Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

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Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

Here, centromeric histone marks on a human artificial chromosome are found to resemble the chromatin landscape in transcribed genes, and selective manipulation shows them to govern the incorporation of the centromere-specifying CENP-A histone variant.

Abstract

Kinetochores assemble on distinct ‘centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4–K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.

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Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP.

Daniel Foltz, Lars Jansen, Aaron Bailey … (2009)

The centromere is responsible for accurate chromosome segregation. Mammalian centromeres are specified epigenetically, with all active centromeres containing centromere-specific chromatin in which CENP-A replaces histone H3 within the nucleosome. The proteins responsible for assembly of human CENP-A into centromeric nucleosomes during the G1 phase of the cell cycle are shown here to be distinct from the chromatin assembly factors previously shown to load other histone H3 variants. Here we demonstrate that prenucleosomal CENP-A is complexed with histone H4, nucleophosmin 1, and HJURP. Recruitment of new CENP-A into nucleosomes at replicated centromeres is dependent on HJURP. Recognition by HJURP is mediated through the centromere targeting domain (CATD) of CENP-A, a region that we demonstrated previously to induce a unique conformational rigidity to both the subnucleosomal CENP-A heterotetramer and the corresponding assembled nucleosome. We propose HJURP to be a cell-cycle-regulated CENP-A-specific histone chaperone required for centromeric chromatin assembly.

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The molecular mechanisms underlying pluripotency and lineage specification from embryonic stem cells (ESCs) are largely unclear. Differentiation pathways may be determined by the targeted activation of lineage-specific genes or by selective silencing of genome regions. Here we show that the ESC genome is transcriptionally globally hyperactive and undergoes large-scale silencing as cells differentiate. Normally silent repeat regions are active in ESCs, and tissue-specific genes are sporadically expressed at low levels. Whole-genome tiling arrays demonstrate widespread transcription in coding and noncoding regions in ESCs, whereas the transcriptional landscape becomes more discrete as differentiation proceeds. The transcriptional hyperactivity in ESCs is accompanied by disproportionate expression of chromatin-remodeling genes and the general transcription machinery. We propose that global transcription is a hallmark of pluripotent ESCs, contributing to their plasticity, and that lineage specification is driven by reduction of the transcribed portion of the genome.

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Author and article information

Journal

Journal ID (nlm-ta): EMBO J

Title: The EMBO Journal

Publisher: Nature Publishing Group

ISSN (Print): 0261-4189

ISSN (Electronic): 1460-2075

Publication date (Print): 19 January 2011

Publication date (Electronic): 14 December 2010

Publication date PMC-release: 14 December 2010

Volume: 30

Issue: 2

Pages: 328-340

Affiliations

[1 ]simpleWellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh, Scotland, UK

[2 ]simpleInstituto Gulbenkian de Ciência , Oeiras, Portugal

[3 ]simpleBiomolecular Networks Laboratories Group, Graduate School of Frontier Biosciences, Osaka University , Osaka, Japan

[4 ]simpleLaboratory of Cell Engineering, Kazusa DNA Research Institute , Chiba, Japan

[5 ]simpleLaboratory of Molecular Pharmacology, National Institutes of Health , Bethesda, MD, USA

Author notes

[a ]Wellcome Trust Centre for Cell Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK. Tel.: +44 131 650 7101; Fax: +44 131 650 7100; E-mail: bill.earnshaw@ 123456ed.ac.uk

[*]

Present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA

Article

Publisher Item ID: emboj2010329

DOI: 10.1038/emboj.2010.329

PMC ID: 3025471

PubMed ID: 21157429

SO-VID: 57b83c66-d181-464a-80dd-23ceb45974ea

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Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore

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