Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

MicroRNAs (miRNAs) are 19–22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer[superscript −/−] mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.Leukemia & Lymphoma Society of AmericaUnited States. Public Health Service (Grant R01-GM34277)United States. Public Health Service (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P01-CA42063)National Cancer Institute (U.S.) Cancer Center Support (Grant P30-CA14051

Twenty years of stereotype threat research: A review of psychological mediators

This systematic literature review appraises critically the mediating variables of stereotype threat. A bibliographic search was conducted across electronic databases between 1995 and 2015. The search identified 45 experiments from 38 articles and 17 unique proposed mediators that were categorized into affective/subjective (n = 6), cognitive (n = 7) and motivational mechanisms (n = 4). Empirical support was accrued for mediators such as anxiety, negative thinking, and mind-wandering, which are suggested to co-opt working memory resources under stereotype threat. Other research points to the assertion that stereotype threatened individuals may be motivated to disconfirm negative stereotypes, which can have a paradoxical effect of hampering performance. However, stereotype threat appears to affect diverse social groups in different ways, with no one mediator providing unequivocal empirical support. Underpinned by the multi-threat framework, the discussion postulates that different forms of stereotype threat may be mediated by distinct mechanisms

Effect of wood smoke exposure on vascular function and thrombus formation in healthy fire fighters

Background: Myocardial infarction is the leading cause of death in fire fighters and has been linked with exposure to air pollution and fire suppression duties. We therefore investigated the effects of wood smoke exposure on vascular vasomotor and fibrinolytic function, and thrombus formation in healthy fire fighters. Methods: In a double-blind randomized cross-over study, 16 healthy male fire fighters were exposed to wood smoke (~1 mg/m3 particulate matter concentration) or filtered air for one hour during intermittent exercise. Arterial pressure and stiffness were measured before and immediately after exposure, and forearm blood flow was measured during intra-brachial infusion of endothelium-dependent and -independent vasodilators 4–6 hours after exposure. Thrombus formation was assessed using the ex vivo Badimon chamber at 2 hours, and platelet activation was measured using flow cytometry for up to 24 hours after the exposure. Results: Compared to filtered air, exposure to wood smoke increased blood carboxyhaemoglobin concentrations (1.3% versus 0.8%; P &lt; 0.001), but had no effect on arterial pressure, augmentation index or pulse wave velocity (P &gt; 0.05 for all). Whilst there was a dose-dependent increase in forearm blood flow with each vasodilator (P &lt; 0.01 for all), there were no differences in blood flow responses to acetylcholine, sodium nitroprusside or verapamil between exposures (P &gt; 0.05 for all). Following exposure to wood smoke, vasodilatation to bradykinin increased (P = 0.003), but there was no effect on bradykinin-induced tissue-plasminogen activator release, thrombus area or markers of platelet activation (P &gt; 0.05 for all). Conclusions: Wood smoke exposure does not impair vascular vasomotor or fibrinolytic function, or increase thrombus formation in fire fighters. Acute cardiovascular events following fire suppression may be precipitated by exposure to other air pollutants or through other mechanisms, such as strenuous physical exertion and dehydration.Originally included in thesis in manuscript form.</p

Modulation of Ago-miRNA regulatory networks by cis-sequence elements and target competition

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.regulators of gene expression in a wide range of organisms and biological processes. Each miRNA guides Argonaute (Ago) protein complexes to target and repress hundreds of genes in a sequence-dependent manner. To identify all targets of miRNA regulation, we performed UV crosslinking and immunoprecipitation (CLIP) of Ago complexes in mouse embryonic (ESC) and mesenchymal (MSC) stem cell lines. We also captured the genome-wide miRNA-independent binding footprint of Ago by performing CLIP in cells that lack Dicer, an enzyme required for mature miRNA biogenesis. We surprisingly found that Ago bound a similar set of genes in the absence of Dicer, and this overlap in target genes was due partially to residual, unprocessed miRNAs in the Dicer KO cells. Other potential sites of miRNA-independent Ago interactions, such as histone transcripts and poly-A cleavage and polyadenylation sites, were also identified. One Ago CLIP dataset revealed the enrichment for a G-rich sequence motif at Ago target sites. We later demonstrated that the G-motif is not directly bound to Ago but rather is enriched near miRNA-guided Ago binding sites. Its presence near miRNA target sites is associated with stronger repression of Ago-miRNA targets. Fortuitously, the original Ago CLIP dataset that identified the G-motif was later shown to likely contain target sites of another co-immunoprecipitating RNA binding protein (RBP). Using mass spectroscopy of Ago antibody immunoprecipitations from Ago KO cells, we identified a list of interacting RBPs that could potentially augment Ago-miRNA activity through the G-motif. To investigate target competition in miRNA networks, we related our CLIP analysis of genome-wide, quantitative Ago binding to measurements of absolute miRNA and target RNA concentrations. We found that all miRNAs other than the miR-290 family in ESCs and let-7 family in MSCs were expressed at concentrations below their total target pool. However, 8-12 miRNA families were expressed at near or greater than equimolar ratios with their pool of high affinity targets, and this affinity-partitioned stoichiometry led to significant Ago accumulation and repression of high affinity target sites despite little consequential binding at low affinity sites. Single-cell reporter assays demonstrated that high expressed miRNAs are not susceptible to physiological inductions of competing target transcripts but targets of lower expressed miRNAs are derepressed in a weakly threshold-like manner upon increased target pool levels. In summary, we identify a network of confidently bound targets of miRNA regulation in ESCs and MSCs, reveal the extent of miRNA-independent binding in these two cell types, provide a list of potential miRNA enhancer RBPs, and create a quantitative context for evaluating target competition in miRNA networks.by Andrew D. Bosson.Ph. D

Multivariate Genome-wide Association Study of Rapid Automatised Naming and Rapid Alternating Stimulus in Hispanic American and African–American Youth

Background: Rapid automatised naming (RAN) and rapid alternating stimulus (RAS) are reliable predictors of reading disability. The underlying biology of reading disability is poorly understood. However, the high correlation among RAN, RAS and reading could be attributable to shared genetic factors that contribute to common biological mechanisms. Objective: To identify shared genetic factors that contribute to RAN and RAS performance using a multivariate approach. Methods: We conducted a multivariate genome-wide association analysis of RAN Objects, RAN Letters and RAS Letters/Numbers in a sample of 1331 Hispanic American and African-American youth. Follow-up neuroimaging genetic analysis of cortical regions associated with reading ability in an independent sample and epigenetic examination of extant data predicting tissue-specific functionality in the brain were also conducted. Results: Genome-wide significant effects were observed at rs1555839 (p=4.03×10-8) and replicated in an independent sample of 318 children of European ancestry. Epigenetic analysis and chromatin state models of the implicated 70 kb region of 10q23.31 support active transcription of the gene RNLS in the brain, which encodes a catecholamine metabolising protein. Chromatin contact maps of adult hippocampal tissue indicate a potential enhancer-promoter interaction regulating RNLS expression. Neuroimaging genetic analysis in an independent, multiethnic sample (n=690) showed that rs1555839 is associated with structural variation in the right inferior parietal lobule. Conclusion: This study provides support for a novel trait locus at chromosome 10q23.31 and proposes a potential gene-brain-behaviour relationship for targeted future functional analysis to understand underlying biological mechanisms for reading disability

Country‐level and individual‐level predictors of men's support for gender equality in 42 countries

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