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.

Wound healing is impaired in diabetes resulting in significant morbidity and mortality. Neutrophils are the main leukocytes involved in the early phase of healing. As part of their anti-microbial defense, neutrophils form extracellular traps (NETs) by releasing decondensed chromatin lined with cytotoxic proteins1. NETs, however, can also induce tissue damage. Here we show that neutrophils isolated from type 1 and type 2 diabetic humans and mice were primed to produce NETs (a process termed, NETosis). Expression of peptidylarginine deiminase 4 (PAD4), an enzyme important in chromatin decondensation, was elevated in neutrophils from individuals with diabetes. When subjected to excisional skin wounds, wild-type (WT) mice produced large quantities of NETs in wounds, but this was not observed in Padi4−/− mice. In diabetic mice, higher levels of citrullinated histone H3 (H3Cit, a NET marker) were found in their wounds and healing was delayed. Wound healing was accelerated in Padi4−/− mice as compared to WT mice, and was not compromised by diabetes. DNase 1, which disrupts NETs, accelerated wound healing in diabetic and normoglycemic WT mice. Thus, NETs impair wound healing, particularly in diabetes where neutrophils are more susceptible to NETosis. Inhibiting NETosis or cleaving NETs may improve wound healing and reduce NET-driven chronic inflammation in diabetes.

Chromatin is the physiologically relevant substrate for all genetic processes inside the nuclei of eukaryotic cells. Dynamic changes in the local and global organization of chromatin are emerging as key regulators of genomic function. Indeed, a multitude of signals from outside and inside the cell converges on this gigantic signaling platform. Numerous post-translational modifications of histones, the main protein components of chromatin, have been documented and analyzed in detail. These 'marks' appear to crucially mediate the functional activity of the genome in response to upstream signaling pathways. Different layers of cross-talk between several components of this complex regulatory system are emerging, and these epigenetic circuits are the focus of this review.

Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.

On the histone H3 tail, Lys 9 and Lys 27 are both methylation sites associated with epigenetic repression, and reside within a highly related sequence motif ARKS. Here we show that the chromodomain proteins Polycomb (Pc) and HP1 (heterochromatin protein 1) are highly discriminatory for binding to these sites in vivo and in vitro. In Drosophila S2 cells, and on polytene chromosomes, methyl-Lys 27 and Pc are both excluded from areas that are enriched in methyl-Lys 9 and HP1. Swapping of the chromodomain regions of Pc and HP1 is sufficient for switching the nuclear localization patterns of these factors, indicating a role for their chromodomains in both target site binding and discrimination. To better understand the molecular basis for the selection of methyl-lysine binding sites, we solved the 1.8 Å structure of the Pc chromodomain in complex with a H3 peptide bearing trimethyl-Lys 27, and compared it with our previously determined structure of the HP1 chromodomain in complex with a H3 peptide bearing trimethyl-Lys 9. The Pc chromodomain distinguishes its methylation target on the H3 tail via an extended recognition groove that binds five additional residues preceding the ARKS motif. Chromatin structure contains the molecular imprint underlying cell memory and epigenetic inheritance, and emerging evidence suggests that covalent modifications of histones play a major role as carriers of epigenetic information (Felsenfeld and Groudine 2003). Histone modifications can be highly reversible, such as histone acetylation, or more stable, such as histone (lysine) methylation (Zhang and Reinberg 2001;Lachner and Jenuwein 2002). Thus, a wide range of chromatin-based regulatory options is available. These include dynamic marks permitting rapid changes in gene expression in response to physiological and environmental stimuli as well as more permanent indexing systems required for the passage of heritable patterns of epigenetic information from one cell generation to the next (Fischle et al. 2003). The identification of enzyme systems responsible for the steady-state balance of posttranslational histone modifications, together with the discovery of binding modules that "read" covalent marks on histones, have been key for our present understanding of gene regulation in the context of the chromatin polymer.Bromodomains have been the first modules implicated in the read-out of histone marks. They show affinity for acetylated lysines in histone and nonhistone proteins (for review, see Zeng and Zhou 2002), and local recruitment of bromodomain factors to certain regions of chromatin might function in mediating acetyl-histone-encoded antisilencing (Ladurner et al. 2003). In contrast, a second conserved module found in a variety of chromosomal proteins, the chromodomain, has been implicated in binding to methylated lysines on the histone tails (Bannister et al. 2001;Jacobs et al. 2001;Lachner et al. 2001). Indeed, recently a biochemical pathway of gene repression by heterochromatin assembly, involving methylation of Lys 9 of H3 by SE...

Risks of tumor recurrence after surgical resection have been known for decades, but the mechanisms underlying treatment failures remain poorly understood. Neutrophils, first-line responders after surgical stress, may play an important role in linking inflammation to cancer progression. In response to stress, neutrophils can expel their protein-studded chromatin to form local snares known as neutrophil extracellular traps (NET). In this study, we asked whether as a result of its ability to ensnare moving cells NET formation might promote metastasis after surgical stress. Consistent with this hypothesis, in a cohort of patients undergoing attempted curative liver resection for metastatic colorectal cancer, we observed that increased postoperative NET formation was associated with a >4-fold reduction in disease-free survival. In like manner, in a murine model of surgical stress employing liver ischemia-reperfusion, we observed an increase in NET formation that correlated with an accelerated development and progression of metastatic disease. These effects were abrogated by inhibiting NET formation in mice, through either local treatment with DNAse or inhibition of the enzyme peptidylarginine deaminase (PAD4), which is essential for NET formation. In growing metastatic tumors, we found that intratumoral hypoxia accentuated NET formation. Mechanistic investigations in vitro indicated that mouse neutrophil-derived NET triggered HMGB1 release and activated TLR9-dependent pathways in cancer cells to promote their adhesion, proliferation, migration and invasion. Taken together, our findings implicate NET in the development of liver metastases after surgical stress, suggesting that their elimination may reduce risks of tumor relapse.

We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

An immense number of post-translational modifications on histone proteins have been described and additional sites of modification are still being uncovered. Whereas many direct and indirect connections between certain histone modifications and distinct biological phenomena have now been established, concepts for comprehending the extreme density and variety of these covalent modifications are lacking. Here, we formally introduce localized 'binary switches' and 'modification cassettes' as new concepts in histone biology, elucidating mechanisms that might govern the biological readout of distinct modification patterns. Specifically, our hypotheses provide missing models for the dynamic readout of stable histone modifications and offer explanations for several long-standing questions embedded in the literature. Our ideas might also apply to non-histone proteins and are open to direct experimental examination.