Monoallelic Expression Of IGF2 Research Articles

Uncovering common pathogenic transcriptional dysregulations in Silver-Russell syndrome

Silver-Russell syndrome (SRS) is a rare developmental disorder, presenting with marked intrauterine and postnatal growth retardation. The heterogeneous associated molecular defects in approximately 40% of SRS patients are due to epigenetic changes in the imprinting control region 1 (ICR1) that regulates the monoallelic expression of IGF2 and H19 in cis. Nevertheless a significant number of SRS patients have epigenetic alterations in other imprinted regions instead, like in cases with maternal uniparental disomy for chromosome 7 (upd(7)mat). Using primary fibroblasts from SRS patients with upd(7)mat and a unique case with upd(11)mat, we can show dysregulation of imprinted genes not located on the chromosomes 7 or 11 respectively, but on other chromosomes, suggesting a functional interaction between imprinted genes throughout the genome. 4C analyses for the ICR1 have shown that this interaction is unlikely to be based on chromatin interactions, but rather due to effects of the gene products themselves. The most likely transfactors mediating the transcriptional regulation of other imprinted genes are long non-coding RNAs. To determine which of these function as transfactors for the transcriptional regulation of other imprinted genes, we performed knock-down assays and monitored the transcriptional consequences on other imprinted genes. Our preliminary analyses also show, that common dysregulation patterns for imprinted genes throughout the genome can be described in SRS patients with different epigenetic alterations, suggesting that these patterns are associated with the clinical phenotype. Funded by BMBF 01GM1114D.

Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II

Monoallelic expression of IGF2 is regulated by CCCTC binding factor (CTCF) binding to the imprinting control region (ICR) on the maternal allele, with subsequent formation of an intrachromosomal loop to the promoter region. The N-terminal domain of CTCF interacts with SUZ12, part of the polycomb repressive complex-2 (PRC2), to silence the maternal allele. We synthesized decoy CTCF proteins, fusing the CTCF deoxyribonucleic acid-binding zinc finger domain to CpG methyltransferase Sss1 or to enhanced green fluorescent protein. In normal human fibroblasts and breast cancer MCF7 cell lines, the CTCF decoy proteins bound to the unmethylated ICR and to the IGF2 promoter region but did not interact with SUZ12. EZH2, another part of PRC2, was unable to methylate histone H3-K27 in the IGF2 promoter region, resulting in reactivation of the imprinted allele. The intrachromosomal loop between the maternal ICR and the IGF2 promoters was not observed when IGF2 imprinting was lost. CTCF epigenetically governs allelic gene expression of IGF2 by orchestrating chromatin loop structures involving PRC2.

Expression of the imprinted IGF2 and H19 genes in the endometrium of cases with unexplained infertility

ObjectiveAs genomic imprinting plays a critical role in the development of the placenta, the aim of this study was to detect whether the expression levels of the imprinted genes IGF2 and H19 in the endometrium differ between infertile and fertile women. Study design: Total RNA was extracted from 30 (15 unexplained infertile and 15 fertile) women's endometrial tissue. cDNA was synthesized from total RNAs of each sample. IGF2 and H19 mRNA expression levels were measured quantitatively using the Real Time PCR method. In order to determine the allelic expression of IGF2 and H19, genomic DNA was extracted from endometrial tissues. ResultsWhen compared with the control group, increased mRNA expression of IGF2 was detected (1.5-fold change, P=0.015) in the unexplained infertility group. In contrast, H19 expression was lower in the infertility group as compared to the control group (4-fold change, P<0.0001). Restriction analysis of cDNA-derived PCR product showed that all patients and controls indicated monoallelic expression of IGF2 and H19. ConclusionOur results showed that altered expression of these imprinted genes might affect implantation and that their timely and appropriate activation is important for proper functioning. To understand the molecular epigenetic basis of implantation and placental development, genomic imprinted genes should be further investigated.

