The linker histone H1 family members are a key component of chromatin and bind to the nucleosomal core particle around the DNA entry and exit sites. H1 can stabilize both nucleosome structure and higher-order chromatin architecture. In general, H1 molecules consist of a central globular domain with more flexible tail regions at both their N-and C-terminal ends. The existence of multiple H1 subtypes and a large variety of posttranslational modifications brings about a considerable degree of complexity and makes studying this protein family challenging. Here, we review recent progress in understanding the function of linker histones and their subtypes beyond their role as merely structural chromatin components. We summarize current findings on the role of H1 in heterochromatin formation, transcriptional regulation and embryogenesis with a focus on H1 subtypes and their specific modifications.

BackgroundThe linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown.ResultsIn this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle.ConclusionsWe have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions.

In the absence of serglycin proteoglycans, connective tissue‐type mast cells fail to assemble mature metachromatic secretory granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of secretory granules as well as the fate of the secretory granule proteases in bone marrow‐derived mast cells from serglycin+/+ and serglycin–/– mice. We show that functional secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin–/– mast cell granules. The low levels of mast cell proteases present in serglycin–/– cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into secretory granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin‐dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, β‐hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into secretory vesicles.

T he metabolic syndrome comprises a cluster of risk factors, including obesity, insulin resistance, hepatic steatosis, and dyslipidemia. It is associated with a variety of cardiovascular diseases such as atherosclerosis, myocardial infarction, and stroke.1 Chronic, low-grade inflammation in key metabolic organs such as the liver and visceral adipose tissue (VAT) Background-Costimulatory cascades such as the CD40L-CD40 dyad enhance immune cell activation and inflammation during atherosclerosis. Here, we tested the hypothesis that CD40 directly modulates traits of the metabolic syndrome in diet-induced obesity in mice. Methods and Results-To induce the metabolic syndrome, wild-type or CD40 −/− mice consumed a high-fat diet for 20 weeks. Unexpectedly, CD40−/− mice exhibited increased weight gain, impaired insulin secretion, augmented accumulation of inflammatory cells in adipose tissue, and enhanced proinflammatory gene expression. This proinflammatory and adverse metabolic phenotype could be transplanted into wild-type mice by reconstitution with CD40-deficient lymphocytes, indicating a major role for CD40 in T or B cells in this context. Conversely, therapeutic activation of CD40 signaling by the stimulating antibody FGK45 abolished further weight gain during the study, lowered glucose levels, improved insulin sensitivity, and suppressed adipose tissue inflammation. Mechanistically, CD40 activation decreased the expression of proinflammatory cytokines in T cells but not in B cells or macrophages. Finally, repopulation of lymphocyte-free Rag1 −/− mice with CD40 −/− T cells provoked dysmetabolism and inflammation, corroborating a protective role of CD40 on T cells in the metabolic syndrome. Finally, levels of soluble CD40 showed a positive association with obesity in humans, suggesting clinical relevance of our findings. Conclusions-We present the surprising finding that CD40 deficiency on T cells aggravates whereas activation of CD40 signaling improves adipose tissue inflammation and its metabolic complications. Therefore, positive modulation of the CD40 pathway might describe a novel therapeutic concept against cardiometabolic disease. T-regulatory (T reg ) cells, CD8 + T cells, and related chemokines and cytokines such as RANTES (regulated on activation normal T cell expressed and secreted) and interferon-γ (IFNγ) colocalize within the inflammatory cell compartment in adipose tissue. 7 In lean adipose tissue, the vast majority of T lymphocytes share features of anti-inflammatory, interleukin (IL)-13-, IL-4-, and IL-10-secreting Th2 or T reg cells. 8In obesity, proinflammatory Th1 cells expressing IFNγ overwhelm Th2 cells.9 Th1 cells, in turn, activate proinflammatory cytokine-secreting macrophages and promote their conversion from M2-like, IL-10-secreting, alternatively activated macrophages to classically activated, M1-like macrophages. 10,11Despite description of the kinetics of cellular infiltration and the associated cytokine/chemokine profiles during the development of obesity, the underlying cause modu...

