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Published March 6, 2014 | Supplemental Material + Accepted Version
Journal Article Open

Citrullination regulates pluripotency and histone H1 binding to chromatin

Abstract

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.

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 (http://www.ebi.ac.uk/arrayexpress/) under accession E-MTAB-1975. Competing interests: T.K. is a co-founder of Abcam.

Attached Files

Accepted Version - emss-67559.pdf

Supplemental Material - nature12942-s1.xlsx

Supplemental Material - nature12942-s2.xlsx

Supplemental Material - nature12942-s3.xlsx

Supplemental Material - nature12942-s4.xlsx

Supplemental Material - nature12942-sf1.jpg

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Created:
August 19, 2023
Modified:
October 20, 2023