Intergenic Differentially Methylated Region Research Articles

Effect of epigenetic activating of Dlk1-Dio3 imprinted cluster on miR-370 expression due to folate deficiency during nerve development

Proper Dlk1-Dio3 imprinting plays a critical role in embryogenesis, and folic acid deficiency may affect the imprinting of this locus through epigenetic regulation. However, whether and how folic acid directly impacts the imprinting status of Dlk1-Dio3 to affect neural development remain unclear. Here, we found decreased IG-DMR (intergenic -differentially methylated regions) methylation in the folate-deficient encephalocele in humans, suggesting that abnormal Dlk1-Dio3 imprinting status is related to neural tube defects (NTDs) caused by folate deficiency. Similar results were obtained with folate-deficient embryonic stem cells. By miRNA chip analysis, folic acid deficiency led to changes in multiple miRNAs, including the upregulation of 15 miRNAs located in the Dlk1-Dio3 locus. Real-time PCR confirmed that seven of these miRNAs were upregulated, especially miR-370. In contrast to normal embryonic development, in which expression of miR-370 is highest at E9.5, the abnormally high and sustained expression of miRNA-370 in folate-deficient E13.5 embryos may contribute to NTDs. In addition, we found that DNMT3A (de novo DNA methyltransferases 3A) is a direct target gene of miR-370 in neural cells, and DNMT3A participates in the role of miR-370 in inhibiting cell migration. Finally, in the folate-deficient mouse model, Dlk1-Dio3 epigenetic activation was found in fetal brain tissue, along with the upregulation of miR-370 and the downregulation of DNMT3A. Collectively, our findings demonstrate a pivotal role of folate in the epigenetic regulation of Dlk1-Dio3 imprinting during neurogenesis, revealing an elegant mechanism for the activation of Dlk1-Dio3 locus miRNAs in folic acid deficiency.

Humanization of a tandem repeat in IG-DMR causes stochastic restoration of paternal imprinting at mouse Dlk1-Dio3 domain.

The Dlk1-Dio3 imprinted domain, regulated by an intergenic differentially methylated region (IG-DMR), is important for mammalian embryonic development. Although previous studies have reported that DNA methylation of a tandem repeated array sequence in paternal IG-DMR (IG-DMR-Rep) plays an essential role in the maintenance of DNA methylation in mice, the function of a tandem repeated array sequence in human IG-DMR (hRep) is unknown. Here, we generated mice with a human tandem repeated sequence, which replaced the mouse IG-DMR-Rep. Mice that transmitted the humanized allele paternally exhibited variable methylation status at the IG-DMR and were stochastically rescued from the lethality of IG-DMR-Rep deficiency, suggesting that hRep plays a role in human IG-DMR for the regulation of imprinted expression. Moreover, chromatin immunoprecipitation analysis showed that TRIM28 was enriched in hypermethylated paternal hRep without ZFP57. Our results suggest that hRep contributes to the maintenance of human IG-DMR methylation imprints via the recruitment of TRIM28.

DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST),zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. RPL is defined as loss of two or more pregnancies, affecting 1-2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. In this case-control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. N/A.

Single-base cytosine methylation analysis in fruits of three Capsicum species

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8–89.1%, 77.6–83.9%, and 22.4–25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.

Maternal Low-Fat Diet Programs the Hepatic Epigenome despite Exposure to an Obesogenic Postnatal Diet.

Obesity and metabolic disease present a danger to long-term health outcomes. It has been hypothesized that epigenetic marks established during early life might program individuals and have either beneficial or harmful consequences later in life. In the present study, we examined whether maternal diet alters DNA methylation and whether such modifications persist after an obesogenic postnatal dietary challenge. During gestation and lactation, male Sprague-Dawley rats were exposed to either a high-fat diet (HF; n = 10) or low-fat diet (LF; n = 10). After weaning, all animals were fed a HF diet for an additional nine weeks. There were no differences observed in food intake or body weight between groups. Hepatic DNA methylation was quantified using both methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylation-sensitive restriction enzyme sequencing (MRE-seq). Overall, 1419 differentially methylated regions (DMRs) were identified. DMRs tended to be located in CpG shores and were enriched for genes involved in metabolism and cancer. Gene expression was measured for 31 genes in these pathways. Map3k5 and Igf1r were confirmed to be differentially expressed. Finally, we attempted to quantify the functional relevance of intergenic DMRs. Using chromatin contact data, we saw that conserved DMRs were topologically associated with metabolism genes, which were associated with differential expression of Adh5, Enox1, and Pik3c3. We show that although maternal dietary fat is unable to reverse offspring weight gain in response to a postnatal obesogenic diet, early life diet does program the hepatic methylome. Epigenetic alterations occur primarily in metabolic and cancer pathways and are associated with altered gene expression, but it is unclear whether they bear consequence later in life.