The linker histone H1 plays an essential role in maintaining and establishing higher-order chromatin structure. As with core histones, histone H1 is also extensively covalently modified. We showed previously that phosphorylation of S27 in human histone H1.4 (H1.4S27-P), prevents binding of heterochromatin protein 1 (HP1) family members (officially known as chromobox protein homologs) to the neighboring dimethylated K26. Here, we present the first functional characterization of H1.4S27-P in vivo and in vitro. We show that H1.4S27 phosphorylation is cell-cycle-regulated and its levels peak on metaphase chromosomes. We identify further Aurora B as the kinase phosphorylating H1.4S27. We demonstrate that histone H1.4 is the only somatic linker histone variant targeted by Aurora B and that Aurora B exclusively phosphorylates S27. Adjacent K26 dimethylation can regulate Aurora B activity towards S27, uncovering a crosstalk between these modifications. Finally, our fluorescence recovery after photobleaching (FRAP) analysis on histone H1.4 mutants suggests a role of S27 phosphorylation in the regulation of histone H1.4 mobility and chromatin binding in mitosis.

Objective-Nucleotides such as ATP, ADP, UTP, and UDP serve as proinflammatory danger signals via purinergic receptors on their release to the extracellular space by activated or dying cells. UDP binds to the purinergic receptor Y 6 (P2Y 6 ) and propagates vascular inflammation by inducing the expression of chemokines such as monocyte chemoattractant protein 1, interleukin-8, or its mouse homologsCCL1 (chemokine [C-C motif] ligand 1)/keratinocyte chemokine, CXCL2 (chemokine [C-X-C motif] ligand 2)/macrophage inflammatory protein 2, and CXCL5 (chemokine [C-X-C motif] ligand 5)/LIX, and adhesion molecules such as vascular cell adhesion molecule 1 and intercellular cell adhesion molecule 1. Thus, P2Y 6 contributes to leukocyte recruitment and inflammation in conditions such as allergic asthma or sepsis. Because atherosclerosis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall, we hypothesized a role of P2Y 6 in atherogenesis. Approach and Results-Intraperitoneal stimulation of wild-type mice with UDP induced rolling and adhesion of leukocytes to the vessel wall as assessed by intravital microscopy. This effect was not present in P2Y 6 -deficient mice. Atherosclerotic aortas of low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks expressed significantly more transcripts and protein of P2Y 6 than respective controls. Finally, P2Y 6 −/− /low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y 6 +/+ /low-density lipoprotein receptor-deficient mice. Bone marrow transplantation identified a crucial role of P2Y 6 on vascular resident cells, most likely endothelial cells, on leukocyte recruitment and atherogenesis. Atherosclerotic lesions of P2Y 6 -deficient mice contained fewer macrophages and fewer lipids as determined by immunohistochemistry. Mechanistically, RNA expression of vascular cell adhesion molecule 1 and interleukin-6 was decreased in these lesions and P2Y 6 -deficient macrophages took up less modified low-density lipoprotein cholesterol. 16 demonstrated that P2Y 6 promotes host defense by augmenting the expression of monocyte chemoattractant protein 1 (MCP-1) and guiding monocytes to the location of infection. Besides the chemokine-driven recruitment, also a direct chemoattractant activity of the UDP-P2Y 6 R axis has been described 17 for leukocytes. Thus, stimulation of the P2Y 6 R has been implicated with chronic inflammatory diseases. Indeed, selective inhibition or knockdown of P2Y 6 R reduced all cardinal features of experimental acute and chronic allergic asthma in mice. 18 Furthermore, P2Y 6 contributes to inflammation of intestinal mucosa promoting IL-8-dependent neutrophil recruitment. Conclusions-We 19Pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid, a synthetic substance blocking several purinergic receptors including P2Y 6 , P2Y 4 , P2Y 11 , and purinergic receptors X (ion channels), reduced atherosclerosis in...

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6Chigh monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe−/− mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6Chigh monocytes and macrophages. SYK inhibition limited Ly6Chigh monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe−/− mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-016-0535-8) contains supplementary material, which is available to authorized users.

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