Investigation of in vitro histone H3 glycosylation using H3 tail peptides

Abstract

Posttranslational modifications (PTMs) on histone tails regulate eukaryotic gene expression by impacting the chromatin structure and by modulating interactions with other cellular proteins. One such PTM has been identified as serine and threonine glycosylation, the introduction of the ß-N-acetylglucosamine (GlcNAc) moiety on histone H3 tail at position Ser10 and Thr32. The addition of the ß-O-GlcNAc moiety on serine or threonine residues is facilitated by the O-GlcNAc transferase (OGT), and can be removed by the action of O-GlcNAcase (OGA). Conflicting reports on histone tail GlcNAc modification in vivo prompted us to investigate whether synthetic histone H3 tail peptides in conjunction with other PTMs are substrates for OGT and OGA in vitro. Our enzymatic assays with recombinantly expressed human OGT revealed that the unmodified and PTM-modified histone H3 tails are not substrates for OGT at both sites, Ser10 and Thr32. In addition, full length histone H3 was not a substrate for OGT. Conversely, our work demonstrates that synthetic peptides containing the GlcNAc functionality at Ser10 are substrates for recombinantly expressed human OGA, yielding deglycosylated histone H3 peptides. We also show that the catalytic domains of human histone lysine methyltransferases G9a, GLP and SETD7 and histone lysine acetyltransferases PCAF and GCN5 do somewhat tolerate glycosylated H3Ser10 close to lysine residues that undergo methylation and acetylation reactions, respectively. Overall, this work indicates that GlcNAcylation of histone H3 tail peptide in the presence of OGT does not occur in vitro.

O-GlcNAcylation: the sweet side of epigenetics

“…emphasized the need to treat previous results on the role of O- GlcNAcylation in epigenetics with caution [ 119 ]. Moreover, certain recent findings raise questions of abundance and occurrence of O -GlcNAcylation on histones as well as presence of other factors for efficient O -GlcNAcylation [ 120 , 121 ]. There is still much to uncover to understand the intricacies of O- GlcNAcylation’s roles in epigenetic regulation and determine how these roles can be targeted to improve human health, underscoring the importance of continued research in this area.…”

Specific regulation of epigenome landscape by metabolic enzymes and metabolites

“…Other acetyl-CoA-producing enzymes have also been reported to form a complex with transcription factors. ACSS2 is recruited to transcriptionally active chromatin by Yin Yang1 (YY1) to activate neuronal gene expression (Mews et al, 2017). YY1 has been thought to recruit KATs, including CBP and p300.…”

“…YY1 has been thought to recruit KATs, including CBP and p300. In this way, ACSS2 provides local acetyl-CoA for histone acetylation at transcriptionally active genes (Mews et al, 2017). In addition, nuclear-translocated ASCC2 interacts with transcription factor EB (TFEB) and fuels histone acetylation from acetate at the promoters of TFEB target genes responsible for lysosomal biogenesis and autophagy (Li et al, 2017b).…”