We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing Tetrahymena thermophila cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in Tetrahymena, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.In eukaryotic cells, DNA is closely associated with histone proteins in the form of chromatin, packaging DNA in a way that remains only partially understood. Although the structure of the nucleosome core is now known in considerable detail (1), how higher order chromatin structures are folded and unfolded to accommodate processes such as transcription, replication, and chromosome segregation remains unclear. During mitosis, DNA is compacted nearly 10,000-fold to ensure proper segregation of the genetic material to daughter cells. Faithful segregation of sister chromatids requires proper condensation of the chromatin during entry into mitosis and decondensation of the fiber during exit from mitosis and is essential for the viability of the cells.Specific posttranslational modifications of histones, particularly acetylation and phosphorylation, correlate well with dynamic aspects of the folding and unfolding of the chromatin fiber (2). For example, hyperphosphorylation of linker histone H1 is temporally coupled with entry into mitosis and has often been presumed to function in mitotic chromatin condensation (3, 4). However, recent experiments show that chromatin condensation can occur in vivo (5) or in vitro (6, 7) in the absence of H1. Furthermore, H1 hyperphosphorylation does not occur in premature chromatin condensation induced by fostriecin (8) or okadaic acid (9). Therefore, the exact function of H1 hyperphosphorylation in mitosis remains unclear.In contrast to H1 hyperphosphorylation, site-specific phosphorylation of core histone H3 at serine 10 seems to occur exclusively during mitosis in mammalian cells (10, 11). Moreover, fostriecin and okadaic acid, which initiate premature chromatin condensation in cell cultures, also induce H3 phosphorylation (8, 9). Similarly, vanadate-induced dephosphorylation of H3 correlates with chromatin decondensation and the rescue of a mitotic mutant that otherwise fails to initiate postmitotic chromatin decondensation (12). Recent studies, using an antibody selective for the Ser-10 phosphorylated H3 amino terminus, have documented a tight correlation b...

BACKGROUND & AIMS: Acute pancreatitis (AP) is characterized by severe inflammation and acinar cell death. Transmembrane protein 173 (TMEM173 or STING) is a DNA sensor adaptor protein on immune cells that recognizes cytosolic nucleic acids and transmits signals that activate production of interferons and the innate immune response. We investigated whether leukocyte STING signaling mediates inflammation in mice with AP. METHODS: We induced AP in C57BL/6J mice (control) and C57BL/6J-Tmem173gt/J mice (STING-knockout mice) by injection of cerulein or placement on choline-deficient DL-ethionine supplemented diet. In some mice, STING signaling was induced by administration of a pharmacologic agonist. AP was also induced in C57BL/6J mice with bone marrow transplants from control or STING-knockout mice and in mice with disruption of the cyclic GMP-AMP synthase (Cgas) gene. Pancreata were collected, analyzed by histology, and acini were isolated and analyzed by flow cytometry, quantitative polymerase chain reaction, immunoblots, and enzyme-linked immunosorbent assay. Bone-marrow–derived macrophages were collected from mice and tested for their ability to detect DNA from dying acinar cells in the presence and absence of deoxyribonuclease (DNaseI). RESULTS: STING signaling was activated in pancreata from mice with AP but not mice without AP. STING-knockout mice developed less severe AP (less edema, inflammation, and markers of pancreatic injury) than control mice, whereas mice given a STING agonist developed more severe AP than controls. In immune cells collected from pancreata, STING was expressed predominantly in macrophages. Levels of cGAS were increased in mice with vs without AP, and cGAS-knockout mice had decreased edema, inflammation, and other markers of pancreatic injury upon induction of AP than control mice. Wild-type mice given bone marrow transplants from STING-knockout mice had less pancreatic injury and lower serum levels of lipase and pancreatic trypsin activity following induction of AP than mice given wildtype bone marrow. DNA from dying acinar cells activated STING signaling in macrophages, which was inhibited by addition of DNaseI. CONCLUSIONS: In mice with AP, STING senses acinar cell death (by detecting DNA from dying acinar cells) and activates a signaling pathway that promotes inflammation. Macrophages express STING and activate pancreatic inflammation in AP.