DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Regulation of transcription occurs in a cell type specific manner orchestrated by epigenetic mechanisms including DNA methylation. Methylation changes may also play a key role in lineage specification during stem cell differentiation. To further our understanding of epigenetic regulation in chondrocytes we characterised the DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) by Infinium 450 K methylation array. Significant DNA hypomethylation was identified during chondrogenic differentiation including changes at many key cartilage gene loci. Integration with chondrogenesis gene expression data revealed an enrichment of significant CpGs in upregulated genes, while characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with methylation profiles of other tissues, including healthy and diseased adult cartilage, identified chondrocyte-specific regions of hypomethylation and the overlap with differentially methylated CpGs in osteoarthritis. Taken together we have associated DNA methylation levels with the chondrocyte phenotype. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis

Defective Leukocyte Adhesion and Chemotaxis Contributes to Combined Immunodeficiency in Humans with Autosomal Recessive MST1 Deficiency.

PURPOSE: To investigate the clinical and functional aspects of MST1 (STK4) deficiency in a profoundly CD4-lymphopenic kindred with a novel homozygous nonsense mutation in STK4. Although recent studies have described the cellular effects of murine Mst1 deficiency, the phenotype of MST1-deficient human lymphocytes has yet to be fully explored. Patient lymphocytes were therefore investigated in the context of current knowledge of murine Mst1 deficiency. METHODS: Genetic etiology was identified by whole exome sequencing of genomic DNA from two siblings, combined with linkage analysis in the wider family. MST1 protein expression was assessed by immunoblotting. The ability of patient lymphocytes to adhere to ICAM-1 under flow conditions was measured, and transwell assays were used to assess chemotaxis. Chemokine receptor expression was examined by flow cytometry and receptor signalling by immunoblotting. RESULTS: A homozygous nonsense mutation in STK4 (c.442C > T, p.Arg148Stop) was found in the patients, leading to a lack of MST1 protein expression. Patient leukocytes exhibited deficient chemotaxis after stimulation with CXCL11, despite preserved expression of CXCR3. Patient lymphocytes were also unable to bind effectively to immobilised ICAM-1 under flow conditions, in keeping with a failure to develop high affinity binding. CONCLUSION: The observed abnormalities of adhesion and migration imply a profound trafficking defect among human MST1-deficient lymphocytes. By analogy with murine Mst1 deficiency and other defects of leucocyte trafficking, this is likely to contribute to immunodeficiency by impairing key aspects of T-cell development and function such as positive selection in the thymus, thymic egress and immune synapse formation in the periphery.This is thepublished version. It first appeared at http://link.springer.com/article/10.1007%2Fs10875-016-0232-2

Allelic expression analysis of the osteoarthritis susceptibility locus that maps to MICAL3

<p>Abstract</p> <p>Background</p> <p>A genome-wide association scan with subsequent replication study that involved over 67,000 individuals of European ancestry has produced evidence of association of single nucleotide polymorphism rs2277831 to primary osteoarthritis (OA) with a P-value of 2.9 × 10^-5. rs2277831, an A/G transition, is located in an intron of <it>MICAL3</it>. This gene is located on chromosome 22q11.21 and the association signal encompasses two additional genes, <it>BCL2L13 </it>and <it>BID</it>. It is becoming increasingly apparent that many common complex traits are mediated by <it>cis</it>-acting regulatory polymorphisms that influence, in a tissue-specific manner, gene expression or transcript stability.</p> <p>Methods</p> <p>We used total and allelic expression analysis to assess whether the OA association to rs2277831 is mediated by an influence on MICAL3, BCL2L13 or BID expression. Using RNA extracted from joint tissues of 60 patients who had undergone elective joint replacement surgery, we assessed whether rs2277831 correlated with allelic expression of either of the three genes by: 1) measuring the expression of each gene by quantitative PCR and then stratifying the data by genotype at rs2277831 and 2) accurately discriminating and quantifying the mRNA synthesised from the alleles of OA patients using allelic-quantitative PCR.</p> <p>Results</p> <p>We found no evidence for a correlation between gene expression and genotype at rs2277831, with P-values of 0.09 for <it>BCL2L13</it>, 0.07 for <it>BID </it>and 0.33 for <it>MICAL3</it>. In the allelic expression analysis we observed several examples of significant (p < 0.05) allelic imbalances, with an allelic expression ratio of 2.82 observed in <it>BCL2L13 </it>(P = 0.004), 2.09 at <it>BID </it>(P = 0.001) and the most extreme case being at <it>MICAL3</it>, with an allelic expression ratio of 5.47 (P = 0.001). However, there was no correlation observed between the pattern of allelic expression and the genotype at rs2277831.</p> <p>Conclusions</p> <p>In the tissues that we have studied, our data do not support our hypothesis that the association between rs2277831 and OA is due to the effect this SNP has on <it>MICAL3, BCL2L13 </it>or <it>BID </it>gene expression. Instead, our data point towards other functional effects accounting for the OA associated signal.</p

microRNA-seq of cartilage reveals an over-abundance of miR-140-3p which contains functional isomiRs

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5' and 3' end, with >99% having one of two seed sequences (5' bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3'UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140- null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140- 3p.1 is more functional than original consensus miR-140-3p seed containing isomiR

Analysis of genetics and DNA methylation in osteoarthritis: What have we learnt about the disease?

AbstractOsteoarthritis (OA) is a chronic musculoskeletal disease characterised by the destruction of articular cartilage, synovial inflammation and bone remodelling. Disease aetiology is complex and highly heritable, with genetic variation estimated to contribute to 50% of OA occurrence. Epigenetic alterations, including DNA methylation changes, have also been implicated in OA pathophysiology. This review examines what genetic and DNA methylation studies have taught us about the genes and pathways involved in OA pathology. The influence of DNA methylation on the molecular mechanisms underlying OA genetic risk and the consequence of this interaction on disease susceptibility and penetrance are also discussed

Increased sex chromosome expression and epigenetic abnormalities in spermatids from male mice with Y chromosome deletions

During male meiosis, the X and Y chromosomes are transcriptionally silenced, a process termed meiotic sex chromosome inactivation (MSCI). Recent studies have shown that the sex chromosomes remain substantially transcriptionally repressed after meiosis in round spermatids, but the mechanisms involved in this later repression are poorly understood. Mice with deletions of the Y chromosome long arm (MSYq–) have increased spermatid expression of multicopy X and Y genes, and so represent a model for studying post-meiotic sex chromosome repression. Here, we show that the increase in sex chromosome transcription in spermatids from MSYq– mice affects not only multicopy but also single-copy XY genes, as well as an X-linked reporter gene. This increase in transcription is accompanied by specific changes in the sex chromosome histone code, including almost complete loss of H4K8Ac and reduction of H3K9me3 and CBX1. Together, these data show that an MSYq gene regulates sex chromosome gene expression as well as chromatin remodelling in spermatids