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Nat Commun
2015 Aug 25;6:7978. doi: 10.1038/ncomms8978.
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Generation of a synthetic GlcNAcylated nucleosome reveals regulation of stability by H2A-Thr101 GlcNAcylation.
Lercher L, Raj R, Patel NA, Price J, Mohammed S, Robinson CV, Schofield CJ, Davis BG.
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O-GlcNAcylation is a newly discovered histone modification implicated in transcriptional regulation, but no structural information on the physical effect of GlcNAcylation on chromatin exists. Here, we generate synthetic, pure GlcNAcylated histones and nucleosomes and reveal that GlcNAcylation can modulate structure through direct destabilization of H2A/H2B dimers in the nucleosome, thus promoting an 'open' chromatin state. The results suggest that a plausible molecular basis for one role of histone O-GlcNAcylation in epigenetic regulation is to lower the barrier for RNA polymerase passage and hence increase transcription.
Figure 1. Modification and refolding of nucleosome complexes with H2A/B-GlcNAc.(a) Graphic representation of a nucleosome structure with H2A Thr-101 in red (adapted from 1KX4)51. (b) Reaction scheme for generation of GlcNAcylated H2A. Cys-101 is converted to dehydroalanine (Dha), which functions as a Michael acceptor for reaction with the GlcNAc-thiol, so replacing the ether linkage with a structurally analogous thioether. (c) Schematic representation of H2A/B dimer and nucleosome refolding (wt H2A in dark green and GlcNAcylated H2A in red). (d) SEC traces for the refolding of H2A/B dimer from wt (blue) and H2A T101GlcNAc (red) and wt H2B. (e) SEC traces for the octamer refolding from wt H3/H4 tetramers and H2A/B-wt (blue) or H2A/B-GlcNAc (red) dimers. (f) SDS–PAGE analysis of the main fractions of the marked peaks (original image shown in Supplementary Fig. 16).
Figure 2. Comparison of stability and composition of nucleosomes refolded using H2A/H2B-wt or H2A/H2B-GlcNAc dimers and wt H3/H4 tetramers.(a) 6% TBE-PAGE gel analysis of salt gradient refolding of wt (left) and GlcNAcylated (right) nucleosomes. (b) Melting profile of wild type and H2A Thr-101 GlcNAcylated nucleosome as assayed by DSF. Experiments were performed in duplicates. (c) Melting profile of wild type and H2A Thr-101 GlcNAcylated nucleosomes reconstituted with different dimer: tetramer ratios (2:1 and 4:1) as monitored by CD at 260 nm. (d) Non-denaturing ESI-MS analysis of wt and GlcNAcylated nucleosomes. Values reported represent the mean value and standard deviation of the observed m/z peaks.
Figure 3. Analysis of OGT function.(a) Genomic snapshots from the UCSC genome browser for OGT ChIP-Seq21 and DNaseI Hypersensitivity by Digital DNaseI from ENCODE/University of Washington (ES-E14 cells)22 in mouse embryonic stem cells. (b) Model for generation and maintenance of open chromatin states by OGT recruitment. When OGT is present (possibly through OGT recruitment, see Supplementary Fig. 15) H2A Thr-101 can be O-GlcNAcylated so causing destabilization of the H2A/H2B dimer in the nucleosome. This promotes hexasome formation and generation of an open chromatin structure.
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