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.

H3K9 methyltransferase EHMT2/G9a controls ERVK-driven noncanonical imprinted genes

Aim: Paternal allele-specific expression of noncanonical imprinted genes in the extraembryonic lineages depends on an H3K27me3-based imprint in the oocyte, which is not a lasting mark. We hypothesized that EHMT2, the main euchromatic H3K9 dimethyltransferase, also has a role in controlling noncanonical imprinting. Methods: We carried out allele-specific total RNA-seq analysis in the ectoplacental cone of somite-matched 8.5 days post coitum embryos using reciprocal mouse crosses. Results: We found that the maternal allele of noncanonical imprinted genes was derepressed from its ERVK promoter in the Ehmt2-/- ectoplacental cone. In Ehmt2-/- embryos, loss of DNA methylation accompanied biallelic derepression of the ERVK promoters. Canonical imprinting and imprinted X chromosome inactivation were generally undisturbed. Conclusion: EHMT2 is essential for repressing the maternal allele in noncanonical imprinting.

H3K9 methyltransferase EHMT2/G9a controls ERVK-driven non-canonical imprinted genes

Unlike regular imprinted genes, non-canonical imprinted genes are known to not depend on gamete-specific DNA methylation difference. Instead, the paternal allele-specific expression of these genes in the extra-embryonic lineages depends on an H3K27me3-based imprint in the oocyte, but this marking is not maintained beyond pre-implantation development. The maintenance of non-canonical imprinting corresponds to maternal allele-specific DNA methylation and paternal allele-specific H3K4me3 at their somatic DMRs, which occur at ERVK repeats. We hypothesized that EHMT2, the main euchromatic H3K9 methyltransferase, also has a role in this process. Using reciprocal mouse crosses and allele-specific RNA-seq analysis, we found that the maternal allele of each known non-canonical imprinted gene was derepressed from its ERVK promoter in the Ehmt2−/− ectoplacental cone of somite-matched 8.5 dpc embryos. In the Ehmt2−/− embryos, loss of DNA methylation accompanied the derepression of both parental alleles of those ERVK promoters. Our study identifies EHMT2 as an essential player that maintains the repressed chromosomal state in non-canonical imprinting.

A novel familial PHP1B variant with incomplete loss-of-methylation at GNAS-A/B and enhanced methylation at GNAS-AS2

Context Pseudohypoparathyroidism type 1B (PHP1B), also referred to as inactivating PTH/PTHrP Signaling Disorder (iPPSD), is characterized by proximal renal tubular resistance to parathyroid hormone (PTH) leading to hypocalcemia, hyperphosphatemia and elevated PTH values. Autosomal dominant PHP1B (AD-PHP1B) with loss-of-methylation at the maternal GNAS A/B:TSS-DMR (transcription start site-differentially methylated region) alone can be caused by maternal deletions involving STX16. Objectives Characterize a previously not reported AD-PHP1B family with loss-of-methylation at GNAS A/B:TSS-DMR, but without evidence for a STX16 deletion on the maternal allele and assess GNAS-AS2:TSS-DMR methylation. Patients and methods DNAs from 24 patients and 10 controls were investigated. AD-PHP1B patients without STX16 deletion from a single family (n=3), AD-PHP1B patients with STX16 deletion (n=9), sporPHP1B (n=10), unaffected controls (n=10), patUPD20 (n=1), and matUPD20 (n=1). Methylation and copy number analyses were performed by pyrosequencing, MS-MPLA, and MLPA, respectively. Results Molecular cloning of PCR-amplified, bisulfite-treated genomic DNA from healthy controls revealed evidence for two distinct GNAS-AS2:TSS-DMR subdomains, named AS2-1 and AS2-2, which showed 16.0±2.3% and 31.0±2.2% methylation, respectively. DNA from affected members of a previously not reported AD-PHP1B family without the known genetic defects revealed incomplete LOM (loss-of-methylation) at GNAS A/B:TSS-DMR, normal methylation at the three well-established maternal and paternal DMRs, and, surprisingly, increased methylation at AS2-1 (32.9±3.5%), but not at AS2-2 (30.5±2.9%). Conclusion The distinct methylation changes at the novel GNAS-AS2:TSS-DMR will help characterize further different PHP1B/iPPSD3 variants and will guide the search for underlying genetic defects, which may provide novel insights into the mechanisms underlying GNAS methylation.

