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.

The architecture of the chromatin fiber, which determines DNA accessibility for transcription and other template-directed biological processes, remains unknown. Here we investigate the internal organization of the 30-nm chromatin fiber, combining Monte Carlo simulations of nucleosome chain folding with EM-assisted nucleosome interaction capture (EMANIC). We show that at physiological concentrations of monovalent ions, linker histones lead to a tight 2-start zigzag dominated by interactions between alternate nucleosomes (i ؎ 2) and sealed by histone N-tails. Divalent ions further compact the fiber by promoting bending in some linker DNAs and hence raising sequential nucleosome interactions (i ؎ 1). Remarkably, both straight and bent linker DNA conformations are retained in the fully compact chromatin fiber as inferred from both EMANIC and modeling. This conformational variability is energetically favorable as it helps accommodate DNA crossings within the fiber axis. Our results thus show that the 2-start zigzag topology and the type of linker DNA bending that defines solenoid models may be simultaneously present in a structurally heteromorphic chromatin fiber with uniform 30 nm diameter. Our data also suggest that dynamic linker DNA bending by linker histones and divalent cations in vivo may mediate the transition between tight nucleosome packing within discrete 30-nm fibers and self-associated higher-order chromosomal forms.chromatin structure ͉ electron microscopy ͉ mesoscopic modeling ͉ Monte Carlo simulations ͉ linker histone T he DNA in eukaryotic chromatin is packed and functionally regulated by histones and nonhistone architectural proteins (1). The primary packing level is represented by an array of repeating units, the nucleosomes, where the DNA is wound around histone octamers (2). Further compaction is achieved through a hierarchy of folding levels, including the 30-nm chromatin fiber (secondary level) and more compact and self-associating tertiary and quaternary forms whose structures are unknown (3-6). Because DNA conformation in chromatin and nucleosome packing are intimately connected to DNA/protein recognition and gene regulation, there has been intense interest in understanding chromatin structure, energetics, and dynamics. Some experimental studies have suggested that nucleosomal arrays fold in a zigzag arrangement with relatively straight linkers and a 2-start nucleosome interaction pattern that brings each nucleosome in proximity to its second nearest neighbor (7-10) consistent with chromatin fibers observed in situ (11). Other evidence suggests that chromatin condensed with linker histones and divalent cations such as Mg 2ϩ can form either zigzag structures with various nucleosome topologies (12, 13) or solenoid-like arrangements. The latter class of structures has bent DNA linkers and predominant interactions between either nearest neighbor nucleosomes (14, 15) and/or between every fifth or sixth nucleosome along the chain (16,17).The picture has became more complex recently with our heighte...

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SUMMARY Structural changes in specific chromatin domains are essential to the orderly progression of numerous nuclear processes, including transcription. We report that the nuclear protein NSBP1 (HMGN5), a recently discovered member of the HMGN nucleosome-binding protein family, is specifically targeted by its C-terminal domain to nucleosomes in euchromatin. We find that the interaction of NSBP1 with nucleosomes alters the compaction of cellular chromatin and that in living cells, NSBP1 interacts with linker histones. We demonstrate that the negatively charged C-terminal domain of NSBP1 interacts with the positively charged C-terminal domain of H5 and that NSBP1 counteracts the linker histone-mediated compaction of a nucleosomal array. Dysregulation of the cellular levels of NSBP1 alters the transcription level of numerous genes. We suggest that mouse NSBP1 is an architectural protein that binds preferentially to euchromatin and modulates the fidelity of the cellular transcription profile by counteracting the chromatin-condensing activity of linker histones.