Loss of imprinting of the Igf2-H19 ICR1 enhances placental endocrine capacity via sex-specific alterations in signalling pathways in the mouse

ABSTRACT Imprinting control region (ICR1) controls the expression of the Igf2 and H19 genes in a parent-of-origin specific manner. Appropriate expression of the Igf2-H19 locus is fundamental for normal fetal development, yet the importance of ICR1 in the placental production of hormones that promote maternal nutrient allocation to the fetus is unknown. To address this, we used a novel mouse model to selectively delete ICR1 in the endocrine junctional zone (Jz) of the mouse placenta (Jz-ΔICR1). The Jz-ΔICR1 mice exhibit increased Igf2 and decreased H19 expression specifically in the Jz. This was accompanied by an expansion of Jz endocrine cell types due to enhanced rates of proliferation and increased expression of pregnancy-specific glycoprotein 23 in the placenta of both fetal sexes. However, changes in the endocrine phenotype of the placenta were related to sexually-dimorphic alterations to the abundance of Igf2 receptors and downstream signalling pathways (Pi3k-Akt and Mapk). There was no effect of Jz-ΔICR1 on the expression of targets of the H19-embedded miR-675 or on fetal weight. Our results demonstrate that ICR1 controls placental endocrine capacity via sex-dependent changes in signalling.

Orientation of mouse H19 ICR affects imprinted H19 gene expression through promoter methylation-dependent and -independent mechanisms

The mouse Igf2/H19 locus is regulated by genomic imprinting, in which the paternally methylated H19 imprinting control region (ICR) plays a critical role in mono-allelic expression of the genes in the locus. Although the maternal allele-specific insulator activity of the H19 ICR in regulating imprinted Igf2 expression has been well established, the detailed mechanism by which the H19 ICR controls mono-allelic H19 gene expression has not been fully elucidated. In this study, we evaluated the effect of H19 ICR orientation on imprinting regulation in mutant mice in which the H19 ICR sequence was inverted at the endogenous locus. When the inverted-ICR allele was paternally inherited, the methylation level of the H19 promoter was decreased and the H19 gene was derepressed, suggesting that methylation of the H19 promoter is essential for complete repression of H19 gene expression. Unexpectedly, when the inverted allele was maternally inherited, the expression level of the H19 gene was lower than that of the WT allele, even though the H19 promoter remained fully hypomethylated. These observations suggested that the polarity of the H19 ICR is involved in controlling imprinted H19 gene expression on each parental allele, dependent or independent on DNA methylation of the H19 promoter.

Loss of imprinting of the Igf2-H19 ICR1 enhances placental endocrine capacity via sex-specific alterations in signalling pathways in the mouse.

Imprinting control region (ICR1) controls the expression of the Igf2 and H19 genes in a parent-of-origin specific manner. Appropriate expression of the Igf2-H19 locus is fundamental for normal fetal development, yet the importance of ICR1 in the placental production of hormones that promote maternal nutrient allocation to the fetus is unknown. To address this, we used a novel mouse model to selectively delete ICR1 in the endocrine junctional zone (Jz) of the mouse placenta (Jz-ΔICR1). The Jz-ΔICR1 mice exhibit increased Igf2 and decreased H19 expression specifically in the Jz. This was accompanied by an expansion of Jz endocrine cell types due to enhanced rates of proliferation and increased expression of pregnancy-specific glycoprotein 23 in the placenta of both fetal sexes. However, changes in the endocrine phenotype of the placenta were related sexually-dimorphic alterations to the abundance of IGF2 receptors and downstream signalling pathways (PI3K-AKT and MAPK). There was no effect of Jz-ΔICR1 on the expression of targets of the H19 embedded miR-675 or on fetal weight. Our results demonstrate that ICR1 controls placental endocrine capacity via sex-dependant changes in signalling.

Grandmaternal smoking during pregnancy is associated with differential DNA methylation in their grandchildren

The idea that information can be transmitted to subsequent generation(s) by epigenetic means has been studied for decades but remains controversial in humans. Epidemiological studies have established that grandparental exposures are associated with health outcomes in their grandchildren, often with sex-specific effects; however the mechanism of transmission is still unclear. We conducted Epigenome Wide Association Studies (EWAS) to test whether grandmaternal smoking during pregnancy is associated with altered DNA methylation (DNAm) in their adolescent grandchildren. We used data from a birth cohort, with discovery and replication datasets of 1225 and 708 individuals (respectively), aged 15-17 years, and tested replication in the same individuals at birth and 7 years. We show for the first time that DNAm at a small number of loci is associated with grandmaternal smoking in humans, and their locations in the genome suggest hypotheses of transmission. We observe and replicate sex-specific associations at two sites on the X chromosome, one located in an imprinting control region and both within transcription factor binding sites (TFBSs). In fact, we observe enrichment for TFBSs among the CpG sites with the strongest associations, suggesting that TFBSs may be a mechanism by which grandmaternal exposures influence offspring DNA methylation. There is limited evidence that these associations appear at earlier timepoints, so effects are not static throughout development. The implication of this work is that effects of smoking during pregnancy may induce DNAm changes in later generations and that these changes are often sex-specific, in line with observational associations.