Effects of programming on the hepatic epigenome by maternal low‐fat diet persist in adult offspring fed an obesogenic diet

Obesity and metabolic disease present a risk to long‐term health outcomes. Epigenetic marks such as DNA methylation are established during early life and have effects on later life through developmental programming. In the present study, we examined whether maternal diet alters DNA methylation and whether such modifications persist after an obesogenic postnatal dietary challenge. During gestation and lactation, male Sprague Dawley rats were exposed to either a high‐fat diet (HF; n = 10) or low‐fat diet (LF; n = 10). After weaning, all animals were fed the HF diet until postnatal week 12. There were no differences observed in food intake or body weight between the two groups of offspring animals. Hepatic DNA methylation was quantified using both Methylated DNA Immunoprecipitation sequencing (MeDIP‐seq) and Methylation‐sensitive Restriction Enzyme sequencing (MRE‐seq). Overall, 1,419 differentially methylated regions (DMRs) were identified. DMRs tended to be located in CpG shores, as opposed to CpG islands, and were enriched for genes involved in metabolism and cancer. Gene expression was measured for 31 candidate genes in these pathways. Among these, Map3k5 and Igf1r were confirmed to be differentially expressed. When the functional relevance of intergenic DMRs was quantified using chromatin contact data, we discovered that conserved DMRs were topologically associated with metabolism genes, which was associated with differential expression of Adh5, Enox1, and Pik3c3. Overall, although maternal dietary fat was unable to reverse offspring weight gain in response to postnatal obesogenic diet, early life diet did program the hepatic methylome. Epigenetic alterations on the hepatic methylome occur primarily in metabolic and cancer pathways and are associated with altered gene expression. Physiological consequences of these epigenetic marks will require future investigations for a better understanding of epigenomic programming.Support or Funding InformationThis project was supported by UIUC Research Board grant #12192, the USDA Cooperative State Research, Education and Extension Service, Hatch project numbers # ILLU‐698‐923 and ILLU‐698‐369, ILLU‐698‐391, and the Data Purchase Program from the University of Illinois Library.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A tandem repeat array in IG-DMR is essential for imprinting of paternal allele at the Dlk1–Dio3 domain during embryonic development

Genomic imprinting is a phenomenon that causes parent-origin-specific monoallelic expression of a small subset of genes, known as imprinted genes, by parentally inherited epigenetic marks. Imprinted genes at the delta-like homolog 1 gene (Dlk1)-type III iodothyronine deiodinase gene (Dio3) imprinted domain, regulated by intergenic differentially methylated region (IG-DMR), are essential for normal development of late embryonic stages. Although the functions of IG-DMR have been reported by generating knockout mice, molecular details of the regulatory mechanisms are not fully understood as the specific sequence(s) of IG-DMR have not been identified. Here, we generated mutant mice by deleting a 216 bp tandem repeated sequence in IG-DMR, which comprised seven repeats of 24 bp motifs, by genome editing technologies. The mutant mice showed that paternal transmission of the deletion allele, but not maternal transmission, induces severe growth retardation and perinatal lethality, possibly due to placental defects. Embryos with a paternally transmitted deletion allele showed biallelic expression of maternally expressed genes and repression of paternally expressed genes. DNA methylation status also showed loss of methylation at IG-DMR and Gtl2-DMR, indicating that the tandem repeat sequence of IG-DMR is one of the functional sequences of IG-DMR, which is required for maintaining DNA methylation imprints of paternal allele at IG-DMR.

Hypermethylation of the non-imprinted maternal MEG3 and paternal MEST alleles is highly variable among normal individuals.

