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.

Loss of imprinting control of the lncRNA H19-fetal mitogen IGF2 gene cluster in the decidual microenvironment of patients with idiopathic spontaneous miscarriages

Miscarriage, the spontaneous loss of a pregnancy before the fetus achieves viability, is a common complication of pregnancy. Decidualization plays a critical role in the implantation of the embryo. To search for molecular factors underlying miscarriage, we explored the role of long noncoding RNAs (lncRNAs) in the decidual microenvironment, where the molecular crosstalk at the feto–maternal interface occurs. By integrating RNA-seq data from recurrent miscarriage patients and decidualized endometrial stromal cells, we identified H19 , a noncoding RNA that exhibits paternally imprinted monoallelic expression in normal tissues, as the most upregulated lncRNA associated with miscarriage. Aberrant upregulation of H19 lncRNA was observed in decidual tissues derived from patients with spontaneous miscarriage as well as decidualized endometrial stromal cells. The maternally imprinted fetal mitogen Igf2, which is usually reciprocally co-regulated with H19 in the same imprinting cluster, was also upregulated. Notably, both genes underwent loss of imprinting, as H19 and IGF2 were actively transcribed from both parental alleles in decidual tissues. Mechanistically, this loss of imprinting in decidual tissues was associated with the loss of the H3K27m3 suppression marker in the IGF2 promoter, CpG hypomethylation at the central CTCF binding site in the imprinting control center (ICR) that is located between IGF2 and H19 , and the loss of CTCF-mediated intrachromosomal looping. These data provide the first evidence that aberrant control of the ICR epigenotype-intrachromosomal looping- H19/IGF2 imprinting pathway may be a critical epigenetic risk factor in the abnormal decidualization related to miscarriage.

Cardiac pathologies in mouse loss of imprinting models are due to misexpression of H19 long noncoding RNA

Maternal loss of imprinting (LOI) at the H19/IGF2 locus results in biallelic IGF2 and reduced H19 expression and is associated with Beckwith-Wiedemann syndrome (BWS). We use mouse models for LOI to understand the relative importance of Igf2 and H19 mis-expression in BWS phenotypes. Here we focus on cardiovascular phenotypes and show that neonatal cardiomegaly is exclusively dependent on increased Igf2. Circulating IGF2 binds cardiomyocyte receptors to hyperactivate mTOR signaling, resulting in cellular hyperplasia and hypertrophy. These Igf2-dependent phenotypes are transient: cardiac size returns to normal once Igf2 expression is suppressed postnatally. However, reduced H19 expression is sufficient to cause progressive heart pathologies including fibrosis and reduced ventricular function. In the heart, H19 expression is primarily in endothelial cells (ECs) and regulates EC differentiation both, in vivo and in vitro. Finally, we establish novel mouse models to show that cardiac phenotypes depend on H19 lncRNA interactions with Mirlet7 microRNAs.

Sex-specific transcriptional differences and loss of gene imprinting in pancreatic neuroendocrine tumors

Pancreatic neuroendocrine tumors (PNETs) occur more frequently in men and are associated with higher mortality in males; however, the molecular basis for these sexual dimorphisms is unclear. Here, we demonstrate that PNETs are associated with the emergence of unique sex-specific transcriptomic differences that are not observed in non-neoplastic pancreatic islet tissues. We also show that while widespread sex-specific differences are present in the DNA methylation landscapes of control pancreatic islets, they are erased in PNETs. This includes a loss of imprinting with regards to many genes. These results implicate an emergence of sex-associated genetic and epigenetic dysregulations in PNETs.

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.

