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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 citrulline1. 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)2 and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis2,3. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response4,5,6,7,8. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal9, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection10. 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 chromatin11, 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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Figure 1: Padi4 expression and activity are features of pluripotent cells.
Figure 2: Citrullination and Padi4 regulate pluripotency during reprogramming and early embryo development.
Figure 3: PADI4 citrullinates Arg 54 on linker histone H1 and affects its binding to nucleosomal DNA.
Figure 4: PADI4 evicts histone H1 from chromatin and affects chromatin condensation.

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Microarray data have been deposited in the ArrayExpress repository (http://www.ebi.ac.uk/arrayexpress/) under accession E-MTAB-1975.

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Acknowledgements

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.

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Tony Kouzarides.

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Competing interests

T.K. is a co-founder of Abcam.

Extended data figures and tables

Extended Data Figure 1 Citrullination and Padi expression profiles in ES, NSO4G and iPS cells; regulation of Padi4 by pluripotency factors in ES cells.

a, Transcript levels for Padi1, Padi2 and Padi3 in ES, NSO4G (NSC) and iPS cells, as assessed by qRT–PCR. Padi6 was undetectable in all three cell types. Expression is normalized to endogenous levels of ubiquitin (UbC). Error bars represent the standard error of the mean of three biological replicates. b, Transcript levels of Padi1, Padi2 and Padi3 in ES cells on switch to 2i containing medium for one passage, as assessed by qRT–PCR. Padi6 was undetectable in both conditions. Expression normalized to UbC. Error bars represent the standard error of the mean of three biological replicates. c, Immunoblot analysis of H3Cit levels in ES, NSO4G and iPS cells. Total H3 is presented as a loading control. d, Immunoblot analysis of total citrullination levels in ES, NSO4G and iPS cells, using an antibody against modified citrulline (ModCit), which recognizes peptidylcitrulline irrespective of amino acid sequence. Total H3 is presented as a loading control. e, ZHBTc4.1 and 2TS22C ES cell lines were treated with 1 μg ml−1 doxycycline for 48 h, resulting in depletion of Oct4 or Sox2 (data not shown). Padi4 mRNA was significantly reduced on Oct4, but not Sox2 knockdown, as assessed by qPCR. Error bars represent standard error of the mean of four biological replicates. f, ChIP-qPCR for Oct4, Sox2, Klf4, RNA polymerase II (PolII), H3K4me3 and H2A on the promoter of Padi4 in mouse ES and NSO4G cells. For each cell condition, the signal is presented as fold enrichment over input and after subtracting background signal from the beads. Error bars represent the standard deviation of three technical qPCR replicates. Asterisks denote difference with ES cells (a) or ES media (b), and 0 h time point (e); NS, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by ANOVA (a) or t-test (b, e).

Extended Data Figure 2 PADI4 overexpression or knockdown in ES cells modulates expression of pluripotency genes.

a, Validation of selected targets from the PADI4 overexpression microarray data set by qRT–PCR. Expression of Pou5f1, Sox2, Klf4 and c-Myc is not affected by PADI4 overexpression. Expression is normalized to UbC. Error bars are presented as standard error of the mean of three biological replicates. b, Transcript levels of mouse Padi4 and human PADI4 in mouse ES cells after transient knockdown with Padi4 or control (ctrl) shRNA, and overexpression of human PADI4 or control vector (pPB ctrl), as assessed by qRT–PCR. Expression normalized to UbC. Error bars represent the standard error of the mean of three biological replicates. c, Transcript levels of mouse Padi4, Tcl1 and Nanog in a mouse ES cell clone stably expressing Padi4 or control (ctrl) shRNA, as assessed by qRT–PCR. Expression is normalized to UbC. Error bars represent the standard error of the mean of three biological replicates. Asterisks denote difference with ctrl (a, b, c) and between samples (b); NS, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by ANOVA (b) or t-test (a, c).

