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ABSTRACTObjectivesTo investigate the functional consequences of the single nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter strongly associated with ankylosing spondylitis (AS).MethodsThe effects of rs4648889 on transcription factor (TF) binding were tested by DNA pull-down and quantitative mass spectrometry. The results were validated by electrophoretic mobility gel shift assays (EMSA), Western blot (WB) analysis of the pulled-down eluates, and chromatin immuno-precipitation (ChIP)-qPCR.ResultsSeveral TFs showed differential allelic binding to a 50bp DNA probe spanning rs4648889. Binding was increased to the AS-risk A allele for IKZF3 (aiolos) in nuclear extracts from CD8+ T-cells (3.7-fold, p<0.03) and several components of the NUcleosome Remodeling Deacetylase (NuRD) complex, including Chromodomain-Helicase-DNA-binding protein 4 (3.6-fold, p<0.05) and Retinoblastoma-Binding Protein 4 (4.1-fold, p<0.02). In contrast, binding of interferon regulatory factor (IRF) 5 was increased to the AS-protective G allele. These results were confirmed by EMSA, WB and ChIP-qPCR.ConclusionsThe association of AS with rs4648889 most likely results from its influence on the binding of this enhancer-like region to TFs, including IRF5, IKZF3 and members of the NuRD complex. Further investigation of these factors and RUNX3-related pathways may reveal important new therapeutic possibilities in AS.
\n \n\n \n \nSeminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and particularly in insects, modifying female physiology and behaviour. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance due to dilution in the female derived sample or rapid processing in females. Here we present a new and complementary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster male reproductive tissue, where Sfps are unprocessed, and highly abundant, and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analysed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data was consistent with 124 previously reported Sfps. We found 8 high confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 31 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.
\n \n\n \n \nBACKGROUND: Oropharyngeal carriage of Neisseria meningitidis is frequent during adolescence, representing a major source of invasive meningococcal disease. This study examined the impact of a serogroup B vaccination (Bexsero, GSK 4CMenB) programme on adolescent N.\u00a0meningitidis carriage using genomic data. METHODS: A total 34,489 oropharyngeal samples were collected as part of a state-wide cluster randomised-controlled trial in South Australia during 2017 and 2018 (NCT03089086). Samples were screened for the presence of N.\u00a0meningitidis DNA by porA PCR prior to culture. Whole genome sequencing was performed on all 1772\u00a0N.\u00a0meningitidis culture isolates and their genomes were analysed. FINDINGS: Unencapsulated meningococci were predominant at baseline (36.3% of isolates), followed by MenB (31.0%), and MenY (20.5%). Most MenB were ST-6058 from hyperinvasive cc41/44, or ST-32 and ST-2870 from cc32. For MenY, ST-23 and ST-1655 from cc23 were prevalent. Meningococcal carriage was mostly unchanged due to the vaccination programme; however, a significant reduction in ST-53 capsule-null meningococci prevalence was observed in 2018 compared to 2017 (OR\u00a0=\u00a00.52; 95% CI: 0.30-0.87, p\u00a0=\u00a00.0106). This effect was larger in the vaccinated compared to the control group (OR\u00a0=\u00a00.37; 95% CI: 0.12-0.98, p\u00a0=\u00a00.0368). INTERPRETATION: While deployment of the 4CMenB vaccination did not alter the carriage of hyperinvasive MenB in the vaccinated population, it altered the carriage of other N.\u00a0meningitidis sequence types following the vaccination program. Our findings suggest 4CMenB vaccination is unlikely to reduce transmission of hyperinvasive N.\u00a0meningitidis strains and therefore ongoing targeted vaccination is likely a more effective public health intervention. FUNDING: This work was funded by GlaxoSmithKline Biologicals SA.
\n \n\n \n \nHepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.
\n \n\n \n \nProgrammed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases.
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