Histone variant H2A.B-H2B dimers are spontaneously exchanged with canonical H2A-H2B in the nucleosome

精子形成に重要なヒストンによるDNAの新たな折りたたみを解明. 京都大学プレスリリース. 2021-02-22.H2A.B is an evolutionarily distant histone H2A variant that accumulates on DNA repair sites, DNA replication sites, and actively transcribing regions in genomes. In cells, H2A.B exchanges rapidly in chromatin, but the mechanism has remained enigmatic. In the present study, we found that the H2A.B-H2B dimer incorporated within the nucleosome exchanges with the canonical H2A-H2B dimer without assistance from additional factors, such as histone chaperones and nucleosome remodelers. High-speed atomic force microscopy revealed that the H2A.B nucleosome, but not the canonical H2A nucleosome, transiently forms an intermediate “open conformation”, in which two H2A.B-H2B dimers may be detached from the H3-H4 tetramer and bind to the DNA regions near the entry/exit sites. Mutational analyses revealed that the H2A.B C-terminal region is responsible for the adoption of the open conformation and the H2A.B-H2B exchange in the nucleosome. These findings provide mechanistic insights into the histone exchange of the H2A.B nucleosome

H4K20me1 and H3K27me3 are concurrently loaded onto the inactive X chromosome but dispensabe for inducing gene silencing

© 2021 EMBO. This is an open access article under the terms of the Creative Commons Attribution License,which permits use, distribution and reproduction in any medium, provided the original work is properly cited.During X chromosome inactivation (XCI), in female placental mammals, gene silencing is initiated by the Xist long non-coding RNA. Xist accumulation at the X leads to enrichment of specific chromatin marks, including PRC2-dependent H3K27me3 and SETD8-dependent H4K20me1. However, the dynamics of this process in relation to Xist RNA accumulation remains unknown as is the involvement of H4K20me1 in initiating gene silencing. To follow XCI dynamics in living cells, we developed a genetically encoded, H3K27me3-specific intracellular antibody or H3K27me3-mintbody. By combining live-cell imaging of H3K27me3, H4K20me1, the X chromosome and Xist RNA, with ChIP-seq analysis we uncover concurrent accumulation of both marks during XCI, albeit with distinct genomic distributions. Furthermore, using a Xist B and C repeat mutant, which still shows gene silencing on the X but not H3K27me3 deposition, we also find a complete lack of H4K20me1 enrichment. This demonstrates that H4K20me1 is dispensable for the initiation of gene silencing, although it may have a role in the chromatin compaction that characterises facultative heterochromatin.This work was supported by Fundação para a Ciência e Tecnologia (S.T.d.R), project grants PTDC/BIA‐ MOL/29320/2017 IC&DT (A. C. R. & S.T.d.R), CEECUIND/01234/207 (S.T.d.R), and SFRH/BD/137099/2018 (A.C.R.), by an ERC Advanced Investigator award ERC‐ADG‐2014 671027 attributed to E.H., Sir Henry Wellcome Postdoctoral Fellowship (J.J.Z.), Japan Society for the Promotion of Science KAKENHI grants (JP17KK0143 and JP20K06484 to Y.S., JP19H04970, JP19H03158 and JP20H05393 to K.M., JP17K17719 to T.H., JP18H05534 to H.Ku, JP18H05527 and JP20H00456 to Y.O., JP17H01417 and JP18H05527 to H.Ki), and Japan Science and Technology Agency (JST) CREST JPMJCR16G1 to T.K., H.Ku, Y.O. and H.Ki, PREST JPMJPR2026 to K.M., and ERATO JPMJER1901 to H.Ku. J.J.Z. is supported by core funding of The Novo Nordisk Foundation Center for Stem Cell Biology (Novo Nordisk Foundation grant number NNF17CC0027852). Open Access funding enabled and organized by Projekt DEAL.info:eu-repo/semantics/publishedVersio

Ru Catalyst Facilitates Total Chemical Protein Synthesis to Investigate the Effect of Epigenetic Modifications Decorated on Linker Histone H1.2 and Heterochromatin Protein 1α (HP1α)

