Human miRNAs: an antiviral defense mechanism

Background miRNAs are short 21-24 nt RNAs that mediate post transcriptional repression of target genes. Various reports have shown that miRNAs are capable of repressing the gene expression levels of different viruses, leading to the suggestion that miRNAs are key mediators of host-virus interaction [1]. HIV-1 is a retrovirus known to cause AIDS, one of the major diseases in humans. The nef gene of the HIV-1 has been shown to be important for virus repression of CD4+ cells and virus progression. It has also been shown earlier that patients infected with nef deleted HIV-1 do not progress from infected to diseased state for longer periods of time, resulting in the Long Term Non-Progressor phenotype [2]. Materials and methods We computationally predicted five endogenously expressed human miRNAs to target the nef gene of HIV-1 retrovirus. On applying other stringency parameters we could focus on two of the five miRNAs viz. hsa-mir-29a and hsa-mir-29b as they were predicted to target the nef gene, at sites highly conserved amongst other clades of HIV-1 [3]. We then created reporter carrying the nef gene inserted downstream of a luciferase reporter. miRNA expression vectors were also made which would express the pri-miRNA when processed and thereby lead to high levels of the miRNA inside the cells. We then identified various cell lines for validating nef as a target for hsa-mir-29a and hsa-mir-29b. Results and discussion Gene reporter assays and ectopic over-expression of miRNAs conclusively showed that human cellular miRNAs hsa-mir-29a and hsa-mir-29b could bring down the nef protein levels and also affect viral replication [4]. These results would provide a better understanding of the mechanisms that could regulate the viral gene expression and human cellular antiviral defense mechanisms whereby miRNAs could serve as potential therapeutics to treat various viral diseases

Human cellular microRNA hsa-miR-29a interferes with viral nef protein expression and HIV-1 replication

protein expression and HIV-1 replicatio

Genetic drivers of heterogeneity in type 2 diabetes pathophysiology.

Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P < 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care

Genetic drivers of heterogeneity in type 2 diabetes pathophysiology

Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P &lt; 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care.</p

A hexanucleotide repeat upstream of eotaxin gene promoter is associated with asthma, serum total IgE and plasma eotaxin levels

Background: Eotaxin (CCL11) is a small protein produced in the lungs of patients with asthma, and is a potent chemoattractant for eosinophils. Aim: To elucidate the role of eotaxin in asthma by an association study of functional and novel eotaxin polymorphisms in case-control and family-based study designs. Methods:Eotaxin +67G/A, -384A/G and -426C/T single-nucleotide polymorphisms and a hexanucleotide (GAAGGA)n repeat 10.9 kb upstream of the gene were genotyped in a cohort of age, sex and ethnically matched patients with asthma (n = 235) and healthy controls (n = 239), and also in a study population of 230 families with asthma recruited from north/northwest India. Total serum IgE (TsIgE) and plasma eotaxin levels were measured using ELISA. Results: +67G/A polymorphism was found to be significantly associated with asthma in case-control (p = 0.009) and family-based studies (p = 0.006). Its functional role, as it was correlated with plasma eotaxin levels (p = 0.006), was also demonstrated. Further, -384C/T single-nucleotide polymorphism was found to be significantly associated with log10 TsIgE (p = 0.016 in case-control and p = 0.018 in families) and eotaxin levels (p = 0.007). Most interestingly, for the first time, a highly significant association of the newly studied (GAAGGA)n hexanucleotide repeat with asthma (p = 3×10-6), log10TsIgE (p = 0.006) and eotaxin levels (p = 0.004) was observed. G_A_C_8 was also identified as an important risk haplotype associated with high TsIgE and plasma eotaxin levels. Conclusions: This study provides further evidence that eotaxin polymorphisms are associated with the development of asthma by regulating eotaxin levels and reinforces towards the scanning of other chemokine genes present at 17q21 locus for their association with asthma and related phenotypes

Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome

<div><p>Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The <i>cis</i>-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human <i>PIK3C2B</i> gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 <i>in vitro</i> and recruits PRC2 complex <i>in vivo</i>. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either <i>YY1</i> or <i>SUZ12</i>. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic <i>Drosophila</i>, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.</p></div

Directinteraction of YY1 with PRE-PIK3C2B.

<p>Labeled 25 merOligo corresponding to repeating unit of PRE-PIK3C2B (5′AGTGAA<u>GCCATCAT</u>GTGAGAATACC3′) was used as the probe in gel mobility shift assay with purified His-YY1. Cold probe 1 is competing unlabeled cognate (Oligo25 mer), 2- (OligoΔYY1) repeating unit without YY1 recognition sequence (5′AGTGAAGTGAGAATACC3′). Each competing oligo was used at a concentration 100× higher than the labeled probe. The super shift with anti-YY1 is shown in the last lane (arrow).</p

Genetic interactions of PRE-PIK3C2B with PRC genes in transgenic flies.

<p>Effect of PRE-PIK3C2B in the background of different PcG mutations on rescue of eye pigmentation is shown: cases of rescue of eye colour are marked with an arrow; trans-heterozygotes show better recovery of eye pigmentation. We have used same conditions of image processing in all the cases.</p

YY1 and SUZ12 dependent transcriptional repression by PRE-PIK3C2B.

<p>A-Expression of endogenous <i>PIK3C2B</i> in HEK cells analysed by qPCR. The expression is increased in presence of siRNAYY1 and siRNASUZ12. Expression in untransfected HEK cells is taken as control. B- Effect on reporter gene expression. The nomenclature for vectors is same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067217#pone-0067217-g001" target="_blank">Figure 1</a>. Ratio of GFP expression in pcDNA-GFP, pc(1 kb)UP or pc(1 kb)DN with and without co-transfection with siRNA YY1 and siRNA SUZ12 is shown on the Y-axis. Error bars, S.E.M of assay in triplicate is shown * p-value<0.05, ** p-value<0.005, n = 3.</p

Interaction of PcG proteins with PRE-PIK3C2B in HEK cells.

<p>The experiments were carried out in HEK293 ells. A-Diagrammatic representation of the endogenous PRE-PIK3C2B region with primers (arrows) used for PCR and the 25 mer repeating unit (filled box). B and C- ChIP with antibodies indicated. Input is 20% of sonicated chromatin. D-Quantitative PCR for PRE-PIK3C2B following ChIP in control cells and cells transfected with siRNAYY1 and siRNA SUZ12. E-Interaction of EZH2 with PRE-PIK3C2B estimated by qPCR in absence and presence of YY1siRNA: F-Localization of EED and the H3K4me3 at PRE-PIK3C2B in absence and presence of siRNAYY1. Error bars: S.E.M of assay in triplicate, * p<0.05, **p-value<0.005,n = 3. The qPCR profile for positive and negative controls are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067217#pone.0067217.s003" target="_blank">Figs. S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067217#pone.0067217.s004" target="_blank">S4</a>.</p