Additional file 1: of SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells

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SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells

Background: Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. AAA is a complex genetic disease but little is known about the role of epigenetics. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes. Results: We assessed global methylation in peripheral blood mononuclear cell DNA from 92 individuals with AAA and 93 controls using enzyme-linked immunosorbent assays, identifying hyper-methylation in those with large AAA and a positive linear association with AAA diameter (P < 0.0001,R2 = 0.3175).We then determined CpG methylation status of regulatory regions in genes located at AAA risk loci identified in genome-wide association studies, using bisulphite next-generation sequencing (NGS) in vascular smooth muscle cells (VSMCs) taken from aortic tissues of 44 individuals (24 AAAs and 20 controls). InIL6R, 2 CpGs were hyper-methylated (P = 0.0145); inERG, 13 CpGs were hyper-methylated (P = 0.0005); inSERPINB9, 6 CpGs were hypo-methylated (P = 0.0037) and 1 CpG was hyper-methylated (P = 0.0098); and inSMYD2, 4 CpGs were hypo-methylated (P = 0.0012).RT-qPCR was performed for each differentially methylated gene on mRNA from the same VSMCs and compared with methylation. This analysis revealed downregulation ofSMYD2andSERPINB9in AAA, and a direct linear relationship betweenSMYD2promoter methylation andSMYD2expression (P = 0.038). Furthermore, downregulation ofSMYD2at the site of aneurysm in the aortic wall was further corroborated in 6 of the same samples used for methylation and gene expression analysis with immunohistochemistry. Conclusions: This study is the first to assess DNA methylation in VSMCs from individuals with AAA using NGS, and provides further evidence there is an epigenetic basis to AAA. Our study shows that methylation status of theSMYD2promoter may be linked with decreasedSMYD2expression in disease pathobiology. In support of our work, downregulatedSMYD2has previously been associated with adverse cardiovascular physiology and inflammation, which are both hallmarks of AAA. The identification of such adverse epigenetic modifications could potentially contribute towards the development of epigenetic treatment strategies in the future

ST2 expression and release by the bronchial epithelium is downregulated in asthma

Background: The airway epithelium plays an important role in wound repair, host defense and is involved in the immunopathogenesis of asthma. Genome wide association studies have described associations between ST2/Interleukin (IL)-33 genes in asthma, but its role in bronchial epithelium is unclear. Methods: ST2 expression was examined in subjects with asthma and healthy controls in bronchial epithelium from biopsies (n = 27 versus n = 9) and brushings (n = 34 versus n = 20) by immunohistochemistry and RNA-Seq. In human primary bronchial epithelial cells ST2 mRNA and protein expression were assessed by qPCR, flow cytometry, Western blotting, and immunofluorescence. IL-33 function in epithelial cells was examined by intracellular calcium measurements, wound healing assays, and synthetic activation by gene array and ELISA. Results: Bronchial epithelial ST2 protein expression was significantly decreased in biopsies in subjects with asthma compared to healthy controls (P =.039). IL1RL1 gene expression in bronchial brushes was not different between health and disease. In vitro primary bronchial epithelial cells expressed ST2 and IL-33 stimulation led to an increase in intracellular calcium, altered gene expression, but had no effect upon wound repair. Epithelial cells released sST2 spontaneously, which was reduced following stimulation with TNFα or poly-IC. Stimulation by TNFα or poly-IC did not affect the total ST2 expression by epithelial cell whereas surface ST2 decreased in response to TNFα, but not poly-IC. Conclusion: In asthma, bronchial epithelium protein expression of ST2 is decreased. Our in vitro findings suggest that this decrease might be a consequence of the pro-inflammatory environment in asthma or in response to viral infection

Lamin A and C are not sumoylated by sumo1.