Extended Data Figure 3 Chromatin immunoprecipitation of H2A, PADI4 and H3Cit at pluripotency loci.

a, Representative ChIP-qPCR for H2A on regulatory regions of Tcl1 and Nanog in mouse ES, NSO4G and iPS cells (corresponding to Fig. 1h). For each cell condition, the signal is presented as fold enrichment over input and after subtracting background signal from the beads. Error bars represent the standard deviation of three technical qPCR replicates. b, ChIP-qPCR for human PADI4 on regulatory regions of Tcl1, Nanog, Klf2 and Kit, which are upregulated by human PADI4 overexpression, in mouse ES cells stably expressing human PADI4. For each cell condition, the signal is presented as fold enrichment over input and after subtracting background signal from the beads. Enhancer regions for Kit located +3.4 kb and +12 kb downstream of transcription termination site. Error bars represent the standard deviation of three technical qPCR replicates. c, Representative ChIP-qPCR for H3Cit on regulatory regions of Tcl1 and Nanog in mouse ES cells stably expressing human PADI4 and treated with 200 μM Cl-amidine for 48 h. For each cell condition, the signal is presented as fold enrichment over input and after subtracting background signal from the beads. Error bars represent the standard deviation of three technical qPCR replicates.

Extended Data Figure 4 Microarray analysis of Padi4 inhibition by Cl-amidine in ES cells.

a, Heat map of the top 70 genes that showed differential expression after Padi4 inhibition in ES cells by with 200 μM Cl-amidine for 48 h, as determined by microarray analysis. Displayed values are normalized log intensities, minus the mean expression of the gene across the two samples. Hierarchical clustering based on correlation. b, Validation of selected targets from the above microarray data set by qRT–PCR. Expression is normalized to UbC. Error bars presented as standard error of the mean of three biological replicates. Asterisks denote difference with Ctrl; *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001 by t-test. c, Gene Ontology for Biological Process (GOBP) analysis for the most regulated gene categories within the microarray data set of Cl-amidine treatment in mouse ES cells. P value is corrected for multiple testing using Benjamini–Hochberg FDR.

Extended Data Figure 5 Padi4 inhibition reduces reprogramming efficiency.

a, Scheme of reprogramming of neural stem cells to pluripotent state. NSO4G cells were retrovirally transduced with Oct4, Klf4 and c-Myc. After 6 days, partially reprogrammed pre-iPS cells arose. For shRNA experiments, pre-iPS cells were stably transfected with control or Padi4 shRNA and then full reprogramming was performed in the presence of 2i/LIF media for 8 days. For Padi4 enzymatic inhibition, pre-iPS cells were immediately changed to 2i/LIF media in the presence of 200 μM Cl-amidine for 8 days. b, Quantification of flow cytometry analysis for the assessment of Oct4–GFP reporter expression in a reprogramming assay using pre-iPS cells stably expressing Padi4 shRNA 4 and control shRNA. Error bars represent standard error of the mean of triplicate samples within a representative from four reprogramming experiments. c, Quantification of Oct4–GFP-positive colonies in the reprogramming assay where pre-iPS cells were Padi4 shRNA 4 versus control (see Fig. 2a), after time-lapse image acquisition with Biostation CT. Error bars represent standard error of the mean of triplicate samples within a representative reprogramming experiment. See Supplementary Video 1 for time-lapse video. d, Immunoblot analysis of H3Cit in pre-iPS cells treated with 2i, after Padi4 knockdown (Padi4 shRNA 4) versus control cells (ctrl shRNA). 2i-containing medium was added on day 2. Gapdh presented as loading control. e, Quantification of flow cytometry analysis for the assessment of Oct4–GFP reporter expression in a reprogramming assay using pre-iPS cells stably expressing Padi4 shRNA 3 and control shRNA. Error bars represent standard error of the mean of triplicate samples. f, qRT–PCR analysis for the expression of Tcl, Nanog and Padi4 mRNAs at the end of the above reprogramming assay (e). Error bars represent standard error of the mean of triplicate samples. g, Quantification of flow cytometry analysis for the assessment of Oct4–GFP reporter expression in a reprogramming assay were treated with 200 μM Cl-amidine. Error bars represent standard error of the mean of triplicate samples within a representative from three reprogramming experiments. h, Quantification of Oct4–GFP-positive colonies in the reprogramming assay where pre-iPS cells were treated with 200 μM Cl-amidine (see Fig. 2c) after time-lapse image acquisition with Biostation CT. Error bars represent standard error of the mean of triplicate samples within a representative reprogramming experiment. i, Immunoblot analysis for the presence of H3Cit at the end of the above reprogramming assay (g). Total histone H3 presented as loading control. Asterisks denote difference with control; NS, not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 by t-test.

