Quantitative Allele-Specific Expression and DNA Methylation Analysis of H19, IGF2 and IGF2R in the Human Placenta across Gestation Reveals H19 Imprinting Plasticity

Abstract

Imprinted genes play important roles in placental differentiation, growth and function, with profound effects on fetal development. In humans, H19 and IGF2 are imprinted, but imprinting of IGF2R remains controversial. The H19 non-coding RNA is a negative regulator of placental growth and altered placental imprinting of H19-IGF2 has been associated with pregnancy complications such as preeclampsia, which have been attributed to abnormal first trimester placentation. This suggests that changes in imprinting during the first trimester may precede aberrant placental morphogenesis. To better understand imprinting in the human placenta during early gestation, we quantified allele-specific expression for H19, IGF2 and IGF2R in first trimester (6–12 weeks gestation) and term placentae (37–42 weeks gestation) using pyrosequencing. Expression of IGF2R was biallelic, with a mean expression ratio of 49∶51 (SD = 0.07), making transient imprinting unlikely. Expression from the repressed H19 alleles ranged from 1–25% and was higher (P<0.001) in first trimester (13.5±8.2%) compared to term (3.4±2.1%) placentae. Surprisingly, despite the known co-regulation of H19 and IGF2, little variation in expression of the repressed IGF2 alleles was observed (2.7±2.0%). To identify regulatory regions that may be responsible for variation in H19 allelic expression, we quantified DNA methylation in the H19-IGF2 imprinting control region and H19 transcription start site (TSS). Unexpectedly, we found positive correlations (P<0.01) between DNA methylation levels and expression of the repressed H19 allele at 5 CpG’s 2000 bp upstream of the H19 TSS. Additionally, DNA methylation was significantly higher (P<0.05) in first trimester compared with term placentae at 5 CpG’s 39–523 bp upstream of the TSS, but was not correlated with H19 repressed allele expression. Our data suggest that variation in H19 imprinting may contribute to early programming of placental phenotype and illustrate the need for quantitative and robust methodologies to further elucidate the role of imprinted genes in normal and pathological placental development.

Imprinted and X-linked non-coding RNAs as potential regulators of human placental function

“…36,37 Biallelic expression of H19 has been observed at higher rates during the first trimester of pregnancy compared with term, 36,38,39 with the early first trimester placenta showing patterns of imprinting plasticity. 30 Together, these studies suggest H19 plays an important regulatory role in early placental development.…”

“…H19 lies within a large imprinted domain (>1 MB), and is predominantly expressed from the maternal chromosome. H19 placental expression is largely monoallelic 30 and is one of the most highly expressed genes in the human placenta. 31 However, the functional roles of H19 are only now beginning to emerge.…”

Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge

“…The resulting methyl-group lies within the major groove of the DNA double-helix (Vargason et al, 2000) and there it can act as a docking site for binding proteins. It is hypothesised that the resulting complexes regulate access of transcription factors to the genome (Burgers et al, 2002;Chen and Riggs, 2011) hence DNA methylation may regulate gene expression during fertilisation, early embryogenesis, X chromosome inactivation (Senner et al, 2011), genomic imprinting (Yagi et al, 2008;Buckberry et al, 2012;Smith et al, 2012;Sun et al, 2012), and cell differentiation (Bar-Nur et al, 2011;Bocker et al, 2011;Tarfiei et al, 2011).…”

Circulating IGF1 and IGF2 and SNP genotypes in men and pregnant and non-pregnant women

“…P1 promoter transcripts of IGF2 are expressed from both parental alleles and IGF2 is expressed bi-allelically in liver from older infants and adults, where imprinting of IGF2 is not closely co-regulated with that of H19 (36, 37). We have recently reported discordant imprinting of IGF2 and H19 in first trimester human placenta at 6 weeks of gestation, where expression of IGF2 is mono-allelic but imprinting of H19 is highly variable (39). Individuals with Beckwith–Wiedemann syndrome and loss of imprinting at this locus, who therefore express maternal and paternal IGF2 alleles, often have pre- and postnatal overgrowth, suggesting increased IGF2 availability (reviewed by (40)).…”