Mitotic chromatin condensation is essential for cell division in eukaryotes. Posttranslational modification of the N-terminal tail of histone proteins, particularly by phosphorylation by mitotic histone kinases, may facilitate this process. In mammals, aurora B is believed to be the mitotic histone H3 Ser10 kinase; however, it is not sufficient to phosphorylate H3 Ser10 with aurora B alone. We show that histone H3 is phosphorylated by vaccinia-related kinase 1 (VRK1). Direct phosphorylation of Thr3 and Ser10 in H3 by VRK1 both in vitro and in vivo was observed. Loss of VRK1 activity was associated with a marked decrease in H3 phosphorylation during mitosis. Phosphorylation of Ser10 by VRK1 is similar to that by aurora B. Moreover, expression and chromatin localization of VRK1 depended on the cell cycle phase. Overexpression of VRK1 resulted in a dramatic condensation of nuclei. Our findings collectively support a role of VRK1 as a novel mitotic histone H3 kinase in mammals.Chromatin congregates to chromosomes during mitosis to facilitate the even segregation of genetic information to two daughter cells. In nucleosomes, the combinational modification of histone tails, the so-called "histone code," controls chromatintemplated processes from gene expression to cell fate decision (20,30). Phosphorylation of the N-terminal tail of histone H3 may be responsible for chromatin condensation (21). During mitosis, the N-terminal tail of histone H3 is phosphorylated at several residues, including Thr3 (5, 36), Ser10 (3,7,17,18), Thr11 (37), and Ser28 (12). A correlation between histone H3 Ser10 phosphorylation and chromatin condensation in Aspergillus nidulans (6) and Tetrahymena thermophila (47) is well established. However, in other species, condensation is not accomplished simply by Ser10 phosphorylation, and additional phosphorylation or modification of histone tails is required (21).A number of studies have shown that members of the aurora kinase family are responsible for phosphorylation of histone H3 (3,7,17,18). Mammals contain three isotypes of aurora kinase designated aurora A, B, and C (11). Among these, aurora B is a strong candidate phosphorylator of Ser10 in histone H3 as is evident from data obtained with hesperadin, the aurora B inhibitor (14), which suppressed Ser10 phosphorylation during mitosis (7, 17). However, residual Ser10 phosphorylation was detected, even upon depletion of aurora B in cells, suggesting the presence of an additional histone H3 kinase (29).NIMA (never in mitosis), the histone H3 Ser10 kinase in Aspergillus nidulans (6, 34), triggers chromatin condensation in cells arrested at the interphase (28). In mammals, Nercc1, the functional ortholog of NIMA, was found to be phosphorylating histone H3 (39). Nucleosomal histone kinase 1 (NHK1) from Drosophila melanogaster is the kinase shown to phosphorylate histone protein in chromatin as a substrate. NHK1 phosphorylated H2A at Thr119 in chromatin but not with free histone as the substrate (1). Recent studies showed that NHK1 participates in mit...

Invariant natural killer T (iNKT), mucosal-associated invariant T (MAIT), and γδ T cells are innate T cells that acquire memory phenotype in the thymus and share similar biological characteristics. However, how their effector differentiation is developmentally regulated is still unclear. Here, we identify analogous effector subsets of these three innate T cell types in the thymus that share transcriptional profiles. Using single-cell RNA sequencing, we show that iNKT, MAIT and γδ T cells mature via shared, branched differentiation rather than linear maturation or TCR-mediated instruction. Simultaneous TCR clonotyping analysis reveals that thymic maturation of all three types is accompanied by clonal selection and expansion. Analyses of mice deficient of TBET, GATA3 or RORγt and additional in vivo experiments corroborate the predicted differentiation paths, while human innate T cells from liver samples display similar features. Collectively, our data indicate that innate T cells share effector differentiation processes in the thymus.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB−/− iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB−/− Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB−/− Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13. IMPORTANCE Salmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Until now, it is known that hypoxia increases the glycolytic enzyme expression at the transcriptional level. Here, we show evidence that hypoxia increases hepatic glucose output and HIF-1 and ATF-2-mediated transactivation of phosphoenolpyruvate carboxykinase (PEPCK), which plays a critical role as a rate-limiting enzyme in gluconeogenesis, gene in liver. HIF-1 directly bound to the specific PEPCK promoter region through its cognate binding element and found as an active complex with coactivator CBP. Additionally, ATF-2 was also involved to regulate hypoxia-dependent PEPCK transcription in the transcriptional complex with HIF-1 and CBP. Interestingly, retinoic acid (RA) signaling induced the recruitment of HIF-1 on the PEPCK promoter, resulting from the functional interaction of HIF-1 and ATF-2 with coactivator CBP. Taken together, these results suggest that hypoxia signaling leads the hepatic glucose production and release via the increased gene expression of gluconeogenic enzymes, possibly playing a role in providing glucose to other tissues, such as endothelial, brain and muscle cells.

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The p53 tumor suppressor protein, a critical modulator of cellular stress responses, is activated through diverse mechanisms that result in its stabilization and transcriptional activation. p53 activity is controlled by transcriptional, translational, and post-translational regulation. The major mechanisms of p53 regulation occur primarily through interactions with HDM2, an E3 ubiquitin ligase that leads to p53 nuclear export and degradation. Here, we demonstrate that hydrogen peroxide-induced oxidative stress elicits down-regulation of HDM2.