TRIM28 regulates Kaiso SUMOylation

Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate DNA methylation of many repetitive elements and several imprinted loci. TRIM28 serves as a scaffold unit that is essential for the formation of stable repressor complexes. In the present study we found that TRIM28 is a binding partner for methyl-DNA binding protein Kaiso. Kaiso is a transcription factor that belongs to the BTB/POZ -zinc finger family. Recent data suggest that deficiency of Kaiso led to reduction of DNA methylation within the imprinting control region of H19/IGF2. Thus, we hypothesized that Kaiso and TRIM28 may cooperate to control methylated genes. We demonstrated that Kaiso interacts with TRIM28 via its two domains: BTB/POZ and three zinc finger domains. When bound to Kaiso’s zinc finger domains TRIM28 weakens their interactions with methylated DNA in vitro. Specific association of TRIM28 with BTB/POZ domain causes Kaiso hyperSUMOylation. Altogether our data describe a putative role of TRIM28 as a regulator of Kaiso activity.

Transient establishment of imprinted DNA methylation of transgenic human IC1 sequence in mouse during the pre-implantation period

Monoallelic gene expression at the Igf2/H19 locus is controlled by paternal allele-specific DNA methylation of the imprinting control region (H19 ICR) that is established during spermatogenesis. We demonstrated that the H19 ICR fragment in transgenic mice acquires allele-specific methylation only after fertilization, which is essential for maintaining its allelic methylation during early embryogenesis. We identified a DNA element required for establishing post-fertilization methylation within a 118 bp (m118) region. A previously generated knock-in mouse whose endogenous H19 ICR was substituted with the human H19 ICR (hIC1; 4.8 kb) sequence revealed that the hIC1 sequence was partially methylated in sperm, although this methylation was lost by the blastocyst stage, which we assume is due to a lack of an m118-equivalent sequence in the hIC1 transgene. To identify a cis sequence involved in post-fertilization methylation within the hIC1 region, we generated three transgenic mouse lines (TgM): one carrying an 8.8 kb hIC1 sequence joined to m118 (hIC1+m118), one with the 8.8 kb hIC1, and one with the 5.8 kb hIC1 sequence joined to m118 (hIC1–3′+m118). We found that the hIC1–3′ region was resistant to de novo DNA methylation throughout development. In contrast, the 5′ portion of the hIC1 (hIC1–5′) in both hIC1+m118 and hIC1 TgM were preferentially methylated on the paternal allele only during preimplantation. As DNA methylation levels were higher in hIC1+m118, the m118 sequence could also induce imprinted methylation of the human sequence. Most importantly, the hIC1–5′ sequence appears to possess an activity equivalent to that of m118.

Epigenetic characterization of the H19/IGF2 locus in calf clones placenta

ABSTRACT: Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals’ placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.

Using a Dielectrophoretic Microfluidic Biochip Enhanced Fertilization of Mouse Embryo in Vitro

Droplet microfluidics has appealed to many interests for its capability to epitomize cells in a microscale environment and it is also a forceful technique for high-throughput single-cell epitomization. A dielectrophoretic microfluidic system imitates the oviduct of mammals with a microchannel to achieve fertilization in vitro (IVF) of an imprinting control-region (ICR) mouse. We applied a microfluidic chip and a positive dielectrophoretic (p-DEP) force to capture and to screen the sperm for the purpose of manipulating the oocyte. The p-DEP responses of the oocyte and sperm were exhibited under applied bias conditions (waveform AC 10 Vpp, 1 MHz) for trapping 1 min. The insemination concentration of sperm nearby the oocyte was increased to enhance the probability of natural fertilization through the p-DEP force trapping. A simulation tool (CFDRC-ACE+) was used to simulate and to analyze the distribution of the electric field. The DEP microfluidic devices were fabricated using poly (dimethylsiloxane) (PDMS) and ITO (indium tin oxide)-glass with electrodes. We discuss the requirement of sperm in a DEP microfluidic chip at varied concentrations to enhance the future rate of fertilization in vitro for an oligozoospermia patient. The result indicates that the rate of fertility in our device is 17.2 ± 7.5% (n = 30) at about 3000 sperms, compatible with traditional droplet-based IVF, which is 14.2 ± 7.5% (n = 28).