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Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis.

ABSTRACT: Post-translational modifications (PTMs) of histones are an essential feature in the dynamic regulation of chromatin. One of these modifications, ubiquitylation, has been speculated to directly influence the stability of the nucleosome, which represents the basic building block of chromatin. Here we report a strategy for the semisynthesis of site-specifically ubiquitylated histone H2A (uH2A). This branched protein was generated through a three-piece expressed protein ligation approach including a traceless ligation at valine. uH2A could be efficiently incorporated into nucleosomes, thereby opening the way to detailed biochemical and biophysical studies on the function of this PTM. Accordingly, we used uH2A, as well as a previously generated ubiquitylated H2B, in chaperone-coupled nucleosome stability assays to demonstrate that the direct effect of ubiquitylated histones on nucleosomal stability is in fact modest.

SUBMITTER: Fierz B

PROVIDER: S-EPMC3535264 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis.

Fierz Beat B Kilic Sinan S Hieb Aaron R AR Luger Karolin K Muir Tom W TW

Journal of the American Chemical Society 20121126 48

Post-translational modifications (PTMs) of histones are an essential feature in the dynamic regulation of chromatin. One of these modifications, ubiquitylation, has been speculated to directly influence the stability of the nucleosome, which represents the basic building block of chromatin. Here we report a strategy for the semisynthesis of site-specifically ubiquitylated histone H2A (uH2A). This branched protein was generated through a three-piece expressed protein ligation approach including a ...[more]

PMID: 23163596

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Project description:The fundamental unit of eukaryotic chromatin is the nucleosome core particle, a protein/DNA complex that binds ∼147 base pairs of DNA to a histone octamer. These histones-H3, H4, H2A, H2B-form the nucleosome core through a stacked interaction in which two H2A-H2B dimers flank the (H3-H4)2 tetramer. In vivo, genetic accessibility can be modulated by the substitution of canonical histones with variant proteins that contain the same structural motif but a different amino acid sequence, such as the transcriptional repression-associated macroH2A variant. Previously, Chakravarthy and Luger published a crystal study that showed that H2A substitution is not necessarily required of both H2A moieties, but that in vitro recombination of nucleosomes in the presence of both macroH2A and H2A histone folds results in a hybrid macroH2A-H2A nucleosome with one dimer of each type. Here, we present molecular dynamics simulations of this hybrid construct and compare the results to our previous study on homogeneous H2A- and macroH2A-containing nucleosomes. We find that the hybrid contains a unique set of dynamics that stabilize the interactions between protein constituents and create an altogether more stable nucleosome, both in terms of protein-DNA and protein-protein binding. While dimer-tetramer interactions are asymmetric, as the difference in moieties would suggest, we observe that it is the canonical dimer that is pulled further into the nucleosome core, resulting in more secure dimer-tetramer bonds and a more stable histone core, and we also find significantly more interaction between the dimer subunits. Together, these models provide evidence for hybrid H2A-macroH2A nucleosome formation being not only possible but actually energetically more favorable than a homogeneous construct, with dynamics that are unique from their homogeneous H2A or macroH2A nucleosome counterparts. These effects of hybrid substitution likely propagate into higher-order chromatin structures to hinder transcriptional activity.

Dataset Information

Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis.

Publications

Stability of nucleosomes containing homogenously ubiquitylated H2A and H2B prepared using semisynthesis.

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