Extended Data Figure 6 Cl-amidine treatment impairs early embryo development.

a, Embryos at 2-cell stage were treated with 200 μM Cl-amidine and snapshots were taken at E3.0, E3.5 and E4.0. 200 μM Cl-amidine embryos arrested at 8-cell stage, whereas control embryos continued development to form blastocysts. Phase contrast images are shown. b, Embryos at 2-cell stage were treated with 10 μM Cl-amidine for 12 h, fixed and stained for H3Cit at the 4-cell stage. Phase contrast, H3Cit (white) and Hoechst 33342 (blue) images are shown. Scale bar, 20 μm. c, Embryos at E3.5 were treated with 10 μM Cl-amidine for 24 h, fixed and stained for H3Cit at E4.5. H3Cit (green) and Hoechst 33342 (blue) images are shown. Bar represents 20 μm. d, Table with quantifications of lineage commitment in E4.5 blastocysts treated with 10 μM Cl-amidine from the 2-cell stage. Asterisks denote difference with control, unpaired t-test; *P < 0.05; n = 3 (50 embryos). e, Embryos were cultured in medium supplemented with 10 μM Cl-amidine from 2-cell stage and through pre-implantation development. E4.5 blastocysts were fixed and stained for Sox17 (primitive endoderm marker, red), Cdx2 (trophectoderm marker, green) and Hoechst 33342 (blue). Scale bar, 20 μm. f, Time-lapse analysis of distribution of inner and outer cells at the 8–16-cell transition, on culturing of embryos with medium containing 10 μM Cl-amidine from 2-cell stage. Error bars represent standard error of the mean. Statistical significance was determined by unpaired t-test or Mann–Whitney U-test upon non-normal distribution. Asterisks denote difference with control; *P ≤ 0.05.

Extended Data Figure 7 TDFA treatment reduces percentage of pluripotent cells in the early embryo.

a, Embryos at 2-cell stage were treated with 100 μM TDFA for 12 h and fixed and stained for H3Cit at 4-cell stage. H3Cit and Hoechst 33342 images are shown. b, Table representing the percentage of cells committed to each embryonic lineage in E4.5 blastocysts on treatment of embryos at 2-cell stage with 100 μM TDFA. Scale bars represent mean percentage (±s.e.m.). Asterisks denote difference with control, Mann–Whitney U-test, *P < 0.05; n = 3 (60 embryos). c, Embryos at 2-cell stage were treated with 100 μM TDFA and fixed at embryonic day E4.5. Phase contrast, Nanog (green), Sox17 (purple), Cdx2 (red) and Hoechst 33342 (blue) images are shown.

Extended Data Figure 8 TSA treatment does not impair early embryo development.

a, Embryos at 2-cell stage were treated with 10 nM TSA for 12 h, and fixed and stained for H3K9ac at 4-cell stage. H3K9ac and Hoechst 33342 images are shown. b, Table representing the percentage of cells committed to each embryonic lineage in E4.5 blastocysts on treatment of embryos at 2-cell stage with 10 nM TSA. Scale bars represent mean percentage (±s.e.m.). Asterisks denote difference with control, unpaired t-test; *P < 0.05; n = 2 (32 embryos). c, Embryos at 2-cell stage were treated with 10 nM TSA and fixed at embryonic day E4.5. Phase contrast, Nanog (green), Sox17 (purple), Cdx2 (red) and Hoechst 33342 (blue) images are shown.

