Little is known about the telomere chromatin dynamics of embryonic stem (ES) cell. Here, we demonstrate localization of histone H3.3 at interphase telomeres and enrichment of Ser31-phosphorylated H3.3 at metaphase telomeres in pluripotent mouse ES cells. Upon differentiation, telomeric H3.3S31P signal decreases, accompanied by increased association of heterochromatin repressive marks and decreased micrococcal nuclease sensitivity at the telomeres. H3.3 is recruited to the telomeres at late S/G2 phase, coinciding with telomere replication and processing. RNAi-depletion of H3.3 induces telomere-dysfunction phenotype, providing evidence for a role of H3.3 in the regulation of telomere chromatin integrity in ES cells. The distinctive changes in H3.3 distribution suggests the existence of a unique and functionally essential telomere chromatin in ES cells that undergoes dynamic differentiation-dependent remodeling during the process of differentiation.[Supplemental material is available online at www.genome.org.]Pluripotent embryonic stem (ES) cells possess an unlimited capacity for self-renewal and the potential to differentiate into multiple lineages. Upon cellular differentiation, they undergo dramatic morphological and molecular changes through selective silencing and activation of specific genes. Recent studies have pointed to epigenetic phenomena as having fundamental roles in the differentiation process (Martens et al. 2005;Meshorer et al. 2006). Examples are the differentiation-dependent increase in the silenced chromatin marks, including H3K9me3, H3K20me2, H4K20me3, and H3K27me3, and a decrease in the level of acetylated H3 and H4. The hyperdynamic binding of structural chromatin proteins is also a hallmark of ES cells, but not of cells that are already lineage committed.While epigenetic factors are essential for maintaining pluripotency, the capacity for continual telomere renewal is also critical. In vertebrates, telomeres consist of tandem arrays of TTAGGG repeats that are bound by a specialized multiprotein complex ''shelterin.'' Telomere length is maintained by telomerase, a reverse transcriptase that adds telomere repeats de novo after each cell division. High telomerase activity is a key factor that maintains telomere self-renewal and proliferative capacity in ES cells. Mammalian telomeres contain epigenetic markers such as H3K9me3 and H4K20me3 that are characteristic of silenced chromatin, and DNA hypermethylation at subtelomeric regions (Garcia-Cao et al. 2004;Gonzalo et al. 2006). Loss of these modifications results in defective telomere functions as shown by aberrantly increased telomere length and chromosomal instability, indicating that these repressive markers are essential for the formation of a compacted telomere chromatin and the regulation of telomere length.The nucleosome consists of an octamer of four core histones, H2A, H2B, H3, and H4, wrapped inside 146 bp of dsDNA. Adjacent nucleosomes are connected via linker DNA bound by the linker histone H1. Higher-order organization of this basic ...

Therapies targeting immune checkpoints have shown great clinical potential in a subset of patients with cancer but may be hampered by a failure to reverse the immunosuppressive tumor microenvironment (TME). As the most abundant immune cells in TME, tumor-associated macrophages (TAM) play nonredundant roles in restricting antitumor immunity. The leucine-rich repeat-containing G-protein-coupled receptor 4 (Lgr4, also known as Gpr48) has been associated with multiple physiologic and pathologic functions. Lgr4 and its ligands R-spondin 1-4 have been shown to promote the growth and metastasis of tumor cells. However, whether Lgr4 can promote tumor progression by regulating the function of immune cells in the tumor microenvironment remains largely unknown. Here, we demonstrate that Lgr4 promotes macrophage M2 polarization through Rspo/Lgr4/Erk/Stat3 signaling. Notably, urethane-induced lung carcinogenesis, Lewis lung carcinoma (LLC), and B16F10 melanoma tumors were all markedly reduced in Lgr4Lyz2 mice, characterized by fewer protumoral M2 TAMs and increased CD8 T lymphocyte infiltration in the TME. Furthermore, LLC tumor growth was greatly depressed when Rspo/Lgr4/Erk/Stat3 signaling was blocked with either the LGR4 extracellular domain or an anti-Rspo1 antibody. Importantly, blocking Rspo-Lgr4 signaling overcame LLC resistance to anti-PD-1 therapy and improved the efficacy of PD-1 immunotherapy against B16F10 melanoma, indicating vital roles of Rspo-Lgr4 in host antitumor immunity and a potential therapeutic target in cancer immunotherapy. This study identifies a novel receptor as a critical switch in TAM polarization whose inhibition sensitizes checkpoint therapy-resistant lung cancer to anti-PD-1 therapy. http://cancerres.aacrjournals.org/content/canres/78/17/4929/F1.large.jpg .

Supplemental Digital Content is available in the text.

