A loss of insulin-like growth factor-2 imprinting is modulated by CCCTC-binding factor down-regulation at senescence in human epithelial cells

The imprinted insulin-like growth factor-2 (IGF2) gene is an auto/paracrine growth factor expressed only from the paternal allele in adult tissues. In tissues susceptible to aging-related cancers, including the prostate, a relaxation of IGF2 imprinting is found, suggesting a permissive role for epigenetic alterations in cancer development. To determine whether IGF2 imprinting is altered in cellular aging and senescence, human prostate epithelial and urothelial cells were passaged serially in culture to senescence. Allelic analyses using an IGF2 polymorphism demonstrated a complete conversion of the IGF2 imprint status from monoallelic to biallelic, in which the development of senescence was associated with a 10-fold increase in IGF2 expression. As a mechanism, a 2-fold decrease in the binding of the enhancer-blocking element CCCTC-binding factor (CTCF) within the intergenic IGF2-H19 region was found to underlie this switch to biallelic IGF2 expression in senescent cells. This decrease in CTCF binding was associated with reduced CTCF expression in senescent cells. No de novo increases in methylation at the IGF2 CTCF binding site were seen. The forced down-regulation of CTCF expression using small interfering RNA in imprinted prostate cell lines resulted in an increase in IGF2 expression and a relaxation of imprinting. Our data suggest a novel mechanism for IGF2 imprinting regulation, that is, the reduction of CTCF expression in the control of IGF2 imprinting. We also demonstrate that altered imprinting patterns contribute to changes in gene expression in aging cells.     

Genomic imprinting of IGF2, p57(KIP2) and PEG1/MEST in a marsupial, the tammar wallaby

Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.     

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.     

多功能转录因子CTCF研究进展

CTCF是一种多功能的真核转录因子,它可以抑制c—myc基因的表达,增强app基因的启动子活性,调控H19／Igf2的印记,作为鸡β-球蛋白等多个基因结构域的绝缘子成分,以及作为X染色体失活的候选蛋白等。CTCF与BORIS的交互表达和雄性生殖细胞的系统发生有关。CTCF的缺失和突变可能导致肿瘤的发生。     

Induced DNA demethylation can reshape chromatin topology at the IGF2-H19 locus

Choriocarcinomas are embryonal tumours with loss of imprinting and hypermethylation at the insulin-like growth factor 2 (IGF2)-H19 locus. The DNA methyltransferase inhibitor, 5-Aza-2′deoxycytidine (5-AzaCdR) is an approved epigenetic cancer therapy. However, it is not known to what extent 5-AzaCdR influences other epigenetic marks. In this study, we set out to determine whether 5-AzaCdR treatment can reprogram the epigenomic organization of the IGF2-H19 locus in a choriocarcinoma cancer cell line (JEG3). We found that localized DNA demethylation at the H19 imprinting control region (ICR) induced by 5-AzaCdR, reduced IGF2, increased H19 expression, increased CTCF and cohesin recruitment and changed histone modifications. Furthermore chromatin accessibility was increased locus-wide and chromatin looping topography was altered such that a CTCF site downstream of the H19 enhancers switched its association with the CTCF site upstream of the IGF2 promoters to associate with the ICR. We identified a stable chromatin looping domain, which forms independently of DNA methylation. This domain contains the IGF2 gene and is marked by a histone H3 lysine 27 trimethylation block between CTCF site upstream of the IGF2 promoters and the Centrally Conserved Domain upstream of the ICR. Together, these data provide new insights into the responsiveness of chromatin topography to DNA methylation changes.     

Recent assembly of an imprinted domain from non-imprinted components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.     

Evolution, function, and regulation of genomic imprinting in plant seed development

Genomic imprinting is an epigenetic phenomenon whereby genetically identical alleles are differentially expressed dependent on their parent-of-origin. Genomic imprinting has independently evolved in flowering plants and mammals. In both organism classes, imprinting occurs in embryo-nourishing tissues, the placenta and the endosperm, respectively, and it has been proposed that imprinted genes regulate the transfer of nutrients to the developing progeny. Many imprinted genes are located in the vicinity of DNA-methylated transposon or repeat sequences, implying that transposon insertions are associated with the evolution of imprinted loci. The antagonistic action of DNA methylation and Polycomb group-mediated histone methylation seems important for the regulation of many imprinted plant genes, whereby the position of such epigenetic modifications can determine whether a gene will be mainly expressed from either the maternally or paternally inherited alleles. Furthermore, long non-coding RNAs seem to play an as yet underappreciated role for the regulation of imprinted plant genes. Imprinted expression of a number of genes is conserved between monocots and dicots, suggesting that long-term selection can maintain imprinted expression at some loci.     

Imprint status of M6P/IGF2R and IGF2 in chickens

Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.     

Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1] and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2 (insulin-like growth factor II) and H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is maintained paternally methylated and maternally unmethylated [6] [7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8] [9] and references therein) to the H19 DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19 DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitro and in vivo.     

Evolutionary diversification of SPANX-N sperm protein gene structure and expression

The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage approximately 7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well.