A Chemical Biology Toolbox for the Study of Protein Methyltransferases and Epigenetic Signaling

Abstract

SUMMARYProtein 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 remarkable potential of individual probes to alter multiple T cell subpopulations with important 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.

The histone methyltransferase DOT1L is essential for humoral immune responses

“…In the first, we compared cell trace violet (CTV)-labeled B cells isolated from Cd23 cre/+ and Dot1l f/f Cd23 cre/+ mice stimulated in vitro with CD40L, IL-4 and IL-5. In the second, we incubated stimulated CTV-labeled wildtype B cells with a small molecule inhibitor to DOT1L (Scheer et al, 2019). In both cases, proliferation was not significantly affected in the absence of DOT1L and nor was cell death (data not shown).…”

“…Inhibition of DOT1L in T helper (Th) cells was shown to result in an increase in both Th1 cells and IFNg production by those cells in vitro (Scheer et al, 2019). B cells also have tailored functional responses to either Th1 cell-biased or Th2 cell-biased stimuli, and although transcription factor expression is associated with dichotomous responses (Piovesan et al, 2017), it is not known whether epigenetic regulators are also important in specialized B cell function.…”

Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich’s ataxia patient cells

“…High levels of specific heterochromatin marks have been previously reported at the first intron of the pathologically silenced FXN gene (for a review of the epigenetic changes associated with FXN see (17)). The epigenetic probe collection from the Structural Genomics Consortium (SGC) comprises a wellcharacterised set of drug-like small molecules which inhibit specific chromatin regulatory proteins and domains including bromodomains, demethylases and methyltransferases (21,22). To identify novel epigenetic targets involved in the regulation of FXN expression, we screened the SGC epigenetic chemical probe set using our FXN-GAA-Luc reporter cell line (15,23) in a 96-well format in duplicate (see Supplementary Table 1) and assayed frataxin-luciferase (FXN-Luc) protein expression by luciferase assay ( Figure 1A).…”

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+T cells

“…Interestingly, inhibition of DOT1L activity in human T cells attenuates graft-versus-host disease in adoptive cell transfer models 26 and it regulates CD4 + T cell differentiation 27 .…”

“…A similar dependency on DOT1L activity and sensitivity to DOT1L inhibitors was recently observed in thymic lymphoma 25 . Interestingly, inhibition of DOT1L activity in human T cells attenuates graft-versus-host disease in adoptive cell transfer models 26 and it regulates CD4 + T cell differentiation 27 .…”