Abstract
The methylation status of DNA methylation regions (DMRs) of the imprinted gene IGF2/Igf2 is associated with neural tube defects (NTDs), which are caused by a failure of the neural tube to fold and close and are the second-most common birth defect; however, the characterization of the expression level of IGF2/Igf2 in neural tissue from human fetuses affected with NTDs remains elusive. More importantly, whether abnormal chromatin structure also influences IGF2/Igf2 expression in NTDs is unclear. Here, we investigated the transcriptional activity of IGF2/Igf2 in normal and NTD spinal cord tissues, the methylation status of different DMRs, and the chromatin structure of the promoter. Our data indicated that in NTD samples from both human fetuses and retinoic acid (RA)-treated mouse fetuses, the expression level of IGF2/Igf2 was upregulated 6.41-fold and 1.84-fold, respectively, compared to controls. H19 DMR1, but not IGF2 DMR0, was hypermethylated in human NTD samples. In NTD mice, h19 DMR1 was stable, whereas the chromatin structure around the promoter of Igf2 might be loosened, which was displayed by higher H3K4 acetylation and lower H3K27 trimethylation. Therefore, the data revealed that IGF2/Igf2 expression can be ectopically up-regulated by dual epigenetic factors in NTDs. In detail, the upregulation of IGF2/Igf2 is likely controlled by hypermethylation of H19 DMR1 in human NTDs, however, in acute external RA-induced NTD mice it is potentially determined by more open chromatin structure.
Highlights
Neural tube defects (NTDs; OMIM 182940) are early, severe and complicated congenital malformations, which result from the failure of the neural tube to close and include cranial NTDs, such as anencephaly; caudal NTDs, such as spina bifida or meningomyelocele; or neural tube closure failure over the entire body axis, called craniorachischisis
We assayed the level of Insulin-like growth factor 2 (IGF2) in the brain tissues of six NTDaffected cases, of whom the clinical phenotypes were two occipital encephalomeningoceles (20-gestation week, female; 22-gestation week, male); one anencephaly and holorachischisis (20-gestation week, female); one anencephaly and parietal encephalomeningocele (22-gestation week, female); one occipital encephalomeningocele and cervical, thoracic spina bifida (37-gestation week, female); and one parietal encephalomeningocele (24-gestation week, male)
The results revealed that the DNA methylation level of the CCCTC-binding factor (CTCF)-binding site in H19 DMR1 in the Watson strand was stable in cases and controls; a CpG site 33bp away was dramatically hypermethylated from 30.8661.19% to 49.6261.62% (P = 2.33e-07, Student’s t-test; Fig. 3A), suggesting that H19 DMR1 is hypermethylated in NTD cases
Summary
Neural tube defects (NTDs; OMIM 182940) are early, severe and complicated congenital malformations, which result from the failure of the neural tube to close and include cranial NTDs, such as anencephaly; caudal NTDs, such as spina bifida or meningomyelocele; or neural tube closure failure over the entire body axis, called craniorachischisis. In 2003, an epidemiological study reported a high prevalence of 138.7 per 10,000 births in the Shanxi province of Northern China [1]; the multifactorial influences that determine the pathogenesis of this disease remain elusive. Several clues have indicated that the inheritance in NTDs can be shown by a parental-specific feature, suggesting that abnormal genomic imprinting might be a pathogenic factor for NTDs [2,3]. The imprinted control region (ICR) of this cluster is the six CCCTC-binding factor (CTCF)-binding site in the DMRs upstream of H19 (namely, H19 DMR1: GenBank accession numbers AF125183; Chr11: 2021103-2021304, UCSC database, Feb2009 (GRCh37/ hg19)). With or without methylated CpG in the ICR influence
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