Identification of a functional genetic variant driving racially dimorphic platelet gene expression of the thrombin receptor regulator, PCTP.

Platelet activation in response to stimulation of the Protease Activated Receptor 4 (PAR4) receptor differs by race. One factor that contributes to this difference is the expression level of Phosphatidylcholine Transfer Protein (PCTP), a regulator of platelet PAR4 function. We have conducted an expression Quantitative Trait Locus (eQTL) analysis that identifies single nucleotide polymorphisms (SNPs) linked to the expression level of platelet genes. This analysis revealed 26 SNPs associated with the expression level of PCTP at genome-wide significance (p \u3c 5×10(-8)). Using annotation from ENCODE and other public data we prioritised one of these SNPs, rs2912553, for functional testing. The allelic frequency of rs2912553 is racially-dimorphic, in concordance with the racially differential expression of PCTP. Reporter gene assays confirmed that the single nucleotide change caused by rs2912553 altered the transcriptional potency of the surrounding genomic locus. Electromobility shift assays, luciferase assays, and overexpression studies indicated a role for the megakaryocytic transcription factor GATA1. In summary, we have integrated multi-omic data to identify and functionalise an eQTL. This, along with the previously described relationship between PCTP and PAR4 function, allows us to characterise a genotype-phenotype relationship through the mechanism of gene expression

Bayesian modelling of high-throughput sequencing assays with malacoda.

NGS studies have uncovered an ever-growing catalog of human variation while leaving an enormous gap between observed variation and experimental characterization of variant function. High-throughput screens powered by NGS have greatly increased the rate of variant functionalization, but the development of comprehensive statistical methods to analyze screen data has lagged. In the massively parallel reporter assay (MPRA), short barcodes are counted by sequencing DNA libraries transfected into cells and the cell\u27s output RNA in order to simultaneously measure the shifts in transcription induced by thousands of genetic variants. These counts present many statistical challenges, including overdispersion, depth dependence, and uncertain DNA concentrations. So far, the statistical methods used have been rudimentary, employing transformations on count level data and disregarding experimental and technical structure while failing to quantify uncertainty in the statistical model. We have developed an extensive framework for the analysis of NGS functionalization screens available as an R package called malacoda (available from github.com/andrewGhazi/malacoda). Our software implements a probabilistic, fully Bayesian model of screen data. The model uses the negative binomial distribution with gamma priors to model sequencing counts while accounting for effects from input library preparation and sequencing depth. The method leverages the high-throughput nature of the assay to estimate the priors empirically. External annotations such as ENCODE data or DeepSea predictions can also be incorporated to obtain more informative priors-a transformative capability for data integration. The package also includes quality control and utility functions, including automated barcode counting and visualization methods. To validate our method, we analyzed several datasets using malacoda and alternative MPRA analysis methods. These data include experiments from the literature, simulated assays, and primary MPRA data. We also used luciferase assays to experimentally validate several hits from our primary data, as well as variants for which the various methods disagree and variants detectable only with the aid of external annotations

Interaction between HIF-1α (ODD) and hARD1 does not induce acetylation and destabilization of HIF-1α

AbstractHypoxia inducible factor-1α (HIF-1α) is a central component of the cellular responses to hypoxia. Hypoxic conditions result in stabilization of HIF-1α and formation of the transcriptionally active HIF-1 complex. It was suggested that mammalian ARD1 acetylates HIF-1α and thereby enhances HIF-1α ubiquitination and degradation. Furthermore, ARD1 was proposed to be downregulated in hypoxia thus facilitating the stabilization of HIF-1α. Here we demonstrate that the level of human ARD1 (hARD1) protein is not decreased in hypoxia. Moreover, hARD1 does not acetylate and destabilize HIF-1α. However, we find that hARD1 specifically binds HIF-1α, suggesting a putative, still unclear, connection between these proteins

PCTP contributes to human platelet activation by enhancing dense granule secretion

PCTP (phosphatidylcholine transfer protein) was discovered recently to regulate aggregation of human platelets stimulated with PAR4 activating peptide (PAR4AP). However, the role of PCTP following thrombin stimulation, the mechanisms by which PCTP contributes to platelet activation, and the role of PCTP with other receptors remained unknown. As mouse platelets do not express PCTP, we treated human platelets with various agonists in the presence of the specific PCTP inhibitor A1. We observed that PCTP inhibition significantly reduced dense granule secretion in response to thrombin, PAR1AP, PAR4AP, convulxin (GPVI agonist) and FcγRIIA crosslinking. In contrast, among these agonists, PCTP inhibition reduced aggregation only to low dose thrombin and PAR4AP. Unlike its effects on dense granule secretion, PCTP inhibition did not reduce alpha granule secretion in response to thrombin or PAR4AP. PCTP inhibition reduced both the increase in cytoplasmic Ca2+ as well as PKC activity downstream of thrombin. These data are consistent with PCTP contributing to secretion of dense granules, and to being particularly important to human PAR4 early signaling events. Future studies will address further these molecular mechanisms and consequences for hemostasis and thrombosis

Characterization of hARD2, a processed hARD1 gene duplicate, encoding a human protein N-α-acetyltransferase