Imprinted genes show parent-specific activity (functional haploidy), which makes them particularly vulnerable to epigenetic dysregulation. Here we studied the methylation profiles of oppositely imprinted genes at single DNA molecule resolution by two independent parental allele-specific deep bisulfite sequencing (DBS) techniques. Using Roche (GSJunior) next generation sequencing technology, we analyzed the maternally imprinted MEST promoter and the paternally imprinted MEG3 intergenic (IG) differentially methylated region (DMR) in fetal cord blood, adult blood, and visceral adipose tissue. Epimutations were defined as paternal or maternal alleles with >50% aberrantly (de)methylated CpG sites, showing the wrong methylation imprint. The epimutation rates (range 2–66%) of the paternal MEST and the maternal MEG3 IG DMR allele, which should be completely unmethylated, were significantly higher than those (0–15%) of the maternal MEST and paternal MEG3 alleles, which are expected to be fully methylated. This hypermethylation of the non-imprinted allele (HNA) was independent of parental origin. Very low epimutation rates in sperm suggest that HNA occurred after fertilization. DBS with Illumina (MiSeq) technology confirmed HNA for the MEST promoter and the MEG3 IG DMR, and to a lesser extent, for the paternally imprinted secondary MEG3 promoter and the maternally imprinted PEG3 promoter. HNA leads to biallelic methylation of imprinted genes in a considerable proportion of normal body cells (somatic mosaicism) and is highly variable between individuals. We propose that during development and differentiation maintenance of differential methylation at most imprinting control regions may become to some extent redundant. The accumulation of stochastic and environmentally-induced methylation errors on the non-imprinted allele may increase epigenetic diversity between cells and individuals.

Overexpression of microRNAs from the Gtl2-Rian locus contributes to postnatal death in mice.

The Dlk1-Dio3 imprinted domain functions in embryonic development but the roles of noncoding RNAs expressed from this domain remain unclear. We addressed this question by generating transgenic (TG) mice harbouring a BAC carrying IG-DMR (intergenic-differentially methylated region), Gtl2-DMR, Gtl2, Rtl1/Rtl1as, and part of Rian. High postnatal lethality (>85%) of the BAC-TG pups was observed in the maternally transmitted individuals (MAT-TG), but not following paternal transmission (PAT-TG). The DNA methylation status of IG-DMR and Gtl2-DMR in the BAC-allele was paternally imprinted similar to the genomic allele. The mRNA-Seq and miRNA-Seq analysis revealed marked expression changes in the MAT-TG, with 1,500 upregulated and 2,131 downregulated genes. The long noncoding RNAs and 12 miRNAs containing the BAC locus were markedly enhanced in the MAT-TG. We identified the 24 target genes of the overexpressed miRNAs and confirmed the downregulation in the MAT-TG. Notably, overexpression of mir770, mir493, and mir665 from Gtl2 in the MAT-TG embryos led to decreased expression of the 3 target genes, Col5a1, Pcgf2, and Clip2. Our results suggest that decreased expression of the 3 target genes concomitant with overexpression of the miRNAs within Gtl2 may be involved in the postnatal death in the MAT-TG. Because this imprinted domain is well conserved between mice and humans, the results of genetic and molecular analysis in mice hold important implications for related human disorders such as Temple syndrome.

Deletion of conserved sequences in IG-DMR at Dlk1-Gtl2 locus suggests their involvement in expression of paternally expressed genes in mice.

Expression regulation of the Dlk1-Dio3 imprinted domain by the intergenic differentially methylated region (IG-DMR) is essential for normal embryonic development in mammals. In this study, we investigated conserved IG-DMR genomic sequences in eutherians to elucidate their role in genomic imprinting of the Dlk1-Dio3 domain. Using a comparative genomics approach, we identified three highly conserved sequences in IG-DMR. To elucidate the functions of these sequences in vivo, we generated mutant mice lacking each of the identified highly conserved sequences using the CRISPR/Cas9 system. Although mutant mice did not exhibit the gross phenotype, deletions of the conserved sequences altered the expression levels of paternally expressed imprinted genes in the mutant embryos without skewing imprinting status. These results suggest that the conserved sequences in IG-DMR are involved in the expression regulation of some of the imprinted genes in the Dlk1-Dio3 domain.

A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron.

