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CTCF maintains differential methylation at the Igf2

Background. An overall coordination between the expressions of genes is required for the proper development of an individual.. Although most genes are expressed from both the constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from only one of the parent

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CTCF maintains differential methylation at the Igf2

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    1. CTCF maintains differential methylation at the Igf2/H19 locus Christopher J. Schoenherr, John M. Levorse & Shirley M. Tilghman Nat Genet. 2003 Jan;33(1):66-9.

    2. Background An overall coordination between the expressions of genes is required for the proper development of an individual.

    3. Background, contd. The imprinted H19 and Igf2 genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of H19, known as the imprinting control region (ICR).

    4. Background, contd.

    5. H19 and Igf2 H19 ( a gene with in vitro growth inhibitory capacity, and encodes fetal liver mRNA) and Igf2 (encodes insulin-like growth factor 2) are part of a cluster of imprinted genes on mouse chromosome 7.

    6. H19 and Igf2, contd. The imprinting of the two genes is mechanistically linked. Deletion of H19 and the ~10-kb region upstream of it leads to biallelic expression of Igf2. Sequences upstream of H19 is important for monoallelic expression of both H19 and Igf2.

    7. CTCF The nuclear protein, CTCF is an evolutionarily conserved zinc finger (ZF) phosphoprotein that binds through combinatorial use of its 11 ZFs to ~50 bp target sites that have remarkable sequence variation.

    8. CTCF, contd. Formation of different CTCFDNA complexes, some of which are methylation-sensitive, results in distinct functions, including gene activation, repression, silencing and chromatin insulation.

    9. CTCF, contd. CTCF is important in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn controls the expression pattern of H19 and Igf2.

    10. Hypothesis The presence of CTCF in male and female germ cells indicates that it could participate in establishing the epigenetic state of the ICR during gametogenesis.

    16. Overall Conclusions CTCF binding is necessary to maintain, but not establish, the differential methylation of the ICR.

    17. The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19. On the paternal chromosome this element is methylated and required for the silencing of H19. On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19. This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.

    18. Chromatin boundaries create independent regions of gene expression by preventing the expansion of heterochromatin and by blocking enhancers from activating inappropriate promoters. An element with enhancer-blocking activity must reside between a promoter and an enhancer to exert its repressive effect. the ICR contains two maternal-specific nuclease hypersensitive regions: HS1, which maps between -4.1 and -3.6 kilobases (kb), and HS2, between -2.7 to -2.1kb upstream of H19. Both are candidate sites for boundary protein binding. the H19 ICR has enhancer-blocking activity and is not a silencer.

    19. both hypersensitive regions of the ICR are required for full enhancer-blocking activity in vivo. In contrast, the sequence between HS1 and HS2 (IVS) had minimal blocking activity. A CpG-rich 45-bp sequence in the human B repeats was found to be 60% identical to two sites in HS1 and three sites in HS2, as reported. CTCF, a zinc-finger protein implicated in both transcriptional activation and repression, is required for the enhancer-blocking function of several vertebrate boundary or insulator elements.

    20. Maternal chromosome-specific hypersensitivity to nuclease digestion has been demonstrated at two regions that are~2.4kb and 3.8kb upstream of the H19 promoter. Also, this region displays paternal chromosome-specific hypermethylation that extends from approximately 4.0 kb to 2.0 kb. Therfore, methylation of this region has been suggested to be responsible for controlling the imprinted expression of H19 and Igf2. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development.

    21. The mouse H19 gene encodes one of the most abundant RNAs in the developing mouse embryo. It is expressed at the blastocyst stage of development, and accumulates to high levels in tissues of endodermal and mesodermal origin. After birth the gene is expressed in all tissues except skeletal muscle. It lacks a common open reading frame in the 2.5-kilobase RNA, but has considerable nucleotide sequence similarity between the genes of rodents and humans. Expression of the gene in transgenic mice results in late prenatal lethality, suggesting that the dosage of its gene product is strictly controlled.

    22. Nature. 1995 May 4;375(6526):34-9. Disruption of imprinting caused by deletion of the H19 gene region in mice. Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman SM. The imprinted H19 gene, which encodes an untranslated RNA, lies at the end of a cluster of imprinted genes in the mouse. Imprinting of the insulin-2 and insulin-like growth factor 2 genes, which lie about 100 kilobases upstream of H19, can be disrupted by maternal inheritance of a targeted deletion of the H19 gene and its flanking sequence. Animals inheriting the H19 mutation from their mothers are 27% heavier than those inheriting it from their fathers. Paternal inheritance of the disruption has no effect, which presumably reflects the normally silent state of the paternal gene. The somatic overgrowth of heterozygotes for the maternal deletion is attributed to a gain of function of insulin-like growth factor 2, rather than a loss of function of H19.

    23. A role for CTCF-mediated enhancer blocking activity has been demonstrated most clearly at the Igf2/H19 locus in mouse and human (10-12). In this imprinted locus the maternally transmitted allele expresses H19 but not Igf2, whereas the paternally transmitted allele expresses Igf2 but not H19. Furthermore the paternal allele is methylated at a site (the ICR or imprinted control region) located between the two genes (Fig. 5). Earlier work had suggested that the ICR might contain an enhancer blocking activity that would prevent downstream endodermal enhancers from activating Igf2. Direct examination reveals that the ICR contains four CTCF binding sites in the mouse ICR and seven in human. Methylation of these sites abolishes CTCF binding. These results indicate that CTCF plays an important role as an insulator protein in allele-specific regulation at this imprinted locus, and that the insulator function can be modulated by DNA methylation, thus making the CTCF sites susceptible to epigenetic regulation. Quite recently a cluster of differentially methylated CTCF sites has been identified at the Xist gene promoter, and it has been suggested that these are enhancer-blocking elements important for X chromosome inactivation (13).

    25. Background Certain loci in the mammalian genome exhibit functional inequivalence of the two alleles. Depending on the parent of origin, some genes are expressed exclusively from the maternal chromosome and others exclusively from the paternal chromosome. Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles.

    26. Strong support in favor of this role also comes from mutant studies in which DNA methyltransferase gene (dnmt) has been deleted. In these mutants, H19 was shown to be expressed in a biallelic manner, whereas Igf2 expression was completely lost (Li et al. 1993 ).

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