Neutrophils trap and kill bacteria by forming highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs). We previously reported that histone hypercitrullination catalyzed by peptidylarginine deiminase 4 (PAD4) correlates with chromatin decondensation during NET formation. However, the role of PAD4 in NET-mediated bacterial trapping and killing has not been tested. Here, we use PAD4 knockout mice to show that PAD4 is essential for NET-mediated antibacterial function. Unlike PAD4+/+ neutrophils, PAD4−/− neutrophils cannot form NETs after stimulation with chemokines or incubation with bacteria, and are deficient in bacterial killing by NETs. In a mouse infectious disease model of necrotizing fasciitis, PAD4−/− mice are more susceptible to bacterial infection than PAD4+/+ mice due to a lack of NET formation. Moreover, we found that citrullination decreased the bacterial killing activity of histones and nucleosomes, which suggests that PAD4 mainly plays a role in chromatin decondensation to form NETs instead of increasing histone-mediated bacterial killing. Our results define a role for histone hypercitrullination in innate immunity during bacterial infection.

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.

No abstract

Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.In eukaryotic cells, 147 bp of DNA is wrapped around a core histone octamer (including two each of histones H3, H2B, H2A, and H4) to form a nucleosome core particle, the basic structural unit of chromatin (52). Posttranslational histone modifications, including methylation, acetylation, phosphorylation, ubiquitination, and citrullination, have been found to play a major role in chromatin functions, such as transcription (5,7,35,39,55). Moreover, specialized protein domains, including chromo-, bromo-, and tudor domains and PHD fingers, have evolved to recognize histone modification "marks" and to regulate nuclear events following histone modifications (33,36,40). Histone Arg methylation is catalyzed by members of the protein Arg methyltransferase (PRMT) family (11,57,61). The methylation of histones H3 and H4 has been correlated with the expression of nuclear receptor target genes and developmentally regulated genes (6, 30). In searching for enzymes that can reverse histone Arg methylation, we and others previously reported that a histone Arg demethylimination enzyme, PAD4 (also called PADI4), can convert both Arg and monomethyl-Arg to citrulline, thereby regulating histone Arg methylation (14,35,62). In contrast to the role of histone Arg methylation in transcriptional activation, the demethylimination and citrullination of histones mediated by PAD4 have been found to play a role in transcriptional repression of nuclear receptor target genes (3,14,62). Several mechanisms can be envisioned to explain the functions of histone demethylimination and cit...

Background: Histone citrullination by PAD4 regulates tumor suppressor gene expression. Results:The novel PAD inhibitor YW3-56 inhibits cancerous growth by perturbing autophagy and regulating the SESN2-mTORC1 signaling axis. Conclusion: YW3-56 regulates the SESN2-mTORC1 autophagy pathway as one of its anticancer mechanisms. Significance: This study identifies a novel function of PAD4 in the autophagy pathway and developed potent PAD inhibitors for future cancer research.

Protein Arg methyltransferases function as coactivators of the tumor suppressor p53 to regulate gene expression. Peptidylarginine deiminase 4 (PAD4/PADI4) counteracts the functions of protein Arg methyltransferases in gene regulation by deimination and demethylimination. Here we show that the expression of a tumor suppressor gene, OKL38, is activated by the inhibition of PAD4 or the activation of p53 following DNA damage. Chromatin immunoprecipitation assays showed a dynamic change of p53 and PAD4 occupancy and histone Arg modifications at the OKL38 promoter during DNA damage, suggesting a direct role of PAD4 and p53 in the expression of OKL38. Furthermore, we found that OKL38 induces apoptosis through localization to mitochondria and induction of cytochrome c release. Together, our studies identify OKL38 as a novel p53 target gene that is regulated by PAD4 and plays a role in apoptosis.

SUMMARY Tumor-suppressor p53 regulates transcription of stress response genes. Many p53 targets remain undiscovered due to uncertainty as to where p53 binds in the genome, and that few genes reside near p53-bound recognition elements (REs). Using ChIP-exo, we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of TFIIB and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes comprised a large portion of a pre-defined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription.

No abstract