IGF2/H19 Imprinting Control Region Research Articles

CSIG-33. BCOR LOSS PROMOTES RETINOBLASTOMA GROWTH AND DISSEMINATION BY MODULATING INSULIN-LIKE GROWTH FACTOR 2

Abstract While loss of RB function is the most common genetic alteration in the pediatric embryonal eye tumor retinoblastoma, mutations leading to BCL-6 corepressor (BCOR) loss of function are the second most common alteration. BCOR mutations are found in more aggressive tumors, and are associated with metastasis and poor prognosis. We investigated the effects of BCOR loss or overexpression in retinoblastoma cells in order to better understand its functional role, and to determine if it represents a new therapeutic target in these tumors. Six established retinoblastoma cell lines were examined, and BCOR protein was most highly expressed in WERI-RB1, but absent in Y79 and two other lines. BCOR knock down in WERI-RB1 cells using shRNA caused markedly increased migration and proliferation. BCOR was also knocked out in these cells using CRSPR, resulting in more than three-fold increased size of intraocular murine xenografts and extensive extraocular extension of tumors. In terms of mechanism, BCOR loss of function induces IGF2 transcription, resulting in an activation of IGF1R and the ERK signaling pathway. In contrast, overexpressing BCOR in Y79 retinoblastoma cells with no baseline protein had opposite effects on IGF2. Targeting IGF2/IGF1R signaling pharmacologically using Lincitinib inhibited proliferation and migration in BCOR-negative retinoblastoma cells. Increased IGF2 transcription was regulated at least in part by hyper methylation of CCCTC-binding factor (CTCF)-binding regions in the IGF2/H19 imprinting control region (ICR), suggesting that BCOR knock down promotes increased methylation at this site. In summary, our studies revealed that BCOR loss promotes the growth and invasion of retinoblastoma cells in vitro and in vivo, at least in part via modulating IGF2 transcription, suggesting new potential therapeutic targets for these aggressive childhood tumors of the eye.

Genetic potential for residual feed intake and diet fed during early- to mid-gestation influences post-natal DNA methylation of imprinted genes in muscle and liver tissues in beef cattle.

Discovery of epigenetic modifications associated with feed efficiency or other economically important traits would increase our understanding of the molecular mechanisms underlying these traits. In combination with known genetic markers, this would provide opportunity to improve genomic selection accuracy in cattle breeding programs. It would also allow cattle to be managed to improve favorable gene expression. The objective of this study was to identify variation in DNA methylation between beef cattle of differential pre-natal nutrition and divergent genetic potential for residual feed intake (RFI). Purebred Angus offspring with the genetic potential for either high (HRFI) or low (LRFI) RFI were prenatally exposed to either a restricted maternal diet of 0.5 kg/d average daily gain (ADG) or a moderate maternal diet of 0.7 kg/d ADG from 30 to 150 d of gestation. We performed DNA methylation analysis of differentially methylated regions (DMR) of imprinted genes (Insulin-like growth factor 2 (IGF2) DMR2, IGF2/H19 imprinting control region (ICR) and IGF2 receptor (IGF2R) DMR2) using post-natal samples of longissimus dorsi (LD) muscle taken from male and female calves at birth and weaning, and of LD muscle, semimembranosus (SM) muscle, and liver samples collected from steers at slaughter (17 months of age). Interestingly, for all three DMR investigated in liver, LRFI steers had higher levels of methylation than HRFI steers. In LD muscle, IGF2/H19 ICR methylation differences for heifers at birth were due to pre-natal diet, while for steers at birth they were mostly the result of genetic potential for RFI with LRFI steers again having higher levels of methylation than HRFI steers. While results from repeated measures analysis of DNA methylation in steers grouped by RFI revealed few differences, in steers grouped by diet, we found higher methylation levels of IGF2 DMR2 and IGF2R DMR2 in LD muscle of restricted diet steers at weaning and slaughter than at birth, as well as increased methylation in LD muscle of restricted diet steers compared with moderate diet steers at weaning and/or slaughter. Our results suggest that differential pre-natal nutrition, and divergent genetic potential for RFI, induces tissue- and sex-specific alterations in post-natal IGF2 and IGF2R methylation patterns and that these patterns can vary with age in Angus beef cattle.

