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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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PAD4 activity is important for the formation of highly decondensed chromatin. (A–C) H4Cit3 and DNA staining of HL-60 granulocytes without treatment (A), with calcium ionophore treatment (B), and with the PAD4 inhibitor Cl-amidine treatment before calcium ionophore treatment (C). The arrows denote decondensed chromatin stained by the H4Cit3 antibody. (D) Western blot assays of histone H3 and H4 citrullination. The histone H3 blot is a loading control. (E–G) Decondensed chromatin (denoted by arrows) was detected in HL-60 cells after Triton X-100 and GST-PAD4 treatment (E and F) but not after the GST-PAD4C645S mutant treatment (G). (H) Western blot assays of histone H3 and H4 citrullination in HL-60 cells after incubation with GST-PAD4 or the GST-PAD4C645S mutant. Ponceau S staining shows the amount of histones. (I) MNase digestion of HL-60 cells treated with GST-PAD4 or the GST-PAD4C645S mutant. Chromatin in GST-PAD4–treated cells (lane 7) is more accessible than that in GST-PAD4C645S–treated cells (lane 8). The red box highlights lanes with a clear difference in MNase digestion. Bars, 30 µm.
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fig3: PAD4 activity is important for the formation of highly decondensed chromatin. (A–C) H4Cit3 and DNA staining of HL-60 granulocytes without treatment (A), with calcium ionophore treatment (B), and with the PAD4 inhibitor Cl-amidine treatment before calcium ionophore treatment (C). The arrows denote decondensed chromatin stained by the H4Cit3 antibody. (D) Western blot assays of histone H3 and H4 citrullination. The histone H3 blot is a loading control. (E–G) Decondensed chromatin (denoted by arrows) was detected in HL-60 cells after Triton X-100 and GST-PAD4 treatment (E and F) but not after the GST-PAD4C645S mutant treatment (G). (H) Western blot assays of histone H3 and H4 citrullination in HL-60 cells after incubation with GST-PAD4 or the GST-PAD4C645S mutant. Ponceau S staining shows the amount of histones. (I) MNase digestion of HL-60 cells treated with GST-PAD4 or the GST-PAD4C645S mutant. Chromatin in GST-PAD4–treated cells (lane 7) is more accessible than that in GST-PAD4C645S–treated cells (lane 8). The red box highlights lanes with a clear difference in MNase digestion. Bars, 30 µm.

Mentions: To test whether PAD4 activity is important for chromatin decondensation, we treated HL-60 granulocytes with Cl-amidine, a newly synthesized PAD4 inhibitor (Luo et al., 2006), before calcium ionophore treatment. Double staining of HL-60 granulocytes with the H4Cit3 antibody and the DNA dye Hoechst was performed to analyze histone citrullination and the formation of NET-like chromatin structure. Without calcium ionophore treatment, each cell had only weak H4Cit3 staining and did not display NET-like chromatin structure (Fig. 3 A). After calcium ionophore treatment, ∼36% of cells were strongly stained with the H4Cit3 antibody (Fig. 3 B). Notably, >8% of cells formed NET-like chromatin structures (Fig. 3 B, arrows). In contrast, Cl-amidine treatment for 15 min before calcium ionophore treatment decreased the number of cells showing positive H4Cit3 staining (Fig. 3 C). Moreover, NET-like chromatin structure was not observed in cells treated with Cl-amidine before calcium ionophore treatment (Fig. 3 C). The effect of PAD4 inhibition by Cl-amidine on histone H3 and H4 citrullination after calcium ionophore treatment was validated by Western blotting (Fig. 3 D, compare lane 2 with lane 3). These results indicate that PAD activity is important for histone citrullination and chromatin decondensation in HL-60 granulocytes after calcium ionophore treatment.


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)

PAD4 activity is important for the formation of highly decondensed chromatin. (A–C) H4Cit3 and DNA staining of HL-60 granulocytes without treatment (A), with calcium ionophore treatment (B), and with the PAD4 inhibitor Cl-amidine treatment before calcium ionophore treatment (C). The arrows denote decondensed chromatin stained by the H4Cit3 antibody. (D) Western blot assays of histone H3 and H4 citrullination. The histone H3 blot is a loading control. (E–G) Decondensed chromatin (denoted by arrows) was detected in HL-60 cells after Triton X-100 and GST-PAD4 treatment (E and F) but not after the GST-PAD4C645S mutant treatment (G). (H) Western blot assays of histone H3 and H4 citrullination in HL-60 cells after incubation with GST-PAD4 or the GST-PAD4C645S mutant. Ponceau S staining shows the amount of histones. (I) MNase digestion of HL-60 cells treated with GST-PAD4 or the GST-PAD4C645S mutant. Chromatin in GST-PAD4–treated cells (lane 7) is more accessible than that in GST-PAD4C645S–treated cells (lane 8). The red box highlights lanes with a clear difference in MNase digestion. Bars, 30 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: PAD4 activity is important for the formation of highly decondensed chromatin. (A–C) H4Cit3 and DNA staining of HL-60 granulocytes without treatment (A), with calcium ionophore treatment (B), and with the PAD4 inhibitor Cl-amidine treatment before calcium ionophore treatment (C). The arrows denote decondensed chromatin stained by the H4Cit3 antibody. (D) Western blot assays of histone H3 and H4 citrullination. The histone H3 blot is a loading control. (E–G) Decondensed chromatin (denoted by arrows) was detected in HL-60 cells after Triton X-100 and GST-PAD4 treatment (E and F) but not after the GST-PAD4C645S mutant treatment (G). (H) Western blot assays of histone H3 and H4 citrullination in HL-60 cells after incubation with GST-PAD4 or the GST-PAD4C645S mutant. Ponceau S staining shows the amount of histones. (I) MNase digestion of HL-60 cells treated with GST-PAD4 or the GST-PAD4C645S mutant. Chromatin in GST-PAD4–treated cells (lane 7) is more accessible than that in GST-PAD4C645S–treated cells (lane 8). The red box highlights lanes with a clear difference in MNase digestion. Bars, 30 µm.
Mentions: To test whether PAD4 activity is important for chromatin decondensation, we treated HL-60 granulocytes with Cl-amidine, a newly synthesized PAD4 inhibitor (Luo et al., 2006), before calcium ionophore treatment. Double staining of HL-60 granulocytes with the H4Cit3 antibody and the DNA dye Hoechst was performed to analyze histone citrullination and the formation of NET-like chromatin structure. Without calcium ionophore treatment, each cell had only weak H4Cit3 staining and did not display NET-like chromatin structure (Fig. 3 A). After calcium ionophore treatment, ∼36% of cells were strongly stained with the H4Cit3 antibody (Fig. 3 B). Notably, >8% of cells formed NET-like chromatin structures (Fig. 3 B, arrows). In contrast, Cl-amidine treatment for 15 min before calcium ionophore treatment decreased the number of cells showing positive H4Cit3 staining (Fig. 3 C). Moreover, NET-like chromatin structure was not observed in cells treated with Cl-amidine before calcium ionophore treatment (Fig. 3 C). The effect of PAD4 inhibition by Cl-amidine on histone H3 and H4 citrullination after calcium ionophore treatment was validated by Western blotting (Fig. 3 D, compare lane 2 with lane 3). These results indicate that PAD activity is important for histone citrullination and chromatin decondensation in HL-60 granulocytes after calcium ionophore treatment.

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