Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Hematology, ENDOTHELIAL AGGREGATION RECEPTOR-1, GENOME-WIDE, PLATELET-AGGREGATION, CPG-SNPS, TRANSCRIPTION FACTORS, GENE, ASSOCIATION, CHROMATIN, IDENTIFICATION, EXPRESSION, Alleles, Antigens, CD34, Cell Differentiation, Cell Proliferation, Chromatin Immunoprecipitation, CpG Islands, DNA Methylation, Enhancer Elements, Genetic, Epigenesis, Genetic, HEK293 Cells, Hematopoietic Stem Cells, Histones, Humans, Introns, Megakaryocytes, Models, Genetic, Polymorphism, Single Nucleotide, Protein Processing, Post-Translational, Receptors, Cell Surface, Transcription, Genetic, 1102 Cardiorespiratory Medicine and Haematology, 1103 Clinical Sciences, 1114 Paediatrics and Reproductive Medicine, Immunology, 3101 Biochemistry and cell biology, 3201 Cardiovascular medicine and haematology, 3213 Paediatrics

Abstract:

Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G-allele of rs12041331, an intronic CpG-SNP, is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A-allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells vs. control HEK-293 cells show a 3-fold higher affinity for the methylated G-allele, compared to non-methylated G or A alleles in a gel EMSA. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first UTR exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared to GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that capacitates PEAR1 enhancer activity through allele-specific DNA methylation.