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Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.

Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA, Allis CD, Coonrod SA - J. Cell Biol. (2009)

Bottom Line: The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes.The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation.Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. yuw12@psu.edu

ABSTRACT
Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.

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Chromatin decondensation induced by PAD4 activation and histone citrullination in HL-60 granulocytes. (A–C) Loss of histone H4Arg3 methylation on the decondensed chromatin (denoted by arrows) after calcium ionophore treatment. (D–F) Increase in H4Cit3 on the decondensed chromatin (denoted by arrows). (G and H) Grayscale images show an increase of H4Cit3 on decondensed chromatin (denoted by arrows). (I and J) Grayscale images show that H4K16 acetylation was not elevated on the decondensed chromatin (denoted by arrows). (K) Changes in histone H3 and H4 citrullination analyzed by Western blotting. Ponceau S staining shows the amount of histones. Bars, 20 µm.
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fig1: Chromatin decondensation induced by PAD4 activation and histone citrullination in HL-60 granulocytes. (A–C) Loss of histone H4Arg3 methylation on the decondensed chromatin (denoted by arrows) after calcium ionophore treatment. (D–F) Increase in H4Cit3 on the decondensed chromatin (denoted by arrows). (G and H) Grayscale images show an increase of H4Cit3 on decondensed chromatin (denoted by arrows). (I and J) Grayscale images show that H4K16 acetylation was not elevated on the decondensed chromatin (denoted by arrows). (K) Changes in histone H3 and H4 citrullination analyzed by Western blotting. Ponceau S staining shows the amount of histones. Bars, 20 µm.

Mentions: In response to DMSO treatment, HL-60 cells differentiate along the granulocyte lineage and express higher levels of PAD4. Previously, we reported that histone H3Arg2, -8, and -17 residues were citrullinated at 0, 27.3, and 6.5% in HL-60 granulocytes after calcium ionophore treatment (Wang et al., 2004). However, the role of histone hypercitrullination remains unclear. In this study, we found that within 15 min after calcium ionophore treatment, a subset of HL-60–derived granulocytes appear to rupture and release long stretches of extensively decondensed chromatin into the extracellular space, forming weblike chromatin structures (Fig. 1 and Fig. S1, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200806072/DC1). In accordance with our previous finding that PAD4 targets histone methylarginine residues for citrullination (Wang et al., 2004), histone H4Arg3 methylation was decreased (Fig. 1 B) and histone H4Cit3 was increased (Fig. 1, E and H) at regions of highly decondensed chromatin, suggesting that chromatin decondensation is associated with PAD activation. The acetylation of histone H4K16 has been correlated with chromatin decondensation (Robinson et al., 2008). In contrast to H4Cit3, this modification was not increased at the decondensed chromatin fibers (Fig. 1, I and J), suggesting that the increased staining of histone citrullination is unlikely to be the result of an increase in antibody accessibility. To further test the increase in histone citrullination, Western blot experiments were performed. After calcium ionophore treatment, both H4Cit3 and histone H3 citrullination (the α-H3Cit antibody is generated against a Cit2-, Cit8-, and Cit17-containing H3 peptide) were significantly increased (Fig. 1 K).


Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation.

Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, Hayama R, Leonelli L, Han H, Grigoryev SA, Allis CD, Coonrod SA - J. Cell Biol. (2009)

Chromatin decondensation induced by PAD4 activation and histone citrullination in HL-60 granulocytes. (A–C) Loss of histone H4Arg3 methylation on the decondensed chromatin (denoted by arrows) after calcium ionophore treatment. (D–F) Increase in H4Cit3 on the decondensed chromatin (denoted by arrows). (G and H) Grayscale images show an increase of H4Cit3 on decondensed chromatin (denoted by arrows). (I and J) Grayscale images show that H4K16 acetylation was not elevated on the decondensed chromatin (denoted by arrows). (K) Changes in histone H3 and H4 citrullination analyzed by Western blotting. Ponceau S staining shows the amount of histones. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654299&req=5

fig1: Chromatin decondensation induced by PAD4 activation and histone citrullination in HL-60 granulocytes. (A–C) Loss of histone H4Arg3 methylation on the decondensed chromatin (denoted by arrows) after calcium ionophore treatment. (D–F) Increase in H4Cit3 on the decondensed chromatin (denoted by arrows). (G and H) Grayscale images show an increase of H4Cit3 on decondensed chromatin (denoted by arrows). (I and J) Grayscale images show that H4K16 acetylation was not elevated on the decondensed chromatin (denoted by arrows). (K) Changes in histone H3 and H4 citrullination analyzed by Western blotting. Ponceau S staining shows the amount of histones. Bars, 20 µm.
Mentions: In response to DMSO treatment, HL-60 cells differentiate along the granulocyte lineage and express higher levels of PAD4. Previously, we reported that histone H3Arg2, -8, and -17 residues were citrullinated at 0, 27.3, and 6.5% in HL-60 granulocytes after calcium ionophore treatment (Wang et al., 2004). However, the role of histone hypercitrullination remains unclear. In this study, we found that within 15 min after calcium ionophore treatment, a subset of HL-60–derived granulocytes appear to rupture and release long stretches of extensively decondensed chromatin into the extracellular space, forming weblike chromatin structures (Fig. 1 and Fig. S1, A–C, available at http://www.jcb.org/cgi/content/full/jcb.200806072/DC1). In accordance with our previous finding that PAD4 targets histone methylarginine residues for citrullination (Wang et al., 2004), histone H4Arg3 methylation was decreased (Fig. 1 B) and histone H4Cit3 was increased (Fig. 1, E and H) at regions of highly decondensed chromatin, suggesting that chromatin decondensation is associated with PAD activation. The acetylation of histone H4K16 has been correlated with chromatin decondensation (Robinson et al., 2008). In contrast to H4Cit3, this modification was not increased at the decondensed chromatin fibers (Fig. 1, I and J), suggesting that the increased staining of histone citrullination is unlikely to be the result of an increase in antibody accessibility. To further test the increase in histone citrullination, Western blot experiments were performed. After calcium ionophore treatment, both H4Cit3 and histone H3 citrullination (the α-H3Cit antibody is generated against a Cit2-, Cit8-, and Cit17-containing H3 peptide) were significantly increased (Fig. 1 K).

Bottom Line: The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes.The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation.Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures.

View Article: PubMed Central - PubMed

Affiliation: Center for Gene Regulation, Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA. yuw12@psu.edu

ABSTRACT
Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.

Show MeSH
Related in: MedlinePlus