BACKGROUND: Protein acetylation is increasingly recognized as an important mechanism regulating a variety of cellular functions. Several human protein acetyltransferases have been characterized, most of them catalyzing ε-acetylation of histones and transcription factors. We recently described the human protein acetyltransferase hARD1 (human Arrest Defective 1). hARD1 interacts with NATH (N-Acetyl Transferase Human) forming a complex expressing protein N-terminal α-acetylation activity. RESULTS: We here describe a human protein, hARD2, with 81 % sequence identity to hARD1. The gene encoding hARD2 most likely originates from a eutherian mammal specific retrotransposition event. hARD2 mRNA and protein are expressed in several human cell lines. Immunoprecipitation experiments show that hARD2 protein potentially interacts with NATH, suggesting that hARD2-NATH complexes may be responsible for protein N-α-acetylation in human cells. In NB4 cells undergoing retinoic acid mediated differentiation, the level of endogenous hARD1 and NATH protein decreases while the level of hARD2 protein is stable. CONCLUSION: A human protein N-α-acetyltransferase is herein described. ARD2 potentially complements the functions of ARD1, adding more flexibility and complexity to protein N-α-acetylation in human cells as compared to lower organisms which only have one ARD

Irisin functions to inhibit malignant growth of human pancreatic cancer cells via downregulation of the PI3K/AKT signaling pathway.

Introduction: Irisin is a newly identified cytokine that has gained increasing attention because of its potential therapeutic applications in metabolic diseases and human cancers. Recently, accumulating evidence indicates that irisin plays an important role in the development and metastasis of various tumors. The aim of this study was to evaluate the effects and underlying mechanisms of irisin on malignant growth of pancreatic cancer cells. Materials and methods: The anti-proliferative effect of irisin was examined using the CCK-8 assay. Irisin-induced apoptosis was determined by the annexin V-FITC/PI staining assay. The effects of irisin on cell migration and invasion were assessed using the scratch-induced wound healing assay and transwell invasion assay, respectively. The expression and phosphorylation of signaling proteins were detected by Western blot analysis. Results: Our results showed that irisin inhibited cell proliferation and induced apoptosis of pancreatic cancer cells in a dose-dependent manner. In addition, irisin decreased the migration and invasion of pancreatic cancer cells. Finally, Western blot analysis revealed that irisin downregulated the PI3K/AKT signaling pathway. Conclusion: Our findings suggest that irisin is a novel therapeutic agent for pancreatic cancer

Functionalization of CD36 Cardiovascular Disease and Expression Associated Variants by Interdisciplinary High Throughput Analysis.

CD36 is a platelet membrane glycoprotein whose engagement with oxidized low-density lipoprotein (oxLDL) results in platelet activation. The CD36 gene has been associated with platelet count, platelet volume, as well as lipid levels and CVD risk by genome-wide association studies. Platelet CD36 expression levels have been shown to be associated with both the platelet oxLDL response and an elevated risk of thrombo-embolism. Several genomic variants have been identified as associated with platelet CD36 levels, however none have been conclusively demonstrated to be causative. We screened 81 expression quantitative trait loci (eQTL) single nucleotide polymorphisms (SNPs) associated with platelet CD36 expression by a Massively Parallel Reporter Assay (MPRA) and analyzed the results with a novel Bayesian statistical method. Ten eQTLs located 13kb to 55kb upstream of the CD36 transcriptional start site of transcript ENST00000309881 and 49kb to 92kb upstream of transcript ENST00000447544, demonstrated significant transcription shifts between their minor and major allele in the MPRA assay. Of these, rs2366739 and rs1194196, separated by only 20bp, were confirmed by luciferase assay to alter transcriptional regulation. In addition, electromobility shift assays demonstrated differential DNA:protein complex formation between the two alleles of this locus. Furthermore, deletion of the genomic locus by CRISPR/Cas9 in K562 and Meg-01 cells results in upregulation of CD36 transcription. These data indicate that we have identified a variant that regulates expression of CD36, which in turn affects platelet function. To assess the clinical relevance of our findings we used the PhenoScanner tool, which aggregates large scale GWAS findings; the results reinforce the clinical relevance of our variants and the utility of the MPRA assay. The study demonstrates a generalizable paradigm for functional testing of genetic variants to inform mechanistic studies, support patient management and develop precision therapies

Racial Difference in Human Platelet PAR4 Reactivity Reflects Expression of PCTP and miR-376c

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. PAR4 thrombin receptor induced platelet aggregation and calcium mobilization were significantly greater in black subjects. Numerous differentially expressed (DE) RNAs were associated with both race and PAR4 reactivity, including phosphatidylcholine transfer protein (PCTP), and platelets from blacks expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked activation of platelets or megakaryocytic cell lines through PAR4 but not PAR1. MiR-376c levels were DE by race and PAR4 reactivity, and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. MiR-376c regulated expression of PC-TP in human megakaryocytes. A disproportionately high number of miRNAs DE by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus, and were lower in platelets from blacks. These results support PC-TP as a regulator of the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race, and emphasize a need to consider race effects when developing anti-thrombotic drugs

Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c.

Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. Platelet aggregation and calcium mobilization induced by the PAR4 thrombin receptor were significantly greater in black subjects. Numerous differentially expressed RNAs were associated with both race and PAR4 reactivity, including PCTP (encoding phosphatidylcholine transfer protein), and platelets from black subjects expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked PAR4- but not PAR1-mediated activation of platelets and megakaryocytic cell lines. miR-376c levels were differentially expressed by race and PAR4 reactivity and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. miR-376c regulated the expression of PC-TP in human megakaryocytes. A disproportionately high number of microRNAs that were differentially expressed by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus and were expressed at lower levels in platelets from black subjects. These results suggest that PC-TP contributes to the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race and emphasize a need to consider the effects of race when developing anti-thrombotic drugs