Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module’s molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes

Additional file 1 of De novo methylation of histone H3K23 by the methyltransferases EHMT1/GLP and EHMT2/G9a

Additional file 1: Figure S1. EHMT1/GLP and EHMT2/G9a de novo methylate H3K18, H3K23, and H3K27, in vitro. (A) Annotated spectra, ions counts and mass error tables for the mass spectrometry analysis of EHMT1/GLP and EHMT2/G9a in vitro HMT reactions (B) Western blot panel of EHMT1/GLP and EHMT2/G9a in vitro HMT reactions for various H3K18 and H3K27 methylation states. H3K4me3 and H3K36me3 were included as negative controls. 50B11 whole cell lysate was used to validate that antibodies for H3K27me1/2, H3K4me3, and H3K36me3, were functional given these modifications were not detected by western blotting in EHMT1/GLP and EHMT2/G9a in vitro HMT reactions. Figure S2. Pharmacologic inhibition of EHMT1/GLP and EHMT2/G9a decreases H3K18 and H3K27 methylation in vitro. (A) UNC0642 dose titration with recombinant SetDB1 and Suv39h2, as measured by the MTase-Glo assay. (B) Western blotting panel showing production and inhibition of various H3K18 and H3K27 methylation states by EHMT1/GLP and EHMT2/G9a. Unlike H3K27me3, H3K27me1 and H3K27me2 were undetectable in this in vitro assay. Figure S3. Pharmacologic inhibition of EHMT1/GLP and EHMT2/G9a decrease H3K18 and H3K27 methylation in vivo. (A) Western blot panel of H3K9, H3K18, H3K23 and H3K27 methylation states in MC38 cells treated with DMSO or UNC0642. (B) Western blot panel of H3K18 and H3K27 methylation states of 50B11 cells treated with DMSO or UNC0642 (C)Western blot panel of H3K18 and H3K27 methylation states of HEK293 cells treated with DMSO or UNC0642. Figure S4. H3K9M, but not H3K23M, decrease H3K23me3 in vivo. (A) MC38 and (B) 50B11 cells lines were transduced with lentivirus carrying plasmids encoding various lysine (K) to methionine (M) mutations at specific residues (e.g. (, 23, 27, etc.) to evaluate their corresponding effect on various histone H3 methylation state in vivo. Figure S5. Genetic ablation of GLP, G9a or both perturbs H3K18 and H3K27 methylation in vivo. Western blot of H3K18 and H3K27 methylation states in mouse ESCs knocking down either EHMT1/GLP, EHMT2/G9a or both and their corresponding effect on H3K18 and h3K27 methylation states. Figure S6. Antibody validation via ELISA and peptide competition assays. Using a 96-well plate ELISA format, ELISAs and peptide competition assays were done to validate H3K9me1, H39me2, H3K9me3, H3K18me1, H3K18me2, H3K18me3, H3K23me1, H3K23me2 H3K23me3, H3K27me1, H3K27me2, and H3M27me3 antibodies for their target methylation state, cross-reactivity with other methylation states of the same epitope and cross-reactivity with methyl-lysines of a different epitope (e.g. K9 antibodies with K23-methyl peptides, etc.)

A Selective Phenelzine Analogue Inhibitor of Histone Demethylase LSD1

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor <i>in vitro</i> and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1–/– cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease

A Selective Phenelzine Analogue Inhibitor of Histone Demethylase LSD1

Lysine-specific demethylase 1 (LSD1) is an epigenetic enzyme that oxidatively cleaves methyl groups from monomethyl and dimethyl Lys4 of histone H3 (H3K4Me1, H3K4Me2) and can contribute to gene silencing. This study describes the design and synthesis of analogues of a monoamine oxidase antidepressant, phenelzine, and their LSD1 inhibitory properties. A novel phenelzine analogue (bizine) containing a phenyl-butyrylamide appendage was shown to be a potent LSD1 inhibitor <i>in vitro</i> and was selective versus monoamine oxidases A/B and the LSD1 homologue, LSD2. Bizine was found to be effective at modulating bulk histone methylation in cancer cells, and ChIP-seq experiments revealed a statistically significant overlap in the H3K4 methylation pattern of genes affected by bizine and those altered in LSD1–/– cells. Treatment of two cancer cell lines, LNCaP and H460, with bizine conferred a reduction in proliferation rate, and bizine showed additive to synergistic effects on cell growth when used in combination with two out of five HDAC inhibitors tested. Moreover, neurons exposed to oxidative stress were protected by the presence of bizine, suggesting potential applications in neurodegenerative disease