<p>(A–C) Western blots of C2C12 nuclear proteins: (A) Untransfected (UNT) or sumo1-YFP transfected, or triple transfected wild-type lamin A-CFP, DsRed2-lamin C and sumo1-YFP probed for lamin A/C. NEM was included with harvesting of selected samples to stabilize sumo conjugation. (B) Western blot A stripped and reprobed for the reversibly sumoylated protein, SP3. (C) UNT or triple transfected wild-type and mutant DsRed2-lamin C, wild-type sumo1-YFP and wild-type Ubc9-HA probed for lamin A/C. (D–E) Immunoprecipitation of endogenous and exogenous lamin A and C. (D) Nuclear extracts of sumo1-HA transfected C2C12 cells were immunoprecipitated using anti-lamin A/C antibody and western blotting was performed for lamin A/C. (E) Nuclear extracts of lamin A-CFP, lamin C-CFP, or empty CFP vector transfected COS7 cells were immunoprecipitated using anti-GFP tag antibody and western blotting was performed for the GFP tags. All protein was harvested in the presence of NEM. Emerin, a known lamin A/C binding partner, was included as a positive control for immunoprecipitation.</p

Genetic factors regulating lung vasculature and immune cell functions associate with resistance to pneumococcal infection

Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms (22 genes out of several hundred located within the QTLs). These candidate genes are known to regulate TGFb signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFbmediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease

Sumo1 localization is disrupted in soleus muscle sections from <i>Lmna</i>^H222P mice.

<p>Fluorescent microscopy images of soleus muscle cross sections from <i>Lmna</i>^+/+ and <i>Lmna</i>^H222P/H222P mice. Top row sections were stained for sumo1 (green). Bottom row sections were stained with only secondary antibody to show background signal of anti-mouse-568 nm antibody (green). All sections were counterstained for DAPI (blue). Each picture presented is representative of the most commonly observed phenotype. Scale bar represents 10 µm. White arrow highlights one cell with nuclear aggregation of sumo1.</p

Mutant lamin A/C expression increases levels of sumoylated proteins.

<p>Western blot analysis of sumo1 in nuclear protein harvested from C2C12 untransfected (UNT) cells or transfected with wild-type (WT) or mutant lamin C-CFP and sumo1-YFP harvested with NEM. Blots were re-probed for GapDH as a loading control.</p

Quantitative analysis of colocalization of sumo1 and mutant lamin A and C.

<p>Quantitative colocalization analysis of sumo1 and lamin A and C was performed using the ImageJ- 1.46r software. Thresholded Manders coefficients tM1 and tM2 and the percentage of pixels intensity above threshold colocalized (% Ch1 or Ch2 int > thresh) in each of the two channels (Ch1 for sumo (green fluorescence) and Ch2 for lamin A and C (red fluorescence) were calculated using the threshold algorithm of Costes et al (2004) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045918#pone.0045918-Costes1" target="_blank">[43]</a>.</p

Number of differentially regulated genes (DEGs) during infection with <i>S. pneumoniae</i>, D39.

<p>The included genes show fold change in expression ≥1.5 and change is significant at p≤0.05. The total number of BALB/cOlaHsd (resistant) and CBA/CaOlaHsd (susceptible) DEGs is indicated below the strain number and amount of commonly and uniquely regulated genes is indicated within the Venn diagram.</p

Phenotype-specific genetic polymorphisms within <i>S. pneumoniae</i> resistance QTLs.

<p>The number of phenotype-associated polymorphisms within the coding region of the QTLs and their consequences. The presented number of SNPs includes the SNPs identified in the EMMA mapping and in the analyses of the full-length sequences within the disease QTLs. No of SNPs– number of phenotype-associated SNPs identified within the gene; Chrom – chromosome, non-synon – SNP causing amino acid change, synon – SNP causing silent mutation, intron – SNP within intron, splicing – SNP within splicing site of the gene. EMMA – indicates whether the genome-wide significant SNPs (p-value< 5×10⁻⁸) were identified within the gene during the EMMA mapping: Y-yes, N-no.</p