209 Large Offspring Syndrome: Effects of in vitro Production on Embryo Epigenetics and Development

In cattle, the use of assisted reproductive technologies (ART) can result in a congenital overgrowth condition known as large/abnormal offspring syndrome (LOS/AOS). The phenotypic characteristics of LOS include; somatic overgrowth, abdominal wall defects, large organs, breathing difficulties, skeletal defects, hypoglycemia, abnormal placentas, difficulty suckling, and perinatal death. LOS can have detrimental effects on the offspring and dam and also pose managerial and financial challenges to the producer. Research from the Rivera laboratory has demonstrated that LOS is an epigenetic syndrome. As in cattle, ART can promote the development of congenital overgrowth in humans, a condition known as Beckwith Wiedemann Syndrome (BWS). For the past 13 years, the Rivera laboratory has been characterizing LOS and we have shown that LOS and BWS are phenotypically and epigenotypically similar. In our studies, using gestation day ~105 Bos taurus taurus x Bos taurus indicus F1 hybrids, we showed global misregulation of imprinted and non-imprinted transcripts, micro RNAs and global misregulation of DNA methylation. In brief, LOS fetuses displayed variable loss-of-imprinting in kidney, liver, muscle and brain, when compared to controls. Biallelic expression of imprinted genes in LOS was associated with tissue-specific hypomethylation of the normally methylated parental allele. Not only was there loss of allele-specific expression of imprinted genes in LOS, but we also observed differential transcript amounts of these genes between control and overgrown fetuses. In addition, a positive correlation was observed between bodyweight and the number of biallelically expressed imprinted genes in LOS fetuses. From this work, we concluded that LOS is a multi-locus loss-of-imprinting condition. Current work, aims to determine if LOS is identifiable during pregnancy using day 55 fetal ultrasonography and day 55 and 105 maternal blood. In addition, we aim to determine how serum supplementation of culture medium can program preimplantation embryos to develop LOS. Findings will be discussed.

Cardiac pathologies in mouse loss of imprinting models are due to misexpression of H19 long noncoding RNA

Maternal loss of imprinting (LOI) at the H19/IGF2 locus results in biallelic IGF2 and reduced H19 expression and is associated with Beckwith Wiedemann syndrome (BWS). We use mouse models for LOI to understand the relative importance of Igf2 and H19 mis-expression in BWS phenotypes. Here we focus on cardiovascular phenotypes and show that neonatal cardiomegaly is exclusively dependent on increased Igf2. Circulating IGF2 binds cardiomyocyte receptors to hyperactivate mTOR signaling, resulting in cellular hyperplasia and hypertrophy. These Igf2-dependent phenotypes are transient: cardiac size returns to normal once Igf2 expression is suppressed postnatally. However, reduced H19 expression is sufficient to cause progressive heart pathologies including fibrosis and reduced ventricular function. In the heart, H19 expression is concentrated predominantly in endothelial cells (ECs) and regulates EC differentiation both, in vivo and in vitro. Finally, we establish novel mouse models to show that cardiac phenotypes depend on H19 lncRNA interactions with let7 microRNA.

Loss of imprinting of the human-specific imprinted gene ZNF597 causes prenatal growth retardation and dysmorphic features: implications for phenotypic overlap with Silver-Russell syndrome

BackgroundZNF597, encoding a zinc-finger protein, is the human-specific maternally expressed imprinted gene located on 16p13.3. The parent-of-origin expression of ZNF597 is regulated by the ZNF597:TSS-DMR, of which only the paternal allele acquires methylation during postimplantation period. Overexpression of ZNF597 may contribute to some of the phenotypes associated with maternal uniparental disomy of chromosome 16 (UPD(16)mat), and some patients with UPD(16)mat presenting with Silver-Russell syndrome (SRS) phenotype have recently been reported.MethodsA 6-year-old boy presented with prenatal growth restriction, macrocephaly at birth, forehead protrusion in infancy and clinodactyly of the fifth finger. Methylation, expression, microsatellite marker, single nucleotide polymorphism array and trio whole-exome sequencing analyses were conducted.ResultsIsolated hypomethylation of the ZNF597:TSS-DMR and subsequent loss of imprinting and overexpression of ZNF597 were confirmed in the patient. Epigenetic alterations, such as UPD including UPD(16)mat and other methylation defects, were excluded. Pathogenic sequence or copy number variants affecting his phenotypes were not identified, indicating that primary epimutation occurred postzygotically.ConclusionWe report the first case of isolated ZNF597 imprinting defect, showing phenotypic overlap with SRS despite not satisfying the clinical SRS criteria. A novel imprinting disorder entity involving the ZNF597 imprinted domain can be speculated.