Sonic Hedgehog Signaling Affected by Promoter Hypermethylation Induces Aberrant Gli2 Expression in Spina Bifida

Abstract

GLI2 is a key mediator of the sonic hedgehog (Shh) signaling pathway and plays an important role in neural tube development during vertebrate embryogenesis; however, the role of gli2 in human folate-related neural tube defects remains unclear. In this study, we compared methylation status and polymorphisms of gli2 between spina bifida patients and a control group to explore the underlying mechanisms related to folate deficiency in spina bifida. No single nucleotide polymorphism was found to be significantly different between the two groups, although gli2 methylation levels were significantly increased in spina bifida samples, accompanied by aberrant GLI2 expression. Moreover, a prominent negative correlation was found between the folate level in brain tissue and the gli2 methylation status (r = -0.41, P = 0.014), and gli2 hypermethylation increased the risk of spina bifida with an odds ratio of 12.45 (95 % confidence interval: 2.71-57.22, P = 0.001). In addition, we established a cell model to illustrate the effect of gli2 expression and the accessibility of chromatin affected by methylation. High gli2 and gli1 mRNA expression was detected in 5-Aza-treated cells, while gli2 hypermethylation resulted in chromatin inaccessibility and a reduced association with nuclear proteins containing transcriptional factors. More meaningful to the pathway, the effect gene of the Shh pathway, gli1, was found to have a reduced level of expression along with a decreased expression of gli2 in our cell model. Aberrant high methylation resulted in the low expression of gli2 in spina bifida, which was affected by the change in chromatin status and the capacity of transcription factor binding.

Methylome analysis for spina bifida shows SOX18 hypomethylation as a risk factor with evidence for a complex (epi)genetic interplay to affect neural tube development

“…Interestingly, a recent study of Petrovic et al suggests that Hedgehog signalling up-regulates SOX18 expression, which promotes migration [ 24 ]. Hedgehog signalling is known to play an important role in neural tube development [ 24 , 25 ]. Evidence for a role for SOX18 in neural tube formation is also supported by our zebrafish studies that showed strongly malformed tails with abnormal neural tube development in embryos with high levels of sox18 after injection of its mRNA.…”

Folate deficiency disturbs hsa‐let‐7 g level through methylation regulation in neural tube defects

“…It has suggested that insufficient folate intake is a cause of NTDs [33]. Impaired DNA methylation is believed to participate in this process [16,34]. Numerous investigations have indeed indicated that the aberrant methylation of miRNA around the promoter regions contributes to dysregulation of its expression [35].…”

“…Epidemiological studies have indicated that lower concentrations of folate, which in turn disturb one-carbon metabolism, during pregnancy are related to an increased risk of NTDs [13][14][15]. Previous studies by our group showed that disturbed epigenetic modifications in the context of folic acid deficiency are involved in the pathogenesis of NTDs, including DNA methylation, histone methylation and micro-RNA (miRNA) regulation [16][17][18]. Intracellular folic acid is a key factor acting as a source of adequate methyl groups for the methylation of DNA and proteins, which explains why folic acid deficiency leads to changes in cytosine methylation and histone methylation that could induce NTDs.…”

Spina Bifida: Morphological Features, Molecular Regulations and Signal Pathways

“…This indicates that in the brain tissue embryos with spina, the SHH pathway is suppressed. Data of Lu et al come to a conclusion of epigenetic silencing of GLI2 may have an important role in the development of spina bifida during embryogenesis [59]. Cladia et al suggested that TNIP1 is a new potential associated gene to spina bifida.…”