Extracellular nucleotides are important messengers involved in series crucial physiological functions through the activation of P2 purinergic receptors. The detailed function and mechanism of the P2Y family in regulating immune response against invaded pathogens still remains unknown. In this study, the activation of purinoreceptor P2Y6 by UDP was found to play a crucial role in promoting host defense against invaded bacteria through monocytes/macrophages recruitment. The expression level of P2Y6 was much higher than other purinoreceptors in RAW264.7 cells, bone marrow macrophages, and peritoneal macrophages determined by real-time PCR. The supernatant of UDP (P2Y6-specific agonist)-treated RAW264.7 cells exhibited direct chemotaxis to monocytes/macrophages in vitro through Boyden Chambers assay. Meanwhile, the releasing of MCP-1 (MCP-1/CCL2) was enhanced obviously by UDP both in mRNA and protein level. Furthermore, the activation of P2Y6 receptor by UDP also promotes ERK phosphorylation and AP-1 activation in a concentration- and time-dependent manner in RAW264.7 cells. This UDP-induced activation could be inhibited by P2Y6 selectivity antagonist (MRS2578), MEK inhibitor (U0126), and MCP-1 blocking Ab, respectively. Moreover, i.p. injection with UDP resulted in a more efficacious clearance of invaded Escherichia coli and lower mortality in peritonitis mouse model. Together, our studies demonstrate that P2Y6 receptor could be a novel mediator in upregulating innate immune response against the invaded pathogens through recruiting monocytes/macrophages.

Mutation of the human genome ranges from single base-pair changes to whole-chromosome aneuploidy. Karyotyping, fluorescence in situ hybridization, and comparative genome hybridization are currently used to detect chromosome abnormalities of clinical significance. These methods, although powerful, suffer from limitations in speed, ease of use, and resolution, and they do not detect copy-neutral chromosomal aberrations--for example, uniparental disomy (UPD). We have developed a high-throughput approach for assessment of DNA copy-number changes, through use of high-density synthetic oligonucleotide arrays containing 116,204 single-nucleotide polymorphisms, spaced at an average distance of 23.6 kb across the genome. Using this approach, we analyzed samples that failed conventional karyotypic analysis, and we detected amplifications and deletions across a wide range of sizes (1.3-145.9 Mb), identified chromosomes containing anonymous chromatin, and used genotype data to determine the molecular origin of two cases of UPD. Furthermore, our data provided independent confirmation for a case that had been misinterpreted by karyotype analysis. The high resolution of our approach provides more-precise breakpoint mapping, which allows subtle phenotypic heterogeneity to be distinguished at a molecular level. The accurate genotype information provided on these arrays enables the identification of copy-neutral loss-of-heterozygosity events, and the minimal requirement of DNA (250 ng per array) allows rapid analysis of samples without the need for cell culture. This technology overcomes many limitations currently encountered in routine clinical diagnostic laboratories tasked with accurate and rapid diagnosis of chromosomal abnormalities.

Accumulating evidence shows that innate immune responses are associated with extracellular nucleotides, particularly ATP. In this article, we demonstrate extensive protection of ATP/P2X7 signaling in a host against viral infection. Interestingly, we observed a significant increase in ATP as a danger signal in vesicular stomatitis virus (VSV)-infected cell supernatant and VSV-infected mice in an exocytosis- and pannexin channel-dependent manner. Furthermore, extracellular ATP reduces the replication of VSV, Newcastle disease virus, murine leukemia virus, and HSV in vivo and in vitro through the P2X7 receptor. Meanwhile, ATP significantly increases IFN-β expression in a concentration- and time-dependent manner. Mechanistically, ATP facilitates IFN-β secretion through P38/JNK/ATF-2 signaling pathways, which are crucial in promoting antiviral immunity. Taken together, these results demonstrate the protective role of extracellular ATP and P2X7 in viral infection and suggest a potential therapeutic role for ATP/P2X7 in viral diseases.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by genomic instability and the early development of many types of cancer. Missense mutations have been identified in the BLM gene (encoding a RecQ helicase) in affected individuals, but the molecular mechanism and the structural basis of the effects of these mutations remain to be elucidated. We analysed five disease-causing missense mutations that are localized in the BLM helicase core region: Q672R, I841T, C878R, G891E and C901Y. The disease-causing mutants had low ATPase and helicase activities but their ATP binding abilities were normal, except for Q672, whose ATP binding activity was lower than that of the intact BLM helicase. Mutants C878R, mapping near motif IV, and G891E and C901Y, mapping in motif IV, displayed severe DNA-binding defects. We used molecular modelling to analyse these mutations. Our work provides insights into the molecular basis of BLM pathology, and reveals structural elements implicated in coupling DNA binding to ATP hydrolysis and DNA unwinding. Our findings will help to explain the mechanism underlying BLM catalysis and interpreting new BLM causing mutations identified in the future.

Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low-resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation-Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy-number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty-two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost-effective, high-throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage.