Dysregulated H19/Igf2 expression disrupts cardiac-placental axis during development of Silver Russell Syndrome-like mouse models
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PROVIDER: PRJNA819477 | ENA |
REPOSITORIES: ENA
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Project description:Abstract: Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell Syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 ICR (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects (VSDs) and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix (ECM) and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis. Methods: E12.5 hearts were lysed with Collagenase, Dispase II, and DNase I. Cardiac endothelial cells were collected using MACS CD31 microbeads and RNA was isolated using RNeasy Micro kit. After confirming RNA integrity using Bioanalyzer, mRNA library was generated from 25ng RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and Ultra II RNA Library Prep Kit. Library quality was assessed by Bioanalyzer and TapeStation. Sequencing was performed on NovaSeq 6000. Quality of raw fastq reads was assessed using FastQC version 0.11.5. Reads were aligned to the GRCm38/mm10 reference using STAR version 2.4.0i with default parameters and maximum fragment size of 2000 bp. Properly paired primary alignments were retained for downstream analysis using Samtools version 1.9. Count matrices were generated using FeatureCounts version 1.6.2 against RefSeq gene annotation and read into DESeq2 to perform normalization and statistical analysis.
2022-11-25 | GSE199377 | GEO
Project description:The insulator model explains the workings of the H19 and Igf2 domain in the soma, where insulation of the Igf2 promoter from its enhancers occurs by CTCF in the maternally inherited unmethylated chromosome but not the paternally inherited methylated allele. The molecular mechanism that targets paternal methylation imprint establishment to the imprinting control region (ICR) in the male germline is unknown. We tested the function of a prospermatogonia-specific broad low-level transcription in this process using mouse genetics. Paternal imprint establishment was abnormal when transcription was stopped at the entry point to the ICR. The germline epimutation persisted into the paternal allele of the soma, resulting in reduced Igf2 in fetal organs, and reduced fetal growth, consistent with the insulator model and IGF2’s role as fetal growth factor. In summary, broad low-level transcription through the ICR initiates paternal imprint establishment at the H19/Igf2 ICR in the male germ line, with implications for Silver-Russell syndrome. The superseries contains RNAseq and ChIPseq data from 15.5 dpc fetal male germ cells that carry the RNA terminator cassette and also from control wild type mice.
2023-08-02 | GSE236416 | GEO
Project description:Silver-Russell syndrome(SRS, OMIM 180860) is an imprinting disordermanifesting in fetal and postnatal growth retardation. Loss of methylation (LOM) is detected in the imprinting control region 1 (ICR1) that controls the H19/IGF2 domain, in about 65% of SRS cases. The cause of this epigenetic deficiency is unknown. It may affect the process of imprint establishment --de novo methylation of ICR1 in the male germ line. In the paralogous mouse locus this process involves DNA methyltransferase DNMT3A and H3K36me2 histone methyltransferase NSD1, but the mechanism that targets these enzymes to this ICR is unknown. Based on our genome-wide deep-sequencing data of transcription and DNA methylation, we hypothesized that low-level transcription is required in prospermatogonia for targeting H3K36me2 and de novo methylation to ICR. We truncated this transcript at the entry point to ICR by gene-targeting. In response, the establishment of DNA methylation imprint was incomplete. The sporadic LOM persisted from the male germ line into the paternal allele of the soma, resulting in reduced Igf2, and increased, biallelic H19 RNA in the fetal organs, consistent with the insulator model. Fetus weight was also variably reduced upon paternal transmission of this mutation, consistent with the role of IGF2 as a fetal growth factor. We identify low-level transcription-through as the mechanism that initiates paternal imprint establishment at the H19/Igf2 ICR in the male germ line. The findings predict that RNA-dependent imprint establishment may be affected in human fetuses resulting in sporadic hypomethylation of the ICR1 in SRS.
2023-08-02 | GSE202875 | GEO
Project description:Changes in microRNA expression in Igf2-p and H19-m mouse embryos (E9.5) were determined in order to assess whether perturbation of miR-483* and miR-675 in Igf2-p and H19-m mutants was likely to have contributed to a modification of tumour phenotype. The Igf2 gene contains miR-483* but the targeted deletion of Igf2-p in these mice spares the region encoding this microRNA. The H19 gene contains miR-675 and its expression was mono-allelelic in heterozygous H19-m mice as evidenced by a significant reduction in miR-675 in these mice relative to WT.
2011-12-01 | E-MTAB-873 | biostudies-arrayexpress
Project description:Exposure to mixtures of toxic metals is known to cause adverse health effects through epigenetic alterations. Here we aimed to examine the unexplored area of aberrant DNA methylation in the H19/IGF2 domain following combined toxic metal exposure. An in vitro epigenotoxicity assay using the human normal liver epithelial cell line THLE-3 was conducted. When THLE-3 cells were exposed to specific concentrations of either organic arsenic or MeHgCl, an increase in the H19 lncRNA levels and a marked reduction in the IGF2 mRNA levels were observed. In contrast, combined exposures coupled with CdCl2 resulted in the transcriptional repression of H19 and transcriptional activation of IGF2. It should be noted that the correlation between the dysregulated expression of H19/IGF2 and the hypermethylated CpG sites within the H19 differentially methylated region (DMR) was statistically significant. Furthermore, we performed transcriptomic analysis of the hepatocytes exposed to toxic metal combinations indicating enrichment of pro-inflammatory and anti-proliferative pathways compared to the unexposed cells. Our results suggest that hazardous metal mixtures may trigger epigenetic aberrations at the H19/IGF2 locus. We propose that altered CpG methylation in the H19 DMR could be a candidate biomarker for hepatic epigenotoxicity, in part, due to environmental exposure.
2024-10-27 | GSE280026 | GEO
Project description:Exposure to mixtures of toxic metals is known to cause adverse health effects through epigenetic alterations. Here we aimed to examine the unexplored area of aberrant DNA methylation in the H19/IGF2 domain following combined toxic metal exposure. An in vitro epigenotoxicity assay using the human normal liver epithelial cell line THLE-3 was conducted. When THLE-3 cells were exposed to specific concentrations of either organic arsenic or MeHgCl, an increase in the H19 lncRNA levels and a marked reduction in the IGF2 mRNA levels were observed. In contrast, combined exposures coupled with CdCl2 resulted in the transcriptional repression of H19 and transcriptional activation of IGF2. It should be noted that the correlation between the dysregulated expression of H19/IGF2 and the hypermethylated CpG sites within the H19 differentially methylated region (DMR) was statistically significant. Furthermore, we performed transcriptomic analysis of the hepatocytes exposed to toxic metal combinations indicating enrichment of pro-inflammatory and anti-proliferative pathways compared to the unexposed cells. Our results suggest that hazardous metal mixtures may trigger epigenetic aberrations at the H19/IGF2 locus. We propose that altered CpG methylation in the H19 DMR could be a candidate biomarker for hepatic epigenotoxicity, in part, due to environmental exposure.
2024-10-27 | GSE280025 | GEO
Project description:The in-vitro analysis of the hypomethylation of the imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). For excluding mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and expression of IGF2 and H19 were measured. Microarray expression analysis was performed. Single cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from patients and controls (Cnormo). IGF2 expression was below the detection limit of the qRT-PCR assay, while H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in supernatants of SRShypo and Cnormo. Cell proliferation was diminished in SRShypo compared to Cnormo (p=.035). Microarray analysis revealed gene expression changes in SRS clones predicting a decrease of cell proliferation and a delay of mitosis. The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal any functional differences towards the normomethylated clones with respect to IGF2 and H19 expression. Furthermore, a clear difference to the clones from healthy individuals was not detected except from a lower proliferation rate arisen from impaired cell cycle progression. 16 samples: 8 SRShypo, 4 SRSnormo, 4 Cnormo
2015-01-01 | E-GEOD-61120 | biostudies-arrayexpress
Project description:The in-vitro analysis of the hypomethylation of the imprinting control region 1 (ICR1) within the IGF2/H19 locus is challenged by the mosaic distribution of the epimutation in tissues from children with Silver-Russell syndrome (SRS). For excluding mosaicism, clonal cultures of skin fibroblasts from four children with SRS and three controls were analyzed. Cell proliferation, IGF-II secretion, and expression of IGF2 and H19 were measured. Microarray expression analysis was performed. Single cell expansion established severely ICR1 hypomethylated clones (SRShypo) and normomethylated clones (SRSnormo) from patients and controls (Cnormo). IGF2 expression was below the detection limit of the qRT-PCR assay, while H19 expression was detectable, without differences between fibroblast clones. Cell count-related IGF-II release was comparable in supernatants of SRShypo and Cnormo. Cell proliferation was diminished in SRShypo compared to Cnormo (p=.035). Microarray analysis revealed gene expression changes in SRS clones predicting a decrease of cell proliferation and a delay of mitosis. The analysis of severely ICR1 hypomethylated clonal fibroblasts did not reveal any functional differences towards the normomethylated clones with respect to IGF2 and H19 expression. Furthermore, a clear difference to the clones from healthy individuals was not detected except from a lower proliferation rate arisen from impaired cell cycle progression.
2015-01-01 | GSE61120 | GEO
Project description:Imprinted genes occur in discrete clusters that are coordinately regulated by shared DNA elements. H19 and Igf2 are linked imprinted genes that play critical roles in development. Loss of imprinting at the IGF2/H19 locus is associated with Beckwith Wiedemann Syndrome (BWS). Here we use comprehensive genetic and genomic analyses to follow muscle development in a mouse model of BWS to dissect the separate and shared roles for Igf2 and H19 in the disease phenotype. We show that LOI results in defects in muscle differentiation and hypertrophy and we identify primary downstream targets of Igf2 (MAPK signaling) and of H19 (AKT/mTOR signaling). Moreover, we demonstrate instances where H19 and Igf2 misexpression work separately, cooperatively, and also antagonistically to establish the disease phenotype. This study underscores the fact that LOI phenotypes are multigenic and complex interactions contribute to disease outcomes.
2017-09-25 | GSE89136 | GEO
Project description:Genomewide methylation analysis in Silver Russell syndrome patients compared to healthy controls
2015-01-12 | GSE55491 | GEO