For epigenetics research, preparing homogeneous proteins bearing site-specific posttranslational modifications (PTMs) is essential to understand the behavior of chromatin. Total chemical protein synthesis is a very powerful method to obtain target proteins with various modifications at site-specific positions. To produce large proteins efficiently, one-pot ligation of multiple peptide fragments was previously reported through repetitive deprotection of protecting groups for N-terminal Cys with palladium complexes. However, this method demanded more than a catalytic amount of metal complexes, and, in general, it had been challenging to achieve catalytic cycles of metal complexes especially for reactions on proteins. Here, we report an efficient and facile method of chemical protein synthesis using Ru catalyst. The use of 10–20 mol% of Ru complexes enabled us to remove the protecting groups on peptides or proteins under peptide ligation conditions, and this complex showed more than 50-fold activity compared to the previous palladium complexes due to the great stability toward thiol moieties. By using this Ru catalyst, we accomplished total chemical synthesis of linker histone H1.2 (212 amino acids) and heterochromatin protein 1a (HP1a) (191 amino acids), which are important components of heterochromatin, through one-pot multiple peptide ligation. This method prompted the preparation of H1.2 and HP1a bearing various patterns of PTMs. Moreover, we found that R53Cit at H1.2 reduced its binding affinity toward nucleosomes and four consecutive phosphorylations at N-terminus HP1a controlled its binding ability against DNA. We envisage that homogeneously modified proteins prepared by our method would facilitate epigenetics research and be applied for the elucidation of various biological phenomena

A chemical catalyst enabling histone acylation with endogenous Acyl-CoA

Post-translational modifications (PTMs) of proteins, e.g., epigenetic acetylation of lysine residues in histones, are crucial to cellular functions and related to diseases. Chemical tools to directly introducing epigenetic lysine acetylation hold promise for elucidating the PTM’s functions and treating diseases. Although several chemical catalysts introducing protein acetylation in live cells were reported, there is no precedent promoting in-cell acetylation of epigenetically important but often low-reactive histone proteins using endogenous acetyl-CoA, as histone acetyltransferases (HATs) do. Herein, we developed a chemical catalyst mBnA enabling selective in-cell histone lysine acylation (H2BK120ac) using endogenous acyl-CoA as a sole acyl donor. A hydroxamic acid of proper electronic characteristics as a nucleophilic catalytic site combined with a thiol-thioester exchange process enabled mBnA to activate low concentration of acyl-CoAs in cells, promoting histone lysine acylations (acetylation and malonylation). This chemical catalyst will be a small-molecule surrogate to HAT and thus a unique tool to synthetic epigenetics

Integral approach to biomacromolecular structure by analytical-ultracentrifugation and small-angle scattering

溶液中の蛋白質構造を正確に評価するための新規解析法を開発 --構造評価の妨げとなる凝集の影響を実験データから除去--. 京都大学プレスリリース. 2020-06-09.Currently, a sample for small-angle scattering (SAS) is usually highly purified and looks monodispersed: The Guinier plot of its SAS intensity shows a fine straight line. However, it could include the slight aggregates which make the experimental SAS profile different from the monodispersed one. A concerted method with analytical-ultracentrifugation (AUC) and SAS, named as AUC-SAS, offers the precise scattering intensity of a concerned biomacromolecule in solution even with aggregates as well that of a complex under an association-dissociation equilibrium. AUC-SAS overcomes an aggregation problem which has been an obstacle for SAS analysis and, furthermore, has a potential to lead to a structural analysis for a general multi-component system

A chemical catalyst enabling histone acylation with endogenous acyl-CoA

Abstract Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease

Live-Cell Epigenome Manipulation by Synthetic Histone Acylation Catalyst System

Chemical modifications of histones play a pivotal role in the epigenome and regulation of gene expression, and their abnormality is tightly linked to numerous disease states in humans. Therefore, chemical tools to manipulate epigenome hold promise for both therapy and the elucidation of epigenetic mechanisms. We previously developed the chemical catalyst LANA-DSH, which binds to nucleosomes via a LANA peptide ligand, and selectively acylates proximal histone H2BK120 to the catalyst moiety by acti- vating acyl-CoAs. Thus far, however, histone acylation by a chem- ical catalyst system in living cells has not yet been demonstrated. Here, we report a chemical catalyst system, composed of a nucleo- some-binding catalyst (PEG-LANA-DSH) and a cell-permeable thioester acyl donor (NAC-acyl), that can promote regioselective lysine acylation of histones in living cells. Whereas LANA-DSH is rapidly decomposed in cells, attachment of polyethylene glycol (PEG) to the LANA moiety can prevent this undesired degradation. Increasing the size of PEG conferred LANA with greater in-cell stability, but reduced catalytic activity, indicating that there is an optimum PEG length balancing stability and catalytic activity. The optimized PEG-LANA-DSH catalyst 11 efficiently promoted H2BK120 acetylation in living cells, which subsequently sup- pressed ubiquitination of H2BK120, a mark regulating various chromatin functions, such as transcription and DNA damage re- sponse. Thus, our chemical catalyst system will be useful as a unique tool to manipulate the epigenome for therapeutic purposes or further understanding epigenetic mechanisms.</div

Solution Structure of Variant H2A.Z.1 Nucleosome Investigated by Small-Angle X-ray and Neutron Scatterings

Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome