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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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Histone citrullination on highly decondensed chromatin in peripheral blood neutrophils treated with TNF-α. (A–C) H4Cit3 staining before TNF-α treatment. (D–F) Approximately 10% of cells were stained with H4Cit3 after 15-min TNF-α treatment. The white arrows denote decondensed chromatin. (G–L) Representative images of NETs stained by both DNA dye and H4Cit3 antibody. Bars, 20 µm.
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fig2: Histone citrullination on highly decondensed chromatin in peripheral blood neutrophils treated with TNF-α. (A–C) H4Cit3 staining before TNF-α treatment. (D–F) Approximately 10% of cells were stained with H4Cit3 after 15-min TNF-α treatment. The white arrows denote decondensed chromatin. (G–L) Representative images of NETs stained by both DNA dye and H4Cit3 antibody. Bars, 20 µm.

Mentions: Blood neutrophils are known to form highly decondensed chromatin structures, termed NETs, after bacterial infection (Brinkmann et al., 2004; Fuchs et al., 2007). To analyze whether histone hypercitrullination and chromatin decondensation occur in blood neutrophils, we treated neutrophils with the proinflammatory cytokine TNF-α. Before TNF-α treatment, very low levels of H4Cit3 antibody staining were observed in neutrophils (Fig. 2, A–C). After TNF-α treatment for 15 min, ∼10% of neutrophils (>300 cells counted from three independent experiments) showed a dramatic increase in histone citrullination (Fig. 2, D–F), suggesting that PAD4 is activated after TNF-α treatment in a subset of blood neutrophils. A close examination of the decondensed chromatin with NET morphology found that high levels of histone citrullination are associated with decondensed chromatin (Fig. 2, G–L). While we were preparing this manuscript, a study showed that various cytokines and extracellular signals such as TNF, lipopolysaccharide, and H2O2 induced histone citrullination and chromatin decondensation in neutrophils (Neeli et al., 2008). Collectively, these experiments relate histone hypercitrullination to chromatin decondensation during NET formation.


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)

Histone citrullination on highly decondensed chromatin in peripheral blood neutrophils treated with TNF-α. (A–C) H4Cit3 staining before TNF-α treatment. (D–F) Approximately 10% of cells were stained with H4Cit3 after 15-min TNF-α treatment. The white arrows denote decondensed chromatin. (G–L) Representative images of NETs stained by both DNA dye and H4Cit3 antibody. Bars, 20 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Histone citrullination on highly decondensed chromatin in peripheral blood neutrophils treated with TNF-α. (A–C) H4Cit3 staining before TNF-α treatment. (D–F) Approximately 10% of cells were stained with H4Cit3 after 15-min TNF-α treatment. The white arrows denote decondensed chromatin. (G–L) Representative images of NETs stained by both DNA dye and H4Cit3 antibody. Bars, 20 µm.
Mentions: Blood neutrophils are known to form highly decondensed chromatin structures, termed NETs, after bacterial infection (Brinkmann et al., 2004; Fuchs et al., 2007). To analyze whether histone hypercitrullination and chromatin decondensation occur in blood neutrophils, we treated neutrophils with the proinflammatory cytokine TNF-α. Before TNF-α treatment, very low levels of H4Cit3 antibody staining were observed in neutrophils (Fig. 2, A–C). After TNF-α treatment for 15 min, ∼10% of neutrophils (>300 cells counted from three independent experiments) showed a dramatic increase in histone citrullination (Fig. 2, D–F), suggesting that PAD4 is activated after TNF-α treatment in a subset of blood neutrophils. A close examination of the decondensed chromatin with NET morphology found that high levels of histone citrullination are associated with decondensed chromatin (Fig. 2, G–L). While we were preparing this manuscript, a study showed that various cytokines and extracellular signals such as TNF, lipopolysaccharide, and H2O2 induced histone citrullination and chromatin decondensation in neutrophils (Neeli et al., 2008). Collectively, these experiments relate histone hypercitrullination to chromatin decondensation during NET formation.

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