Unusual deletion of the maternal 11p15 allele in Beckwith–Wiedemann syndrome with an impact on both imprinting domains

Background Whereas duplications in 11p15.5 covering both imprinting centers (ICs) and their subordinated genes account for up to 1% of Beckwith–Wiedemann and Silver–Russell syndrome patients (BWS, SRS), the deletions in 11p15.5 reported so far only affect one of the ICs. In these cases, not only the size and gene content had an impact on the phenotype, but also the sex of the contributing parent influences the clinical signs of the deletion carrier. Results We here report on the first case with a heterozygous deletion within the maternal allele affecting genes which are regulated by both ICs in 11p15.5 in a BWS patient, and describe the molecular and clinical consequences in case of its maternal or paternal inheritance. Conclusions The identification of a unique deletion affecting both 11p15.5 imprinting domains in a BWS patient illustrates the complexity of the regulation mechanisms in these key imprinting regions.

Maternal allele mutation: Slippage synthesis furnishing evolutionary trend

Gain or loss of repeat motifs leads to an allelic mismatch in the disputed child which is a deviation from the Mendelian inheritance, thereby leading to a paternal mismatch of putative father or exclusion of mother in case of maternal allelic mismatch. This allelic mismatch at one or more loci is a major cause of forensic inferences. The biological samples of the case were genotyped using Powerplex®- Fusion 5C system kit and Investigator® Argus X-12 QS kit as per recommendations of the manufacturer. In this case, identification of a putrefied dead body with 22 autosomal STR loci, primarily analyzed by Powerplex®- Fusion 5C system kit divulged a maternal mismatch at locus D13S317. Alleles at the locus D13S317 allegedly belonging to the father of the deceased and the mother were observed as 10/11, 11/11 and 10/10 respectively. To rule out allelic mismatch at this particular locus, 12 X-STR loci were amplified, in which all the maternal alleles of deceased completely matched with the mother. This case study indicates the extension of one microsatellite repeat motif (TATC) at locus D13S317 in the population of Rajasthan. The reported mutation rate was 0.14% and 0.04% at locus D13S317 in paternal and maternal meiosis respectively.

Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity. This disorder is caused by the absence of paternally expressed gene products from chromosome 15q11–q13. We previously demonstrated that knocking out ZNF274, a Kruppel-associated box-A-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS induced pluripotent stem cells (iPSCs). However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here, we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared with ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

Dopa decarboxylase is a genetic hub of parental control over offspring behavior

SUMMARYDopa decarboxylase (DDC) regulates the synthesis of monoaminergic neurotransmitters and is linked to psychiatric and metabolic disorders. Ddc exhibits complex genomic imprinting effects that have not been functionally studied. Here, we investigate different noncanonical imprinting effects at the cellular level with a focus on Ddc. Using allele-specific reporter mice, we found Ddc exhibits dominant expression of the maternal allele in subpopulations of cells in 14 of 52 brain regions, and dominant paternal or maternal allele expression in adrenal cell subpopulations. Maternal versus paternal Ddc allele null mutations differentially affect offspring social, foraging and exploratory behaviors. Machine learning analyses of naturalistic foraging in Ddc−/+ and +/− offspring uncovered finite behavioral sequences controlled by the maternal versus paternal Ddc alleles. Additionally, parental Ddc genotype is revealed to affect behavior independent of offspring genotype. Thus, Ddc is a hub of maternal and paternal influence on behavior that mediates diverse imprinting and parental effects.HIGHLIGHTSDopa decarboxylase (Ddc) allelic expression resolved at the cellular levelCells differentially express maternal versus paternal Ddc allelesMaternal and paternal Ddc alleles control distinct behavioral sequencesParental Ddc genotype affects offspring independent of mutation transmissioneTOCAllelic reporter mice and machine learning analyses reveal dopa decarboxylase is affected by diverse imprinting and parental effects that shape finite behavioral sequences in sons and daughters.

Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay, and hyperphagia/obesity. This disorder is caused by the absence of paternally-expressed gene products from chromosome 15q11-q13. We previously demonstrated that knocking out ZNF274, a KRAB-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS iPSCs. However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared to ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

Allelic H3K27me3 to allelic DNA methylation switch maintains noncanonical imprinting in extraembryonic cells

Faithful maintenance of genomic imprinting is essential for mammalian development. While germline DNA methylation–dependent (canonical) imprinting is relatively stable during development, the recently found oocyte-derived H3K27me3-mediated noncanonical imprinting is mostly transient in early embryos, with some genes important for placental development maintaining imprinted expression in the extraembryonic lineage. How these noncanonical imprinted genes maintain their extraembryonic-specific imprinting is unknown. Here, we report that maintenance of noncanonical imprinting requires maternal allele–specific de novo DNA methylation [i.e., somatic differentially methylated regions (DMRs)] at implantation. The somatic DMRs are located at the gene promoters, with paternal allele–specific H3K4me3 established during preimplantation development. Genetic manipulation revealed that both maternal EED and zygotic DNMT3A/3B are required for establishing somatic DMRs and maintaining noncanonical imprinting. Thus, our study not only reveals the mechanism underlying noncanonical imprinting maintenance but also sheds light on how histone modifications in oocytes may shape somatic DMRs in postimplantation embryos.