Dataset Information

Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader-Willi Syndrome.

ABSTRACT: The role of epigenetic regulation is in large parts connected to cancer, but additionally, its therapeutic claim in neurological disorders has emerged. Inhibition of histone H3 lysine N-methyltransferase, especially G9a, has been recently shown to restore candidate genes from silenced parental chromosomes in the imprinting disorder Prader-Willi syndrome (PWS). In addition to this epigenetic approach, pitolisant as G-protein coupled histamine H3 receptor (H3R) antagonist has demonstrated promising therapeutic effects for Prader-Willi syndrome. To combine these pioneering principles of drug action, we aimed to identify compounds that combine both activities, guided by the pharmacophore blueprint for both targets. However, pitolisant as selective H3R inverse agonist with FDA and EMA-approval did not show the required inhibition at G9a. Pharmacological characterization of the prominent G9a inhibitor A-366, that is as well an inhibitor of the epigenetic reader protein Spindlin1, revealed its high affinity at H3R while showing subtype selectivity among subsets of the histaminergic and dopaminergic receptor families. This work moves prominent G9a ligands forward as pharmacological tools to prove for a potentially combined, symptomatic and causal, therapy in PWS by bridging the gap between drug development for G-protein coupled receptors and G9a as an epigenetic effector in a multi-targeting approach.

SUBMITTER: Reiner D

PROVIDER: S-EPMC7419559 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader-Willi Syndrome.

Reiner David D Seifert Ludwig L Deck Caroline C Schüle Roland R Jung Manfred M Stark Holger H

Scientific reports 20200811 1

The role of epigenetic regulation is in large parts connected to cancer, but additionally, its therapeutic claim in neurological disorders has emerged. Inhibition of histone H3 lysine N-methyltransferase, especially G9a, has been recently shown to restore candidate genes from silenced parental chromosomes in the imprinting disorder Prader-Willi syndrome (PWS). In addition to this epigenetic approach, pitolisant as G-protein coupled histamine H3 receptor (H3R) antagonist has ...[more]

PMID: 32782417

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Project description:Methylation of lysine 9 (K9) in the N-terminus tail of histone H3 (H3) in chromatin is associated with transcriptionally silenced genes and is mediated by histone methyltransferases. Murine G9a is a 1263 amino acid H3-K9 methyltransferase that possesses characteristic SET domain and ANK repeats. In this paper, we have used a series of green fluorescent protein-tagged deletion constructs to identify two nuclear localization signals (NLS), the first NLS embedded between amino acids 24 and 109 and the second between amino acids 394 and 401 of murine G9a. Our data show that both long and short G9a isoforms were capable of entering the nucleus to methylate chromatin. Full-length or N-terminus-deleted G9a isoforms were also catalytically active enzymes that methylated recombinant H3 or synthetic peptides representing the N-terminus tail of H3. In vitro methylation reactions using N-terminus tail peptides resulted in tri-methylation of K9 that remained processive, even in G9a enzymes that lacked an N-terminus region by deletion. Co-expression of G9a and H3 resulted in di- and tri-methylation of H3-K9, while siRNA-mediated knockdown of G9a in HeLa cells resulted in reduction of global H3-K9 di- and tri-methylation. A recombinant deletion mutant enzyme fused with maltose-binding protein (MBP-G9aDelta634) was used for steady-state kinetic analysis with various substrates and was compared with full-length G9a (G9aFL). Turnover numbers of MBP-G9aDelta634 for various substrates was approximately 3-fold less compared with G9aFL, while their Michaelis constants (K(m)) for recombinant H3 were similar. The K(AdoMet)m for MBP-G9aDelta634 was approximately 2.3-2.65 microM with various substrates. Catalytic efficiencies (kcat/K(m)) for both MBP-G9aDelta634 and G9aFL were similar, suggesting that the N-terminus is not essential for catalysis. Furthermore, mutation of conserved amino acids R1097A, W1103A, Y1120A, Y1138A and R1162A, or the metal binding C1168A in the catalytic region, resulted in catalytically impaired enzymes, thereby confirming the involvement of the C-terminus of G9a in catalysis. Thus, distinct domains modulate nuclear targeting and catalytic functions of G9a.

Dataset Information

Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader-Willi Syndrome.

Publications

Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H<sub>3</sub> receptor for Prader-Willi Syndrome.

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