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Citrullination regulates pluripotency and histone H1 binding to chromatin

Christophorou, Maria A. and Castelo-Branco, Gonçalo and Halley-Stott, Richard P. and Oliveira, Clara Slade and Loos, Remco and Radzisheuskaya, Aliaksandra and Mowen, Kerri A. and Bertone, Paul and Silva, José C. R. and Zernicka-Goetz, Magdalena and Nielsen, Michael L. and Gurdon, John B. and Kouzarides, Tony (2014) Citrullination regulates pluripotency and histone H1 binding to chromatin. Nature, 507 (7490). pp. 104-108. ISSN 0028-0836. PMCID PMC4843970. doi:10.1038/nature12942.

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[img] Image (JPEG) (Extended Data Figure 1 : Citrullination and Padi expression profiles in ES, NSO4G and iPS cells; regulation of Padi4 by pluripotency factors in ES cells) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 2 : PADI4 overexpression or knockdown in ES cells modulates expression of pluripotency genes) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 3 : Chromatin immunoprecipitation of H2A, PADI4 and H3Cit at pluripotency loci) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 4 : Microarray analysis of Padi4 inhibition by Cl-amidine in ES cells) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 5 : Padi4 inhibition reduces reprogramming efficiency) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 6 : Cl-amidine treatment impairs early embryo development) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 7 : TDFA treatment reduces percentage of pluripotent cells in the early embryo) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 8 : TSA treatment does not impair early embryo development) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 9 : Mass spectrometry data for citrullinated and unmodified H1.2) - Supplemental Material
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[img] Image (JPEG) (Extended Data Figure 10 : Mass spectrometry spectra for citrullinated H1.5; PADI4 treatment of differentiated nuclei leads to H1 citrullination and chromatin decompaction) - Supplemental Material
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[img] MS Excel (Supplementary Table 1. The file contains the primer sequences) - Supplemental Material
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[img] MS Excel (Supplementary Table 2. This file contains the complete microarray data for PADI4 over-expression in ES cells) - Supplemental Material
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[img] MS Excel (Supplementary Table 3. This file contains complete microarray data for PADI4 inhibition in ES cells) - Supplemental Material
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[img] MS Excel (Supplementary Table 4. This file contains the complete Mass Spec dataset) - Supplemental Material
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[img] Video (QuickTime) (Video 1. Time-lapse video of reprogramming experiments) - Supplemental Material
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Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

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Zernicka-Goetz, Magdalena0000-0002-7004-2471
Additional Information:© 2014 Nature Publishing Group. Received 06 September 2012. Accepted 06 December 2013. Published 26 January 2014. This work was funded by programme grants from Cancer Research UK (T.K.) and EMBL (P.B., R.L.). R.P.H.-S. and J.B.G. are supported by the Medical Research Council (G1001690) and the Wellcome Trust. G.C.-B. was funded by EMBO (Long-Term Post-Doctoral Fellowship), European Union (FP7 Marie Curie Intra-European Fellowship for Career Development) and Swedish Research Council. M.A.C. was funded by an EMBO Long-Term Post-Doctoral Fellowship and a Human Frontier Science Programme Long-Term Post-Doctoral Fellowship. C.S.O. was supported by FAPESP (Foundation for Research Support of the State of São Paulo) and mouse embryo work was supported by the Wellcome Trust programme grant to M.Z.-G. M.L.N. was partly supported by the Novo Nordisk Foundation Center for Protein Research, the Lundbeck Foundation, and by and the European Commission’s 7th Framework Programme HEALTH-F7-2010-242129/SYBOSS. K.A.M. was funded by NIH grant AI099728. We would like to thank S. Lestari, A. Cook and C. Hill for technical assistance; P. Thompson for providing the TDFA compound; GSK Epinova for Cl-amidine; T. Bartke for the gift of histone octamers and help with nucleosome pull-down assays; A. Finch for help with FPLC chromatography; A. Jedrusik for help with embryo work; R. Walker at the Flow Cytometry Core Facility at Wellcome Trust Centre for Stem Cell Research, University of Cambridge and T. Theunissen for help with the flow cytometry; and members of the Kouzarides laboratory for critical discussions of the work. 2TS22C cells were provided by H. Niwa. The ChIP grade H1.2 antibody was a gift from A. Skoultchi. Maria A. Christophorou & Gonçalo Castelo-Branco - These authors contributed equally to this work. Author Contributions: M.A.C., G.C.-B. and T.K. conceived the idea for this project, designed experiments and wrote the manuscript with the help of all the authors. G.C.-B. and M.A.C. performed ES-cell transductions, established transgenic pre-iPS and ES cell lines and performed gene expression analyses. M.A.C. carried out mutagenesis, protein isolation, biochemical and chromatin immunoprecipitation experiments, and performed citrullination analyses with the help of K.A.M.; G.C.-B. performed reprogramming experiments, with the help of J.S. and A.R.; M.A.C. prepared proteomic samples and M.L.N. performed mass spectrometric analyses. R.P.H.-S. and M.A.C. performed PADI4 treatments of permeabilized cells and subsequent chromatin compaction analyses, with the help of J.B.G. C.S.O. and M.Z.-G. designed and performed mouse embryo experiments. R.L. and P.B. performed bioinformatic analyses of microarray data. T.K. supervised the study. Data deposits: Microarray data have been deposited in the ArrayExpress repository ( under accession E-MTAB-1975. Competing interests: T.K. is a co-founder of Abcam.
Funding AgencyGrant Number
European Molecular Biology Organization (EMBO)UNSPECIFIED
Medical Research Council (UK)G1001690
Marie Curie FellowshipUNSPECIFIED
Swedish Research CouncilUNSPECIFIED
Human Frontier Science ProgramUNSPECIFIED
Fundação de Amparo à Pesquisa do Estado de Sao Paulo (FAPESP)UNSPECIFIED
Novo Nordisk FoundationUNSPECIFIED
Lundbeck FoundationUNSPECIFIED
European Research Council (ERC)HEALTH-F7-2010-242129/SYBOSS
Issue or Number:7490
PubMed Central ID:PMC4843970
Record Number:CaltechAUTHORS:20190405-170316424
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:94551
Deposited By: George Porter
Deposited On:10 Apr 2019 15:31
Last Modified:16 Nov 2021 17:05

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