Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events

Abstract

interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events" (2013 AbstractHolocarboxylasesynthetase (HCS) catalyzes the binding of the vitamin biotin to histonesH3 and H4, thereby creating rare histonebiotinylation marks in the epigenome. These marksco-localize with K9-methylated histone H3 (H3K9me), an abundant gene repression mark. The abundance of H3K9me marks in transcriptionally competent loci decreases when HCS is knocked down and when cells are depleted of biotin. Here we tested the hypothesis that the creation of H3K9me marks is at least partially explained by physical interactions between HCS and histone-lysine Nmethyltransferases. Using a novel in silico protocol, we predicted that HCS-interacting proteins contain a GGGG(K/R)G(I/M)R motif. Thismotif, with minor variations, is present in the histonelysine N-methyltransferase EHMT1. Physical interactions between HCS and the N-terminal, ankyrin, and SET domains in EHMT1 were confirmed using yeast-two-hybrid assays, limited proteolysis assays, and co-immunoprecipitation. The interactions were stronger between HCS and the N-terminus in EHMT1 compared with the ankyrin and SET domains, consistent with the localization of the HCS-binding motif in the EHMT1 N-terminus. HCS has the catalytic activity to biotinylate K161 within the binding motif in EHMT1. Mutation of K161 weakenedthe physical interaction between EHMT1 and HCS, but it is unknown whether this effect was caused by loss of biotinylation or loss of the motif. Importantly, HCS knockdown decreased the abundance of H3K9me marks in repeats, suggesting that HCS plays a role in creating histone methylation marks in these loci. We conclude that physical interactionsbetween HCS and EHMT1 mediate epigenomic synergies between biotinylation and methylation events.

Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyltransferase 1 in the transcriptional repression of long-terminal repeats

“…This observation goes beyond our previous studies, which suggest that HLCS interacts physically with the core histones H3 and H4 and the histone methyltransferase EHMT-1. 8,17 The interactions between HLCS and histones may account for the binding of HLCS to chromatin, but does not explain the punctuate pattern of HLCS localization that was observed in previous studies, 6,7 simply because core histones can be found in all nucleosomes in chromatin. Likewise, the interactions between HLCS and EHMT-1 are unlikely to mediate the initial positioning of HLCS, based on previous observations that HLCS knockdown causes a loss of EHMT-1 dependent H3K9me marks.…”

“…In contrast, when DNMT1 was incubated lysine-9 methyltransferase EHMT-1. 8,17 Lysine-9 methylated histone H3 (H3K9me) is an abundant gene repression mark and EHMT-1 is one of the enzymes capable of creating this mark. 18 HLCS physically interacts with EHMT-1 and catalyzes the biotinylation of lysine (K) residues in EHMT-1, which strengthens the interactions between the proteins in vitro.…”

“…18 HLCS physically interacts with EHMT-1 and catalyzes the biotinylation of lysine (K) residues in EHMT-1, which strengthens the interactions between the proteins in vitro. 17 Both biotin depletion and loss of HLCS cause a decrease of H3K9me marks. [9][10][11]19 Third, when DNA methylation marks are erased by 5-azacytidine treatment in human cell cultures, the abundance of histone biotinylation marks decreases considerably in LTRs; this is an uni-directional effect and biotin depletion has no effect on the abundance of DNA methylation marks.…”

“…Full-length human HLCS (NM_000411) was subcloned from plasmid pET41a (+)-HLCS 44 into vector p3XFLAG-CMV-26 (Sigma-Aldrich, Cat# E7283) using NotI and XbaI, thereby creating plasmid FLAG/Myc-HLCS for studies of HLCS overexpression and its effects on H3K9me marks and LTR transcriptional activity. Plasmid pCMV-myc-HLCS was created as described previously 17 and was used for overexpression of Myc-tagged HLCS in co-immunoprecipitation studies. Full-length human MeCP2 (GenBank Accession:NM_004992) was subcloned from plasmid pGADT7-MeCP2 (unpublished) into vector pCMV-HA (Clontech, Cat# 635690) using Sfil and XhoI for overexpression of HA-tagged MeCP2.…”

“…Proteins were separated by gel electrophoresis and stained using Coomassie blue. 17 In control experiments MeCP2 and DNMT1 were incubated with GST peptide to formally eliminate the possibility that the GST tag protected the proteins against proteolytic digestion.…”