In this study, we characterize the molecular and functional features of a novel protein called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly proliferating tissues and increased in a subset of ovarian carcinoma patients, which statistically correlated with poor prognosis and residual disease. These observations implied that SPOC1 might play a role in cellular proliferation and oncogenesis. Here we show that the endogenous SPOC1 protein is labile, primarily chromatin associated and its expression as well as localization are regulated throughout the cell cycle. SPOC1 is dynamically regulated during mitosis with increased expression levels and biphasic localization to mitotic chromosomes indicating a functional role of SPOC1 in mitotic processes. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in defects in mitotic chromosome condensation, alignment and aberrant sister chromatid segregation. Finally, we have been able to show, using micrococcal nuclease (MNase) chromatin-digestion assays that SPOC1 expression levels proportionally influence the degree of chromatin compaction. Collectively, our findings show that SPOC1 modulates chromatin structure and that tight regulation of its expression levels and subcellular localization during mitosis are crucial for proper chromosome condensation and cell division.

Mouse ´Macrophage ´CD163 ´Scavenger receptor ´DexamethasoneCD163 is a glycoprotein belonging to the scavenger receptor cysteine-rich superfamily (SRCR) expressed on cells of the monocyte/macrophage lineage. The protein is induced by the anti-inflammatory mediator, dexamethasone, and is proposed to be associated with the downregulatory phase of inflammatory reactions. However, the biological properties of the protein are poorly characterized. In the present report, the mouse CD163 cDNA (mCD163) was cloned from dexamethasone-treated peritoneal macrophages using a reverse transcription-PCR-based screening method. The predicted polypeptide sequence of the type I transmembrane glycoprotein consists of a 38-amino acid signal peptide, nine SRCR domains, one transmembrane domain, and a short cytoplasmic tail. Sequence variance analysis of all mouse and human CD163-SRCR D.J. Schaer ( ) ) ´P. Linnscheid ´H. Staege ´G. Schoedon

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

Deactivation of macrophage functions plays an important role in human infectious and inflammatory diseases. In this study, differential-display RT-PCR was used to analyze the gene expression of human mononuclear phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone (DEX), in the absence and presence of infection with Listeria monocytogenes (Listeria). Two novel differentially expressed mRNA species were discovered: FIND (IL-Four INDuced) was upregulated with IL-4 but down-regulated with DEX, and is predicted to code for an M(r) 53,000 transmembrane protein. LIND (Listeria INDuced) was induced by Listeria infection, and is predicted to code for an M(r) 39,000 nuclear or cytoplasmic protein containing three coiled-coil domains. In addition, we report several novel effects of deactivators and infection on the expression of known genes: (1) IL-4 caused pronounced upregulation of ABCG2, coding for an ATP-binding cassette transporter highly expressed in the placenta, which mediates multidrug resistance of cancer cells, but is otherwise of unknown function; (2) both DEX and IL-4 downregulated osteopontin, an important factor of host resistance against intracellular infections; (3) inhibition of the CC-chemokine I-309 mRNA expression by all three deactivators in the presence of Listeria infection, and (4) upregulation by Listeria infection of the interferon-stimulated gene ISG20 of unknown function, whose product localizes with nuclear dots/PML bodies.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.DOI: http://dx.doi.org/10.7554/eLife.10607.001

It is unclear whether the distinct nuclear morphologies of human A dark (Ad) and A pale (Ap) spermatogonia are manifestations of different stages of germ cell development or phases of the mitotic cycle, or whether they may reflect still unknown molecular differences. According to the classical description by Clermont, human dark type A spermatogonium (Ad) may contain one, sometimes two or three nuclear 'vacuolar spaces' representing chromatin rarefaction zones. These structures were readily discerned in paraffin sections of human testis tissue during immunohistochemical and immunofluorescence analyses and thus represented robust morphological markers for our study. While a majority of the marker proteins tested did not discriminate between spermatogonia with and without chromatin rarefaction zones, doublesex-and mab-3-related transcription factor (DMRT1), tyrosine kinase receptor c-Kit/CD117 (KIT) and proliferation-associated antigen Ki-67 (KI-67) appeared to be restricted to subtypes which lacked the rarefaction zones. Conversely, exosome component 10 (EXOSC10) was found to accumulate within the rarefaction zones, which points to a possible role of this nuclear domain in RNA processing.

HIPK2 shows overlapping localization with p53 in promyelocytic leukemia (PML) nuclear bodies (PMLNBs) and functionally interacts with p53 to increase gene expression. Here we demonstrate that HIPK2 and the PML-NB resident protein Sp100 synergize for the activation of p53-dependent gene expression. Sp100 and HIPK2 interact and partially colocalize in PML-NBs. The cooperation of HIPK2 and Sp100 for the induction of p21 Waf1 is completely dependent on the presence of p53 and the kinase function of HIPK2. Downregulation of Sp100 levels by expression of siRNA does not interfere with p53-mediated transcription, but obviates the enhancing effect of HIPK2. In summary, these experiments reveal a novel function for Sp100 as a coactivator for HIPK2-mediated p53 activation.

Human spermatogonia (Spg) and their fetal precursors express fibroblast growth factor receptor 3 (FGFR3). To further elucidate the role of FGFR3 in the control of Spg self-renewal, proliferation, and/or differentiation, and to narrow down the FGFR3-positive cell type(s) in the normal adult human testis, tissue sections and whole mount preparations of seminiferous tubules were analyzed combining immunofluorescence and confocal fluorescence microscopy. FGFR3 protein was chiefly observed in cellular membranes and cytoplasmic vesicles of a subpopulation of type A Spg, which comprised the chromatin rarefaction zone-containing type A(dark). Cytoplasmic expression of FGFR3 and nuclear expression of proliferation-associated antigen KI-67 were mutually exclusive. Similarly, FGFR3-positive Spg were negative for Doublesex and Mab-3 related transcription factor 1 (DMRT1). By contrast, undifferentiated embryonic cell transcription factor 1 (UTF1) and survival time-associated PHD finger in ovarian cancer 1 protein (SPOC1) were co-expressed in the nuclei of FGFR3-positive Spg. Whole mounted seminiferous tubules illustrated the clonogenic arrangement of the FGFR3/UTF1 double-positive Spg, which mainly occurred as pairs or quadruplets and, different from the KIT-positive Spg, showed no overlap with KI-67 labeled clusters. Taken together, in the adult human testis, FGFR3 expression is a feature of small clones of rarely dividing type A Spg which resemble "undifferentiated" Spg, including the spermatogonial stem cells.