A chemical biology toolbox to study protein methyltransferases and epigenetic signaling

Abstract

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4⁺ T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.

Fig. 1

Summary of chemical probes. a Phylogenetic trees of human PR and SET domain lysine methyltransferases (upper tree), and the β-barrel fold enzymes (lower tree). Trees are annotated to show chemical probes in this collection that inhibit PKMTs (turquoise circle), a Rossman fold PKMT (dark red square), monomethyl and asymmetric dimethyl PRMTs (blue triangle), symmetric dimethyl PRMTs (orange triangle); and methyltransferase protein complexes (purple star). The number of annotations adjacent to each target is equal to the number of chemical probes for that target. b Detailed coverage of the major histone H3 and H4 methyl marks modulated by this collection of chemical probes. The methylated lysine (K) and arginine (R) residues are annotated in bold font. The PMTs that write the marks are shown with green (PKMTs) or blue (PRMTs) borders, along with the chemical probes that inhibit these PMTs. Also included are non-histone substrates (gray ovals) of PRMT5, SETD7, and SMYD2. c Selectivity of each chemical probe has been assessed against 34 SAM-dependent methyltransferases. SKI-73 and SGC3027 are pro-drugs and selectivity was determined on the respective active components. See also Supplementary Tables 1-3. SAM S-adenosylmethionine, SAH S-adenosylhomocysteine, me methyl, me2a asymmetric dimethyl, me2s symmetric dimethyl, me2/3 di- and tri-methyl marks are written by the same enzyme

Fig. 2

Structural mechanisms of PMT inhibition by chemical probes. a Inhibitors of G9a (PDB: 3HNA (GLP with H3K9 substrate) and 4NVQ (A-366)); SUV420H2 (PDB: 4AU7 (mSUV420H2 with H4 substrate) and SUV420H1 (PDB: 5CPR (A-196)); SETD7 (PDB: 1O9S (H3 substrate) and 4JLG (PFI-2)); PRMT5 (PDB: 4GQB (H4 substrate) and 5C9Z (GSK591)); SMYD2 (PDB: 3TG5 (p53 substrate) and 5ARG (BAY-598)); SMYD3 (PDB: 5EX0 (MAP3K2 substrate) and (BAY-6035)) all bind in the substrate (peptide) binding pocket. b SGC0946 binds in the SAM-binding pocket of DOT1L thereby preventing cofactor binding (PDB ID: 3QOW (SAM), 4ER6 (SGC0946)). LLY-283 also occupies the SAM-binding pocket of PRMT5-MEP50 complex (PDB ID: 4GQB (SAM analog) and 6CKC (LLY-283)). c Three distinct modes of allosteric inhibition of protein methyltransferases. SGC707 binds to PRMT3 in an allosteric site that prevents productive formation of the enzyme’s activation helix (PDB ID: 4RYL). OICR-9429 binds to WDR5 and inhibits MLL1 activity by disrupting WDR5-MLL1-RBBP5 complex (PDB ID: 4QL1). A-395 binds to the EED subunit of the PRC2 complex thereby preventing binding of activating peptides (PDB ID: 5K0M)

Fig. 3

Affinity Reagents for Chemoproteomics. a Volcano plot of MTM7172-enriched proteome (labeled blue) from HEK293 cell lysate, with targets significantly (FDR=0.05, S0 = 0.2) competed by 20 μM MS023 with respect to 20 μM MS094 negative control. Proteins marked in purple indicate known interactors of the identified direct targets of MS023 close to the significance threshold. b Volcano plot of (A-395)-NH₂-enriched proteome (labeled blue) from G401 cell lysate, with targets significantly (FDR = 0.05, S0 = 0.2) competed by 20 μM A-395 with respect to 20 μM A-395N negative control. c Volcano plot of SGC2077-enriched proteome (labeled blue) from Jurkat cell lysate, with targets significantly (FDR = 0.05, S0 = 0.2) competed by 20 µM SGC0946, with respect to DMSO control. d STRING network evaluation of targets significantly competed by MS023. Lines in the STRING evaluation represent evidenced interactions, with line thickness indicating confidence (high to low). e STRING network evaluation of targets significantly competed by A-395. The chemical structures of the chemical biology reagents, MTM7172, (A-395)-NH₂, and SGC2077, are shown in Supplementary Table 2. See also Supplementary Fig. 1

Fig. 4

Differential Effects of PMT Inhibition on Murine and Human Th1 Cell Differentiation. a, b CD4⁺ T cells from the spleen and peripheral lymph nodes of IFN-γ-YFP reporter mice were enriched and polarized under Th0 (IL-2) or Th1 cell conditions in the absence (Th0) or presence of indicated probes (1 μM; red) or their controls (where available; black). a Flow cytometric analysis of intracellular YFP reporter signal (representing IFN-γ expression) was detected at day 4. b Secreted IFN-γ was analyzed by ELISA in the supernatant of the same experiment. Each data point represents one of three biological replicates and the data shown is representative of three independent experiments. c, d CD4⁺ T cells from the blood of three healthy human donors were cultured under Th0 or Th1 cell-polarizing conditions in the presence or absence of indicated probes or their controls. c Flow cytometric analysis of intracellular IFN-γ was detected at day 4. d Secreted IFN-γ was analyzed by ELISA in the supernatant of the same experiment. Each data point represents one of three donors. Dotted lines visualize the mean frequency of IFN-γ-positive Th1 cells in the absence of probes (a, c) or the mean concentration of IFN-γ in the supernatant (b, d). Significant differences are indicated with an asterisk and were calculated using one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). e Western blot analysis of the effect of indicated inhibitors (red) or control compounds (black) on the trimethylation of H3K27 and dimethylation of H3K79 in CD4⁺ T cells under Th1, Th2, Th17, Treg cell-polarizing conditions. Please see Supplementary Figs. 5a-5d for information on the MW markers. Data shown is representative of 2 independent experiments. Error bars represent SEM. See also Supplementary Figs. 2-5

Fig. 5

DOT1L-dependent H3K79me2 is dynamically regulated at lineage-specific promoters. a Analysis of H3K79me2 in CD4⁺ T cells under Th1 polarizing conditions in the presence of the DOT1L inhibitor SGC0946 (1 µM) or the control compound SGC0649 (1 µM) with time as measured by western blot. b Analysis of IFN-γ production by FACS under the same conditions as in a. Significant differences are indicated with an asterisk and were calculated using one-way ANOVA (*p ≤ 0.05, **p ≤ 0.01). Data shown are representative of three independent experiments. c DOT1L-dependent regulation of gene expression in Th1 cells. MA-plot (M (log ratio); A (mean average)) comparing SGC0649-treated and SGC0946-treated IFN-γ⁺ CD4⁺ T cells after Th1 polarization of up and down genes. Dots represent genes that show >1.5-fold increase (FDR 0.01) or decrease in SGC0946-treated CD4⁺ T cells compared to SGC0649-treated CD4⁺ T cells. Average expression is the mean of three biological replicates. Error bars represent SEM. See also Supplementary Figs. 3 and 5

Fig. 6

Overview of PMT Inhibition on Murine CD4⁺ T cell Differentiation. Data is presented as fold-increase (black) or decrease (red) of cytokine production (IFN-γ, IL-13, and IL-17A for Th1, Th2, and Th17 cells, respectively) or FOXP3 expression (Treg cells) over that of the control compound-treated (or untreated) cells. Data shown are from two to three individual experiments with three biological replicates each. *Significant cell death: *p < 0.05 compared to DMSO control. See also Supplementary Figs. 6 and 7