASS-like proteins at enhancers is dispensable for development and viability

Abstract

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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Figure 1: The catalytic activity of Trr is dispensable for viability.
Figure 2: Enhancer chromatin modification is specifically affected by mutations altering Trr catalytic activity.
Figure 3: Gene expression is only modestly affected by Trr-dependent enhancer methylation.
Figure 4: Subtle phenotypes of lines expressing trr catalytic mutants.
Figure 5: MLL3 and MLL4 catalytic activity is not required for enhancer function in mESCs.

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Acknowledgements

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).

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Contributions

A.S. and H.-M.H. conceived and initiated the project. R.R. performed Drosophila ChIP–seq studies, conducted genetic tests and stress response assays, and wrote the manuscript. C.C.S. and A.K. assisted with Drosophila experiments. Y.T. performed Trr/WRAD reconstitution and in vitro methyltransferase assays. K.C. and M.A.M. generated mutant mESCs, and L.W., A.P., and C.C.S. assisted with mammalian studies. Sequencing data were analyzed by C.K.C. and E.T.B. while libraries were generated and sequenced by E.J.R. and S.A.M. Mass spectrometry was performed and results analyzed by N.A.A. and N.L.K. Cohesin genetic interaction data were contributed by M.G. and D.D. Critical feedback and advice were provided by E.R.S. throughout the course of this project.

Corresponding author

Correspondence to Ali Shilatifard.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Confirmation of trr alleles by sequencing.

(a) Chromatograms confirming the correct mutations incorporated into trr[1] 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[1] 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[1] 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).

Supplementary Figure 3 Minimal gene expression changes in Drosophila adult brains.

(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.

Supplementary Figure 5 Uncropped western blot images corresponding to Figure 1.

(a) Uncropped western blot images corresponding to Figure 1c. (b) Uncropped western blot images of transcription factors corresponding to Figure 1d. (c) Uncropped western blot images of histone modifications corresponding to Figure 1d.

Supplementary Figure 6 Uncropped western blot images corresponding to Figure 5.

(a) Uncropped western blot images corresponding to Figure 5b. (b) Uncropped western blot images corresponding to Figure 5c.

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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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