Beckwith–Wiedemann‐like macroglossia and 18q23 haploinsufficiency

Beckwith-Wiedemann syndrome (BWS) is an overgrowth condition with tumor proclivity linked to a genetic imbalance of a complex imprinted region in 11p15.5. A female child with features fitting in with the BWS diagnostic framework and an apparent loss of imprinting (LOI) of the IGF2 gene in 11p15.5 was also reported to have a de novo chromosome 18q segmental deletion (Patient 1), thus pointing at the location of a possible trans-activating regulator element for maintenance of IGF2 imprinting and providing one of the few examples of locus heterogeneity of BWS. A second child with de novo 18q23 deletion and features of macroglossia, naevus flammeus, bilateral inguinal hernia and transient neonatal hypoglycemia, thus also fitting in with the BWS diagnostic framework, is here fully reported (Patient 2). In this child, an analysis of the BWS1 locus precluded any paternal isodisomy and showed a normal imprinting pattern (mono-allelic expression of IGF2 and normal H19 and CDKN1OT1/LIT1 methylation index). In Patients 1 and 2, deletions were shown to overlap, defining a minimal region of haplo-insufficiency of 3.8-5.6 Mb in 18q23. We conclude that this region provides a candidate location for an original macroglossia condition with strong overlap with BWS, but without obvious upstream functional relationship with the BWS1 locus in 11p15.5. Because this minimal region of haplo-insufficiency falls into a common region of deletion in 18q- syndrome, we inferred that this macroglossia condition would follow a recessive pattern of inheritance.

The chicken beta-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain.

Imprinting of the mouse insulin-like growth factor 2 (Igf2) and H19 genes is regulated by an imprinting control region (ICR). The hypomethylated maternal copy functions as a chromatin insulator through the binding of CTCF and prevents Igf2 activation in cis, while hypermethylation of the paternal copy inactivates insulator function and leads to inactivation of H19 in cis. The specificity of the ICR sequence for mediating imprinting and chromatin insulation was investigated by substituting it for two copies of the chicken beta-globin insulator element, (Ch beta GI)(2), in mice. This introduced sequence resembles the ICR in size, and in containing CTCF-binding sites and CpGs, but otherwise lacks homology. On maternal inheritance, the (Ch beta GI)(2) was hypomethylated and displayed full chromatin insulator activity. Monoallelic expression of Igf2 and H19 was retained and mice were of normal size. These results suggest that the ICR sequence, aside from CTCF-binding sites, is not uniquely specialized for chromatin insulation at the Igf2/H19 region. On paternal inheritance, the (Ch beta GI)(2) was also hypomethylated and displayed strong insulator activity--fetuses possessed very low levels of Igf2 RNA and were greatly reduced in size, being as small as Igf2-null mutants. Furthermore, the paternal H19 allele was active. These results suggest that differential ICR methylation in the female and male germ lines is not acquired through differential binding of CTCF. Rather, it is likely to be acquired through a separate or downstream process.

Insulin-like growth factor II in uterine smooth-muscle tumors: maintenance of genomic imprinting in leiomyomata and loss of imprinting in leiomyosarcomata.

Uterine leiomyosarcomata, malignant smooth-muscle tumors that may arise from existing leiomyomata or directly from the myometrium, have been shown to oversecrete insulin-like growth factor II (IGF-II). It has been shown recently that the IGF-II gene (IGF2) is maternally imprinted in both mice and humans. Only the paternal allele of IGF2 is usually expressed, except in adult liver and in the central nervous system in which biallelic expression is seen. Recently, loss of IGF2 imprinting has been reported in Wilms' tumor and in several other malignancies, and it has been suggested that biallelic expression of the gene leads to overexpression of IGF-II peptide and increased mitogenic activity. Using the ApaI restriction enzyme polymorphism in exon 9 of IGF2, we examined the expression of IGF2 in five informative uterine leiomyosarcomata and found that IGF2 was expressed biallelically in two tumors. In contrast, five samples of normal myometrium and six informative benign uterine leiomyomata showed monoallelic expression of IGF2. The incidence of heterozygosity at this locus was markedly lower in patients with leiomyosarcoma (6%) than in the healthy population (42% heterozygous; P < 0.01). H19, a gene that is imprinted in normal tissues, was expressed monoallelically in all informative normal and neoplastic uterine tumors. In contrast, the IGF-II receptor gene was expressed from both alleles in all tissues examined, confirming the finding that the human IGF-II receptor gene is not imprinted in humans. This study provides further evidence that relaxation of IGF2 genomic imprinting occurs not only in childhood tumors but also in adult-onset tumors, and it suggests a novel epigenetic mechanism for oncogenesis throughout life.