Allele-specific DNA methylation reinforces PEAR1 enhancer activity.
Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (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 with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. 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 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with 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 reinforces PEAR1 enhancer activity through allele-specific DNA methylation.
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology.
Benedetta
0000-0002-3649-7931
B
Izzi
Department Development and Regeneration, Stem Cell Institute, and.
Mariaelena
M
Pistoni
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology.
Katrien
K
Cludts
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology.
Pinar
P
Akkor
Laboratory for Translational Genetics, Vesalius Research Center, Department of Oncology, Flemish Institute of Biotechnology, University of Leuven, Leuven, Belgium.
Diether
D
Lambrechts
Department Development and Regeneration, Stem Cell Institute, and.
Catherine
0000-0001-7564-4079
C
Verfaillie
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology.
Peter
P
Verhamme
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology.
Kathleen
0000-0002-4381-2442
K
Freson
Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology.
Marc F
0000-0002-6474-3933
MF
Hoylaerts
D000483:Alleles; D018952:Antigens, CD34 / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D044127:Epigenesis, Genetic; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism; D008957:Models, Genetic; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D018952:Antigens, CD34; D006657:Histones; C502809:PEAR1 protein, human; D011956:Receptors, Cell Surface