Extended Data Figure 9 Mass spectrometry data for citrullinated and unmodified H1.2.

a, Histogram demonstrating the mass accuracies of all fragment ion masses used for identifying citrullinated peptides in our HCD MS/MS spectra. >490,000 y- and b-ion masses are depicted. The average absolute mass accuracy for all of these fragment ions is 3.97 p.p.m. b, Scatter plot representing SILAC ratios in ES cells cultured in 13C6 l-lysine (HEAVY) and LIGHT medium separately, to assess the extent and quality of SILAC labelling. No significant outliers are observed, indicating equal labelling. c, Peptide coverage of histone H1 by LC-MS analysis. Detected peptides are highlighted in light green and cover >60% of H1. Whereas all arginine residues of histone H1 (highlighted in dark green) were accounted for by the analysis, Arg 54 was the only one found citrullinated. d, Fragmentation spectra of the unmodified LysC peptide ERSGVSLAALKK surrounding Arg 54 of H1.2 (unmodified counterpart of citrullinated peptide depicted in Fig. 3d). The y and b series indicate fragments at amide bonds of the peptide. e, Fragment ion table (expected and observed masses for detected y and b ions) for the identified H1R54 citrullination of peptide ERSGVSLAALKK on histone H1.2 (as shown in Fig. 3d). All measured fragment ions were detected with mass accuracies <10 p.p.m., unambiguously identifying that the detected peptide sequence harbours a citrullination at position Arg 54. f, Theoretical and measured b- and y-ion fragment masses for the corresponding unmodified and heavy SILAC labelled H1.2 peptide, as presented in d above.

Extended Data Figure 10 Mass spectrometry spectra for citrullinated H1.5; PADI4 treatment of differentiated nuclei leads to H1 citrullination and chromatin decompaction.

a, MS spectrum of histone H1.5 in a SILAC proteomic screen for identification of PADI4 substrates. Linker histone H1.5 is deiminated by PADI4, as identified by a highly increased SILAC ratio of the heavy labelled identified peptide (marked by a red dot). b, Fragmentation spectra of the doubly charged LysC peptide ERGGVSLPALK surrounding Arg 54 of H1.5. The y and b series indicate fragments at amide bonds of the peptide, unambiguously verifying the citrullinated peptide. c, Mutation of Arg 54 renders histone H1.2 refractory to deimination. Immunoblot analysis of recombinant histone H1.2 using an antibody that detects all deimination events (ModCit). Wild-type and Arg 54-mutant H1.2 were treated with recombinant PADI4, in the presence of activating calcium. Only wild-type H1.2 can be deiminated, indicating that Arg 54 is the only substrate of PADI4 in H1.2. No-calcium reactions presented as negative controls. Total H1.2 presented as loading control. d, Schematic representation of the position of Arg 54 within the globular domain linker histone H1.2. e, Immunoblot analysis of the ‘Pellet’ fraction of C2C12 permeabilized cells treated with recombinant PADI4. Presence of H3Cit species indicates PADI4 activity. Total H3 is presented as a control for equal use of starting material in the two experimental conditions. f, Immunofluorescence analysis of C2C12 nuclei after treatment with recombinant PADI4. Presence of H3Cit species indicates PADI4 activity. DNA is visualized by staining with DAPI. g, Fragmentation spectra of the citrullination site Arg 54 on the evicted H1.2 peptide ERSGVSLAALK (corresponding to Fig. 4b). The evicted histone H1 is citrulinated at Arg 54. h, Theoretical and measured b- and y-ion fragment masses for the citrullinated H1.2 peptide (peptide sequence ERSGVSLAALK) evicted after treatment of C2C12 cells with recombinant human PADI4 (corresponding to Fig. 4b). i, Micrococcal nuclease digestion of C2C12 nuclei after treatment with recombinant PADI4, as described in Fig. 4a. M, size marker.

Supplementary information

Supplementary Table 1

The file contains the primer sequences. (XLSX 12 kb)

Supplementary Table 2

This file contains the complete microarray data for PADI4 over-expression in ES cells. (XLSX 0 kb)

Supplementary Table 3

This file contains complete microarray data for PADI4 inhibition in ES cells. (XLSX 7756 kb)

Supplementary Table 4

This file contains the complete Mass Spec dataset. (XLSX 31 kb)

Time-lapse video of reprogramming experiments

Phase contrast and fluorescence time-lapse videos for the assessment of Oct4-GFP reporter expression after reprogramming of pre-iPS cells stably expressing Padi4 and Ctrl shRNAs. (MOV 1463 kb)

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Christophorou, M., Castelo-Branco, G., Halley-Stott, R. et al. Citrullination regulates pluripotency and histone H1 binding to chromatin. Nature 507, 104–108 (2014). https://doi.org/10.1038/nature12942

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