Paternal obesity impairs hepatic gluconeogenesis of offspring by altering Igf2/H19 DNA methylation

Over the past four decades, the global prevalence of obesity has increased rapidly in all age ranges. Emerging evidence suggests that paternal lifestyle and environmental exposure have a crucial role in the health of offspring. Therefore, the current study investigated the impact of paternal obesity on the metabolic profile of offspring in a male mouse model of obesity. Female offspring of obese fathers fed a high-fat diet (HFD) (60% kcal fat) showed hyperglycemia because of enhanced gluconeogenesis and elevated expression of phosphoenolpyruvate carboxykinase (PEPCK), which is a key enzyme involved in the regulation of gluconeogenesis. Methylation of the Igf2/H19 imprinting control region (ICR) was dysregulated in the liver of offspring, and the sperm, of HFD fathers, suggesting that epigenetic changes in germ cells contribute to this father-offspring transmission. In addition, we explored whether H19 might regulate hepatic gluconeogenesis. Our results showed that overexpression of H19 in Hepa1-6 cells enhanced the expression of PEPCK and gluconeogenesis by promoting nuclear retention of forkhead box O1 (FOXO1), which is involved in the transcriptional regulation of Pepck. Thus, the current study suggests that paternal exposure to HFD impairs the gluconeogenesis of offspring via altered Igf2/H19 DNA methylation.

The effect of laser-assisted hatching on the methylation and expression pattern of imprinted gene IGF2/H19 in mouse blastocysts and offspring

This study aimed to determine the effects of drilling and thinning treatment of laser-assisted hatching on the expression and methylation of imprinted gene IGF2/H19 in embryos and offspring. The prehatching blastocysts with treatment of drilling or thinning, or control prehatching blastocysts, were transplanted in surrogate uteri. The DNA methylation of IGF2/H19 imprinting control region (ICR) and the expression of IGF2 and H19 were respectively evaluated using bisulfite conversion-mediated sequencing and real-time PCR. The drilling group showed a significant increase in the development rate of hatched blastocysts in comparison with the control and thinning group. DNA methylation level of IGF2/H19 ICR of hatched blastocysts in the thinning group was 27.33% in comparison with the 38.67% and 36% observed in the control and drilling group. The thinning treatment increased the DNA methylation level of IGF2/H19 ICR in the placenta in comparison with the control and drilling group. The drilling and thinning treatment decreased the expression level of H19 mRNA in prehatching and hatched blastocysts as well as placenta, while a significant increase in the expression level of H19 mRNA of offspring was observed in the thinning group. The thinning treatment increased the expression level of IGF2 mRNA of prehatching blastocysts and offspring and a significant decrease in placenta, while the drilling treatment resulted in a significant increase in the expression level of IGF2 mRNA of hatched blastocysts and placenta. These observations suggested that drilling used for hatching of in vitro cultured mouseblastocysts may improvethe production of offspring.

Grandmaternal betaine supplementation enhances hepatic IGF2 expression in F2 rat offspring through modification of promoter DNA methylation.

We reported previously that maternal betaine promotes hepatic insulin-like growth factor (IGF2) expression in F1 offspring rats through hypermethylation of the IGF2/H19 imprinting control region (ICR). It remains unknown whether this acquired trait can be transmitted to the F2 generation. This study aimed to determine whether dietary betaine supplementation to grand dams affects the hepatic IGF2 expression in F2 rat offspring and how it is related to alterations in DNA methylation. F2 rat offspring derived from grand dams fed basal or betaine-supplemented diet (10 g kg-1 ) were examined at weaning. Serum IGF2 concentration was measured with enzyme-linked immunosorbent assay (ELISA). Hepatic expression of IGF2, together with other proliferation and apoptosis markers, was determined by using quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry. The methylation status of the IGF2/H19 ICR and the promoters of IGF2 gene were detected by methylated DNA immunoprecipitation quantitative polymerase chain reaction (MeDIP-qPCR). The maternal betaine-induced up-regulation of hepatic IGF2 expression in F1 rat offspring was transmitted to the F2 generation. The F2 rats from the betaine group demonstrated enhanced hepatic IGF2 expression at both mRNA and protein levels, in association with higher serum IGF2 concentration. No alterations were observed in the ICR methylation of the IGF2/H19 locus, and hypomethylation was detected in promoters of IGF2 gene in betaine group. These results indicate that maternal betaine enhances hepatic IGF2 expression in F2 rat offspring through modification of DNA methylation on IGF2 promoters. © 2019 Society of Chemical Industry.