Genomic imprinting is an epigenetic regulation that leads to gene expression in a parent-of-origin specific manner. AFF3, the central component of the Super Elongation Complex-like 3 (SEC-L3), is enriched at both the intergenic-differentially methylated region (IG-DMR) and the Meg3 enhancer within the imprinted Dlk1-Dio3 locus to regulate the allele-specific gene expression in this locus. The localization of AFF3 to IG-DMR requires ZFP57. However, how AFF3 functions at the Meg3 enhancer in maintaining allele-specific gene expression remains unclear. Here, we demonstrate that AFF3 is associated with the Krüppel-like zinc finger protein ZFP281 in mouse embryonic stem (ES) cells. ZFP281 recruits AFF3 to the Meg3 enhancer within the imprinted Dlk1-Dio3 locus, thus regulating the allele-specific expression of the Meg3 polycistron. Our genome-wide analyses further identify ZFP281 as a critical factor generally associating with AFF3 at enhancers and functioning together with AFF3 in regulating the expression of a subset of genes. Our study suggests that different zinc finger proteins can recruit AFF3 to different regulatory elements and differentially regulate the function of AFF3 in a context-dependent manner.

Parent-of-Origin DNA Methylation Dynamics during Mouse Development

Parent-specific differentially methylated regions (DMRs) are established during gametogenesis and regulate parent-specific expression of imprinted genes. Monoallelic expression of imprinted genes is essential for development, suggesting that imprints are faithfully maintained in embryos and adults. Totest this hypothesis, we targeted a reporter for genomic methylation to the imprinted Dlk1-Dio3 intergenic DMR (IG-DMR) to assess the methylation of both parental alleles at single-cell resolution. Biallelic gain or loss of IG-DMR methylation occurred in a small fraction of mouse embryonic stem cells, significantly affecting developmental potency. Mice carrying the reporter in either parental allele showed striking parent-specific changes in IG-DMR methylation, causing substantial and consistent tissue- and cell-type-dependent signatures in embryos and postnatal animals. Furthermore, dynamics in DNA methylation persisted during adult neurogenesis, resulting in inter-individual diversity. This substantial cell-cell DNA methylation heterogeneity implies that dynamic DNA methylation variations in the adult may be of functional importance.

Type 3 iodothyronine deiodinase in neonatal goats: molecular cloning, expression, localization, and methylation signature.

Type 3 iodothyronine deiodinase (DIO3) is an important enzyme in the metabolism of thyroid hormones. It plays critical roles in fetal development and neonatal growth and is especially important for brain development in mammals. In the present study, we profiled the expression pattern and methylation signature of the DIO3 gene in goats. The complete coding sequence of caprine DIO3 encoded a protein of 301 amino acids and harbored an internal selenocysteine-encoding TGA codon. The DIO3 messenger RNA (mRNA) was predominantly expressed in the neonatal goat liver (P < 0.01), while expression in other tissues was quite low, with the lowest levels in the lung. In in situ hybridization, the DIO3 mRNA was predominantly localized in the liver and the lowest content was detected in the lung. The DIO3 transcript was widely localized in neurons and the neuropil. Methylation profiling of the DIO3 CpG island showed a significant difference between the 5' region (CpGs_1∼24) and the 3' region (CpG_25∼51) of the coding region. Furthermore, no significant difference in methylation status was observed among the six tested tissues with levels in the range of 29.11-33.12%. The CpG islands in the intergenic-differentially methylated region (IG-DMR) showed significantly different methylated levels among tissues, and the highest methylated level was observed in lung (CpG island 1, 69.34%) and longissimus dorsi (LD) (CpG island 2, 52.62%) tissues. Our study lays a foundation for understanding DIO3 function and the diseases caused by altered methylation profiles of the DIO3 gene.

Abstract A1-24: DNA methylation sequencing of prostate tumors: Possible molecular mechanisms for cancer aggressiveness

Abstract A major challenge in the clinical management of prostate cancer is the inability to definitively diagnose indolent versus aggressive cases. Contributing to this challenge is a lack of basic science understanding of the molecular basis behind aggressiveness subtypes in prostate cancer. DNA methylation is the epigenetic addition of a methyl group to the DNA base cytosine and has been found to regulate cell proliferation and environmental adaptation. We hypothesized that DNA methylation changes are a mechanism by which an aggressive cancer attains phenotypes that distinguish it from indolent cases via disruption of regulatory networks. This hypothesis was tested by comparing DNA methylation between benign prostate and both low grade (Gleason score 6) and high grade (Gleason score 8 to 10) groups. Methylome-wide next generation sequencing was performed on formalin-fixed paraffin embedded (FFPE) samples from radical prostatectomy cases using MBD-isolated genome sequencing (MiGS). Global clustering analyses showed some separation between the three groups, but also indicated some heterogeneity. Specific differentially methylated regions (DMRs) were detected between the three groups. The two most prevalent were hypermethylation specific to high grade (491 DMRs at 4% false discovery rate) and hypermethylation shared between both cancer subtypes (1334 DMRs at 1% false discovery rate). Statistical and computational analysis of this data set was non-trivial and the software developed to perform the analysis has been made available. High grade specific DMRs are abundant in intergenic and gene body contexts, and occur less frequently in promoters and CpG islands than DMRs shared between both subtypes. Intergenic DMRs are enriched for putative functional elements (ChIP-seq peaks and DNaseI hypersensitive sites from the ENCODE project) suggesting that these changes may have functional impacts as local or regional enhancers regulating multiple genes. To analyze this in more detail, DNA-protein binding sites for specific factors were compared between 5 spatial contexts. Differential patterns of enrichment were observed for regulatory binding factors between promoters, intergenic regions, and gene 3' ends. A de novo motif search revealed 8 frequent sequence motifs in intergenic DMRs that are not similar to any known binding motifs, indicating possible binding sites for as yet unknown factors. For DMRs that are proximal to genes, an interaction network based on curated pathway data was built using the GeneMANIA Cytoscape plugin. Gene ontology enrichment was observed for “positive regulation of cell motility” (Q-value=1.4e-07, 22 of 296 genes in term) and “regulatory region DNA binding” (Q-value=6.7e-07, 18 of 255 genes in term) within this network. Interestingly, each GO term appears as a distinct cluster on the interaction network. While the specific regulatory action of these DMRs and exact target genes in aggressive cancer remains to be determined experimentally, this work has established the presence of focal hypermethylation that distinguishes indolent and aggressive prostate cancer. The high frequency of intergenic but functionally enriched genomic contexts and functional enrichment of these DMRs at genic sites for transcription factors suggests that they regulate aggressiveness in a complex manner through secondary and tertiary effects. This study provides a first glimpse at molecular differences between indolent and aggressive prostate and future work may lead to the development of biomarkers and treatments based on this knowledge. Citation Format: Jeffrey M. Bhasin, Lars Matkin, Margaret G. Taylor, Byron H. Lee, Cristina Magi-Galluzi, Eric A. Klein, Bo Hu, Yaomin Xu, Angela H. Ting. DNA methylation sequencing of prostate tumors: Possible molecular mechanisms for cancer aggressiveness. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-24.

Kagami-Ogata syndrome: a clinically recognizable upd(14)pat and related disorder affecting the chromosome 14q32.2 imprinted region.

Human chromosome 14q32.2 carries paternally expressed genes including DLK1 and RTL1, and maternally expressed genes including MEG3 and RTL1as, along with the germline-derived DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived MEG3-DMR. Consistent with this, paternal uniparental disomy 14 (upd(14)pat), and epimutations (hypermethylations) and microdeletions affecting the IG-DMR and/or the MEG3-DMR of maternal origin, result in a unique phenotype associated with characteristic face, a small bell-shaped thorax with coat-hanger appearance of the ribs, abdominal wall defects, placentomegaly and polyhydramnios. Recently, the name ‘Kagami–Ogata syndrome' (KOS) has been approved for this clinically recognizable disorder. Here, we review the current knowledge about KOS. Important findings include the following: (1) the facial ‘gestalt' and the increased coat-hanger angle constitute pathognomonic features from infancy through childhood/puberty; (2) the unmethylated IG-DMR and MEG3-DMR of maternal origin function as the imprinting control centers in the placenta and body respectively, with a hierarchical interaction regulated by the IG-DMR for the methylation pattern of the MEG3-DMR in the body; (3) RTL1 expression level becomes ~2.5 times increased in the absence of functional RTL1as-encoded microRNAs that act as a trans-acting repressor for RTL1; (4) excessive RTL1 expression and absent MEG expression constitute the primary underlying factor for the phenotypic development; and (5) upd(14)pat accounts for approximately two-thirds of KOS patients, and epimutations and microdeletions are identified with a similar frequency. Furthermore, we refer to diagnostic and therapeutic implications.

Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

PRC2 Is Required to Maintain Expression of the Maternal Gtl2-Rian-Mirg Locus by Preventing De Novo DNA Methylation in Mouse Embryonic Stem Cells.

Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency.

DNA methylation dynamics of a maternally methylated DMR in the mouse Dlk1–Dio3 domain

The mouse delta-like homolog 1 and type III iodothyronine deiodinase (Dlk1–Dio3) imprinted domain contains three known paternally methylated differentially methylated regions (DMRs): intergenic DMR (IG-DMR), maternally expressed 3-DMR (Gtl2-DMR), and Dlk1-DMR. Here, we report the first maternally methylated DMR, CpG island 2 (CGI-2), is located approximately 800bp downstream of miR-1188. CGI-2 is highly methylated in sperm and oocytes, de-methylated in pre-implantation embryos, and differentially re-methylated during post-implantation development. CGI-2, similarly to Gtl2-DMR and Dlk1-DMR, acquires differential methylation prior to embryonic day 7.5 (E7.5). Both H3K4me3 and H3K9me3 histone modifications are enriched at CGI-2. Furthermore, CCCTC-binding factor (CTCF) binds to both alleles of CGI-2 in vivo. These results contribute to the investigation of imprinting regulation in this domain.

Epimutations of the IG-DMR and the MEG3-DMR at the 14q32.2 imprinted region in two patients with Silver-Russell Syndrome-compatible phenotype.

Maternal uniparental disomy 14 (UPD(14)mat) and related (epi)genetic aberrations affecting the 14q32.2 imprinted region result in a clinically recognizable condition which is recently referred to as Temple Syndrome (TS). Phenotypic features in TS include pre- and post-natal growth failure, prominent forehead, and feeding difficulties that are also found in Silver–Russell Syndrome (SRS). Thus, we examined the relevance of UPD(14)mat and related (epi)genetic aberrations to the development of SRS in 85 Japanese patients who satisfied the SRS diagnostic criteria proposed by Netchine et al and had neither epimutation of the H19-DMR nor maternal uniparental disomy 7. Pyrosequencing identified hypomethylation of the DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the MEG3-DMR in two cases. In both cases, microsatellite analysis showed biparental transmission of the homologs of chromosome 14, with no evidence for somatic mosaicism with full or segmental maternal isodisomy involving the imprinted region. FISH and array comparative genomic hybridization revealed neither deletion of the two DMRs nor discernible copy number alteration in the 14q32.2 imprinted region. Methylation patterns were apparently normal in other six disease-associated DMRs. In addition, a thorough literature review revealed a considerable degree of phenotypic overlap between SRS and TS, although body asymmetry was apparently characteristic of SRS. The results indicate the occurrence of epimutation affecting the IG-DMR and the MEG3-DMR in the two cases, and imply that UPD(14)mat and related (epi)genetic aberrations constitute a rare but important underlying factor for SRS.

Studies of Molecular Mechanisms Associated With Increased Deiodinase 3 Expression in a Case of Consumptive Hypothyroidism

Consumptive hypothyroidism (CH) is a rare form of hypothyroidism due to increased catabolic activity of type 3 iodothyronine deiodinase (DIO3) that can occur in large tumors. PATIENTs with CH typically present with markedly increased requirements for exogenous thyroid hormone and resolution after removal of the source of ectopic DIO3. DIO3 is encoded by DIO3, an imprinted gene expressed on the paternal allele that is located in a DIO3/delta-like 1 homolog (DLK1) gene locus regulated by a common control region, intergenic differentially methylated region (IGDMR). Because DIO3 is an imprinted gene, loss of imprinting at the IGDMR is thought to play a role in its increased expression; however, the molecular mechanism for DIO3 in CH currently is not known. The aim of the study was to determine the molecular mechanism for CH in an adult patient. The study was conducted in the Department of Endocrinology of a tertiary care center in Singapore. We report the case of an adult Asian female patient with a large intrathoracic fibrous tumor and severe hypothyroidism that resolved after tumor resection. The patient's tumor expressed increased levels of DIO3 and DLK1 mRNA and protein levels. Methylation-specific PCR of the IGDMR showed similar hypomethylation in placenta, thyroid, leukocytes, and tumor. Western blotting showed activation of sonic hedgehog (SHH) and MAPK signaling pathways that can increase DIO3 and DLK1 expression. Loss of imprinting did not account for overexpression of DIO3 in the patient's tumor. Instead SHH and MAPK/ERK pathway activation was associated with systemic thyroid hormone catabolism and growth of the tumor. These findings raise the possibility that other tumors that have increased SHH and MAPK/ERK signaling also may have intratumor or systemic effects on thyroid hormone function.