Early prenatal alcohol exposure alters imprinted gene expression in placenta and embryo in a mouse model.

Prenatal alcohol exposure (PAE) can harm the embryonic development and cause life-long consequences in offspring’s health. To clarify the molecular mechanisms of PAE we have used a mouse model of early alcohol exposure, which is based on maternal ad libitum ingestion of 10% (v/v) ethanol for the first eight days of gestation (GD 0.5–8.5). Owing to the detected postnatal growth-restricted phenotype in the offspring of this mouse model and both prenatal and postnatal growth restriction in alcohol-exposed humans, we focused on imprinted genes Insulin-like growth factor 2 (Igf2), H19, Small Nuclear Ribonucleoprotein Polypeptide N (Snrpn) and Paternally expressed gene 3 (Peg3), which all are known to be involved in embryonic and placental growth and development. We studied the effects of alcohol on DNA methylation level at the Igf2/H19 imprinting control region (ICR), Igf2 differentially methylated region 1, Snrpn ICR and Peg3 ICR in 9.5 embryonic days old (E9.5) embryos and placentas by using MassARRAY EpiTYPER. To determine alcohol-induced alterations globally, we also examined methylation in long interspersed nuclear elements (Line-1) in E9.5 placentas. We did not observe any significant alcohol-induced changes in DNA methylation levels. We explored effects of PAE on gene expression of E9.5 embryos as well as E9.5 and E16.5 placentas by using quantitative PCR. The expression of growth promoter gene Igf2 was decreased in the alcohol-exposed E9.5 and E16.5 placentas. The expression of negative growth controller H19 was significantly increased in the alcohol-exposed E9.5 embryos compared to controls, and conversely, a trend of decreased expression in alcohol-exposed E9.5 and E16.5 placentas were observed. Furthermore, increased Snrpn expression in alcohol-exposed E9.5 embryos was also detected. Our study indicates that albeit no alterations in the DNA methylation levels of studied sequences were detected by EpiTYPER, early PAE can affect the expression of imprinted genes in both developing embryo and placenta.

Essential Role of DNA Methyltransferase 1-mediated Transcription of Insulin-like Growth Factor 2 in Resistance to Histone Deacetylase Inhibitors.

Purpose: Histone deacetylase inhibitors (HDI) are promising anticancer therapies; however, drug resistance limits their efficacy. Here, we investigated the molecular mechanisms underlying HDI resistance, focusing on the mechanism of HDI-mediated induction of insulin-like growth factor 2 (IGF2) based on our previous study.Experimental Design: The methylation status of CCCTC-binding factor (CTCF)-binding sites in the IGF2/H19 imprinting control region (ICR) were determined by methylation-specific PCR and bisulfite sequencing. The effectiveness of single or combinatorial blockade of DNA methyltransferase 1 (DNMT1) and histone deacetylase (HDAC) was evaluated using cell viability assay and patient-derived tumor xenograft (PDX) model.Results: HDAC inhibition by vorinostat increased acetylated STAT3 (K685), resulting in transcriptional upregulation of DNMT1 DNMT1-mediated hypermethylation of CTCF-binding sites in the IGF2/H19 ICR decreased CTCF insulator activity, leading to a transcriptional upregulation of IGF2 and activation of the insulin-like growth factor 1 receptor (IGF-1R) pathway in cells with acquired or de novo vorinostat resistance. Strategies targeting DNMT1 diminished the IGF2 expression and potentiated vorinostat sensitivity in preclinical models of lung cancer with hypermethylation in the H19/IGF2 ICR. The degree of ICR hypermethylation correlated with vorinostat resistance in patient-derived lung tumors and in patients with hematologic malignancies.Conclusions: DNMT1-mediated transcriptional upregulation of IGF2 is a novel mechanism of resistance to HDIs, highlighting the role of epigenetic deregulation of IGF2 in HDI resistance and the potential value of the H19/IGF2 ICR hypermethylation and DNMT1 expression as predictive biomarkers in HDI-based anticancer therapies. Clin Cancer Res; 23(5); 1299-311. ©2016 AACR.

Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection.

The paternal-allele-specific methylation of the Igf2/H19 imprinting control region (ICR) is established during gametogenesis and maintained throughout development. To elucidate the requirement of the germline passage in the maintenance of the imprinting methylation, we established a system introducing a methylated or unmethylated ICR-containing DNA fragment (ICR-F) into the paternal or maternal genome by microinjecting into the paternal or maternal pronucleus of fertilized eggs, and traced the methylation pattern in the ICR-F. When the ICR-F was injected in a methylated form, it was demethylated approximately to half degree at blastocyst stage but was almost completely remethylated at 3 weeks of age. In the case of the unmethylated form, the ICR-F remained unmethylated at the blastocyst stage, but was almost half-methylated at 3 weeks of age. Interestingly, the paternally injected ICR-F was highly methylated compared with maternally injected ICR-F at 3 weeks of age, partially mimicking the endogenous methylation pattern. Moreover, introduction of mutations in the CTCF (CCCTC binding factor) binding sites of the ICR-F, which are known to be important for the maintenance of hypomethylated maternal ICR, induced hypermethylation of the mutated ICR-F in both paternal and maternal pronuclear injected 3-week-old mice. Our results suggest the presence of a protection-against-methylation activity of the CTCF binding site in establishing the preferential paternal methylation during post-fertilization development and the importance of germline passage in the maintenance of the parental specific methylation at H19 ICR.

The effects of maternal anxiety during pregnancy on IGF2/H19 methylation in cord blood.

Compelling evidence suggests that maternal mental health in pregnancy can influence fetal development. The imprinted genes, insulin-like growth factor 2 (IGF2) and H19, are involved in fetal growth and each is regulated by DNA methylation. This study aimed to determine the association between maternal mental well-being during pregnancy and differentially methylated regions (DMRs) of IGF2 (DMR0) and the IGF2/H19 imprinting control region (ICR) in newborn offspring. Maternal depression, anxiety and perceived stress were assessed at 28 weeks of pregnancy in the Barwon Infant Study (n=576). DNA methylation was measured in purified cord blood mononuclear cells using the Sequenom MassArray Platform. Maternal anxiety was associated with a decrease in average ICR methylation (Δ=−2.23% 95% CI=−3.68 to −0.77%), and across all six of the individual CpG units in anxious compared with non-anxious groups. Birth weight and sex modified the association between prenatal anxiety and infant methylation. When stratified into lower (⩽3530 g) and higher (>3530 g) birth weight groups using the median birth weight, there was a stronger association between anxiety and ICR methylation in the lower birth weight group (Δ=−3.89% 95% CI=−6.06 to −1.72%), with no association in the higher birth weight group. When stratified by infant sex, there was a stronger association in female infants (Δ=−3.70% 95% CI=−5.90 to −1.51%) and no association in males. All the linear regression models were adjusted for maternal age, smoking and folate intake. These findings show that maternal anxiety in pregnancy is associated with decreased IGF2/H19 ICR DNA methylation in progeny at birth, particularly in female, low birth weight neonates. ICR methylation may help link poor maternal mental health and adverse birth outcomes, but further investigation is needed.

Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

BackgroundSeveral recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.ResultsWe describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.ConclusionTM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1236-7) contains supplementary material, which is available to authorized users.

Aberrant DNA methylation at Igf2–H19 imprinting control region in spermatozoa upon neonatal exposure to bisphenol A and its association with post implantation loss

Bisphenol A (BPA) is an estrogenic compound commonly used in manufacture of various consumer products. Earlier studies from our group have demonstrated that neonatal exposure of male rats to BPA causes decrease in sperm count and motility, increase in post implantation loss, ultimately leading to subfertility during adulthood. One of the factors contributing for post implantation loss is altered methylation pattern of imprinted genes. The present study was undertaken to investigate the molecular effects of neonatal exposure of male rats to BPA (2.4μg/pup) (F0) on the methylation of H19 imprinting control region (ICR) in resorbed embryo (F1) and compared with spermatozoa of their respective sires (F0). We observed a significant down regulation in the transcript expression of Igf2 and H19 genes in BPA resorbed embryo (F1) as compared to control viable embryo. A significant hypomethylation was observed at the H19 ICR in the spermatozoa as well as in resorbed embryo sired by rats exposed neonatally to BPA. These results indicated that the aberrant methylation at ICR in spermatozoa was inherited by embryo which causes perturbation in the expression of Igf2 and H19, ultimately leading to post implantation loss. This could be one of the possible mechanisms of BPA induced adverse epigenetic effects on male fertility.

DNA methylation of the IGF2/H19 imprinting control region and adiposity distribution in young adults

BackgroundThe insulin-like growth factor 2 (IGF2) and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR) has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity.DNA methylation in peripheral blood (n = 315) at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs), analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC) at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression.ResultsThe principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units) positively correlated with skin fold thickness (at four CpG units) (P-values between 0.04 to 0.001) and subcutaneous adiposity (P = 0.023) at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC.ConclusionsAs greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we hypothesize that the HC may be a more sensitive marker of early life programming of the IGF axis and of fetal physiology than birth size. To verify this, investigations of the dynamics of IGF2/H19 methylation and expression from birth to adolescence are required.

Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith–Wiedemann syndrome and Silver–Russell syndrome

Hyper- and hypomethylation at the IGF2-H19 imprinting control region (ICR) result in reciprocal changes in IGF2-H19 expression and the two contrasting growth disorders, Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS). DNA methylation of the ICR controls the reciprocal imprinting of IGF2 and H19 by preventing the binding of the insulator protein, CTCF. We here show that local changes in histone modifications and CTCF–cohesin binding at the ICR in BWS and SRS together with DNA methylation correlate with the higher order chromatin structure at the locus. In lymphoblastoid cells from control individuals, we found the repressive histone H3K9me3 and H4K20me3 marks associated with the methylated paternal ICR allele and the bivalent H3K4me2/H3K27me3 mark together with H3K9ac and CTCF–cohesin associated with the non-methylated maternal allele. In patient-derived cell lines, the mat/pat asymmetric distribution of these epigenetic marks was lost with H3K9me3 and H4K20me3 becoming biallelic in the BWS and H3K4me2, H3K27me3 and H3K9ac together with CTCF–cohesin becoming biallelic in the SRS. We further show that in BWS and SRS cells, there is opposing chromatin looping conformation mediated by CTCF–cohesin binding sites surrounding the locus. In normal cells, lack of CTCF–cohesin binding at the paternal ICR is associated with monoallelic interaction between two CTCF sites flanking the locus. CTCF–cohesin binding at the maternal ICR blocks this interaction by associating with the CTCF site downstream of the enhancers. The two alternative chromatin conformations are differently favoured in BWS and SRS likely predisposing the locus to the activation of IGF2 or H19, respectively.

The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

Background BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions.Methodology/Principal FindingsHere we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates.ConclusionsThe extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation.

MicroRNA signature in testes-derived male germ-line stem cells

The testis-derived male germ-line stem (GS) cell, the in vitro counterpart of spermatogonial stem cell (SSC), can initiate donor-derived spermatogenesis in recipient testes and therefore, has been viewed as a future therapeutic modality for treatment of male infertility in azoospermic patients and in cancer patients who are expecting chemotherapy. Upon extended in vitro culture, GS cells also generate a second cell type called multipotent adult germ-line stem (maGS) cell which, upon testicular transplantation, produces teratoma instead of initiating spermatogenesis. Here, we show that expressions of both Let-7a and Let-7d were consistently higher while that of miR-294 (embryonic stem cell-cycle-regulating miRNA; ESCC) was lower in GS cells than in maGS cells. Furthermore, among several putative targets of Let-7 identified by in silico bioinformatics, expressions of Igf2 and H19 mRNA targets significantly differed between GS and maGS cells. However, although the CTCF binding factor (a component of DNA methylation machinery at Igf2-H19 cluster) was also a putative target for Let-7, the difference in expressions of Igf2 and H19 between GS and maGS cells was not mediated through a change in DNA methylation. Both GS and maGS cells maintained androgenetic imprinting at the Igf2-H19 imprinting control region and Peg1 differentially methylated region. In conclusion, our study suggests that high Let-7 expression may be a unique property of GS cells and expressions of Let-7 and ESCC miRNAs may serve as miRNA signatures to distinguish them from maGS cells during clinical transplantation, to avoid the likelihood of teratoma formation due to maGS cells generated during extended in vitro culture of GS cells.

Mutational analysis of the poly(ADP-ribosyl)ation sites of the transcription factor CTCF provides an insight into the mechanism of its regulation by poly(ADP-ribosyl)ation.

Poly(ADP-ribosyl)ation of the conserved multifunctional transcription factor CTCF was previously identified as important to maintain CTCF insulator and chromatin barrier functions. However, the molecular mechanism of this regulation and also the necessity of this modification for other CTCF functions remain unknown. In this study, we identified potential sites of poly(ADP-ribosyl)ation within the N-terminal domain of CTCF and generated a mutant deficient in poly(ADP-ribosyl)ation. Using this CTCF mutant, we demonstrated the requirement of poly(ADP-ribosyl)ation for optimal CTCF function in transcriptional activation of the p19ARF promoter and inhibition of cell proliferation. By using a newly generated isogenic insulator reporter cell line, the CTCF insulator function at the mouse Igf2-H19 imprinting control region (ICR) was found to be compromised by the CTCF mutation. The association and simultaneous presence of PARP-1 and CTCF at the ICR, confirmed by single and serial chromatin immunoprecipitation assays, were found to be independent of CTCF poly(ADP-ribosyl)ation. These results suggest a model of CTCF regulation by poly(ADP-ribosyl)ation whereby CTCF and PARP-1 form functional complexes at sites along the DNA, producing a dynamic reversible modification of CTCF. By using bioinformatics tools, numerous sites of CTCF and PARP-1 colocalization were demonstrated, suggesting that such regulation of CTCF may take place at the genome level.

Postnatal Survival of Mice with Maternal Duplication of Distal Chromosome 7 Induced by a Igf2/H19 Imprinting Control Region Lacking Insulator Function

The misexpressed imprinted genes causing developmental failure of mouse parthenogenones are poorly defined. To obtain further insight, we investigated misexpressions that could cause the pronounced growth deficiency and death of fetuses with maternal duplication of distal chromosome (Chr) 7 (MatDup.dist7). Their small size could involve inactivity of Igf2, encoding a growth factor, with some contribution by over-expression of Cdkn1c, encoding a negative growth regulator. Mice lacking Igf2 expression are usually viable, and MatDup.dist7 death has been attributed to the misexpression of Cdkn1c or other imprinted genes. To examine the role of misexpressions determined by two maternal copies of the Igf2/H19 imprinting control region (ICR)—a chromatin insulator, we introduced a mutant ICR (ICRΔ) into MatDup.dist7 fetuses. This activated Igf2, with correction of H19 expression and other imprinted transcripts expected. Substantial growth enhancement and full postnatal viability was obtained, demonstrating that the aberrant MatDup.dist7 phenotype is highly dependent on the presence of two unmethylated maternal Igf2/H19 ICRs. Activation of Igf2 is likely the predominant correction that rescued growth and viability. Further experiments involved the introduction of a null allele of Cdkn1c to alleviate its over-expression. Results were not consistent with the possibility that this misexpression alone, or in combination with Igf2 inactivity, mediates MatDup.dist7 death. Rather, a network of misexpressions derived from dist7 is probably involved. Our results are consistent with the idea that reduced expression of IGF2 plays a role in the aetiology of the human imprinting-related growth-deficit disorder, Silver-Russell syndrome.

Defining Contributions of Paternally Methylated Imprinted Genes at the Igf2-H19 and Dlk1-Gtl2 Domains to Mouse Placentation by Transcriptomic Analysis

Parental genome functions in ontogeny are determined by interactions among transcripts from the maternal and paternal genomes, which contain many genes whose expression is strictly dependent on their parental origin as a result of genomic imprinting. Comprehensive recognition of the interactions between parental genomes is important for understanding genomic imprinting in mammalian development. The placenta is a key organ for exploring the biological significance of genomic imprinting. To decipher the unknown roles of paternally methylated imprinted genes on chromosomes 7 and 12 in mouse placentation, we performed a transcriptomic analysis on placentae in three types of bimaternal conceptuses that contained genomes derived from both non-growing and fully grown oocytes. Furthermore, we used the Ingenuity pathway analysis software to predict key networks and identify functions specific to paternally methylated imprinted genes regulated by the Igf2-H19 imprinting control region and Dlk1-Dio3 imprinting control region. The data suggested that dynamic conversion of the gene expression profile by restoring the expression of paternally methylated imprinted genes resulted in phenotypic improvements in bimaternal placentae. These results provide a framework to further explore the role of epigenetic modifications in paternal genome during mouse placentation.

The Igf2/H19 imprinting control region exhibits sequence-specific and cell-type-dependent DNA methylation-mediated repression

Methylation of CpGs is generally thought to repress transcription without significant influence from the sequence surrounding the methylated dinucleotides. Using the mouse Igf2/H19 imprinting control region (ICR), Igf2r differentially methylated region 2 (DMR2) and bacterial sequences, we addressed how methylation-dependent repression (MDR) from a distance varies with CpG number, density and surrounding sequence. In stably transfected F9 cells, the methylated ICR repressed expression from a CpG-free reporter plasmid more than 1000-fold compared with its unmethylated control. A segment of pBluescript, with a CpG number equal to the ICR's but with a higher density, repressed expression only 70-fold when methylated. A bacteriophage lambda fragment and the Igf2r DMR2 showed minimal MDR activity, despite having CpG numbers and densities similar to or greater than the ICR. By rearranging or deleting CpGs, we identified CpGs associated with three CTCF sites in the ICR that are necessary and sufficient for sequence-specific MDR. In contrast to F9 cells, the methylated ICR and pBS fragments exhibited only 3-fold reporter repression in Hela cells and none in Cos7. Our results show that the strength of MDR from a distance can vary a 1000-fold between different cell types and depends on the sequence surrounding the methylated CpGs, but does not necessarily increase with CpG number or density.

IGF2/H19 imprinting analysis of human germ cell tumors (GCTs) using the methylation‐sensitive single‐nucleotide primer extension method reflects the origin of GCTs in different stages of primordial germ cell development

Previous studies have demonstrated biallelic expression of the imprinted genes H19 and IGF2 and loss of DNA methylation of the SNRPN gene, indicating a common precursor cell of human germ cell tumors (GCTs), namely, the primordial germ cell (PGC). In this study, we applied the methylation-sensitive single-nucleotide primer extension (MS-SNuPE) technique to the analysis of the IGF2/H19 imprinting control region (ICR) in 55 GCTs from representative clinical and histologic subgroups. Most GCTs showed low methylation at the IGF2/H19 ICR. All 8 ovarian GCTs, 9 of 10 testicular seminomas, 7 of 10 testicular nonseminomas (all in adolescents/adults), 6 of 9 testicular yolk sac tumors (YSTs), and 12 of 14 nongonadal GCTs (all in infants/children) were hypomethylated. The highest methylation was observed in three childhood YSTs (boys) and 2 of 4 spermatocytic seminomas. The latter are derived from more advanced stages of germ-cell development. The predominantly low methylation of most of the other GCTs correlates with studies that demonstrated erasure of the methylation imprint of the IGF2/H19 ICR during embryonal PGC migration and development. These findings suggest that the IGF2/H19 methylation status in GCTs might reflect preservation of the physiologic imprinting erasure in PGCs rather than a loss of imprinting in a sense that is accepted for somatic tumors. Furthermore, this study indicates that imprinting control mechanisms other than the proposed CTCF (CCCTC binding factor) boundary model regulate IGF2 expression during this stage of PGC development as well as in GCTs derived from PGC. (c) 2005 Wiley-Liss, Inc.