CTCF maintains differential CTCF maintains differential methylation at the methylation at the Igf2Igf2//H19H19 locus locus

• Christopher J. Schoenherr, John M. Levorse & Christopher J. Schoenherr, John M. Levorse & Shirley M. TilghmanShirley M. TilghmanNat Genet. 2003 Jan;33(1):66-9.Nat Genet. 2003 Jan;33(1):66-9.

BackgroundBackground

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

• Although most genes are expressed from both the Although most genes are expressed from both the constituent alleles of the genome, a small subset of constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from autosomal genes are preferentially expressed from only one of the parental alleles, a phenomenon known only one of the parental alleles, a phenomenon known as genomic imprinting.as genomic imprinting.

• Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles.

BackgroundBackground, contd., contd.

• The imprinted The imprinted H19 H19 and and Igf2Igf2 genes are considered genes are considered paradigms of genomic imprinting as their monoallelic paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of expression pattern is coordinated by a short stretch of sequence located upstream of sequence located upstream of H19H19, known as the , known as the imprinting control region (ICR).imprinting control region (ICR).

BackgroundBackground, contd., contd.

• This region shows differential methylation, with This region shows differential methylation, with hypermethylation specifically on the paternal allele.hypermethylation specifically on the paternal allele.

• On the maternal allele this region acts as an insulator On the maternal allele this region acts as an insulator and harbours maternal specific hypersensitive sites. and harbours maternal specific hypersensitive sites.

• The hypersensitive sites were identified as the result The hypersensitive sites were identified as the result of association of the vertebrate insulator protein CTCF of association of the vertebrate insulator protein CTCF with the region.with the region.

H19H19 and and Igf2Igf2• H19H19 ( a gene with in vitro growth inhibitory capacity, ( a gene with in vitro growth inhibitory capacity,

and encodes fetal liver mRNA) and and encodes fetal liver mRNA) and Igf2 Igf2 (encodes (encodes insulin-like growth factor 2) are part of a cluster of insulin-like growth factor 2) are part of a cluster of imprinted genes on mouse chromosome 7.imprinted genes on mouse chromosome 7.

• The genes exhibit reciprocity in allele-specific The genes exhibit reciprocity in allele-specific expression. Only the maternal allele of expression. Only the maternal allele of H19H19 is is expressed whereas for expressed whereas for Igf2Igf2, it is the paternal allele , it is the paternal allele that is active.that is active.

• Expression of the two genes in endodermal tissues is Expression of the two genes in endodermal tissues is dependent on a common set of enhancers located dependent on a common set of enhancers located between 7 and 9 kb downstream of the between 7 and 9 kb downstream of the H19H19 promoter. promoter.

H19H19 and and Igf2Igf2, contd., contd.

• The imprinting of the two genes is mechanistically The imprinting of the two genes is mechanistically linked. Deletion of linked. Deletion of H19H19 and the ~10-kb region and the ~10-kb region upstream of it leads to biallelic expression of upstream of it leads to biallelic expression of Igf2Igf2. .

• Sequences upstream of Sequences upstream of H19H19 is important for is important for monoallelic expression of both monoallelic expression of both H19H19 and and Igf2Igf2..

CTCFCTCF

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

CTCF, contd.CTCF, contd.

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

CTCF, contd.CTCF, contd.

• CTCF is important in the formation of an epigenetically CTCF is important in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn regulated chromatin insulator at the ICR, which in turn controls the expression pattern of controls the expression pattern of H19H19 and and Igf2Igf2..

• Disrupting the spectrum of target specificities by ZF Disrupting the spectrum of target specificities by ZF

mutations or by abnormal selective methylation of mutations or by abnormal selective methylation of targets is associated with cancer. targets is associated with cancer.

• CTCF plays an imprortant role in the maintenance of CTCF plays an imprortant role in the maintenance of the methylation profile of the regionthe methylation profile of the region..

HypothesisHypothesis

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

• CTCF binding may CTCF binding may maintainmaintain the unmethylated the unmethylated state of the maternal ICR throughout state of the maternal ICR throughout development. development.

Introducing mutant ICR into ES cells

Methods:

Homologous recombination

Southern blot

Electrophoretic mobility shift assay probes: oligos for the four wt CTCF sites Competitors: mutant or wild-type ICR

Fig1. Targeting of the mutant and wild-type ICR and the relative binding affinity of mutant CTCF sites.

Conclusion: Mutant CTCF sites in the ICR have markedly less CTCF binding affinity

CTCF binding maintains the unmethylated state of the maternal ICR

Fig2. Methylation analysis of the mutant ICR

Methods:

Southern blot

Genomic DNA from neonatal liver was digested by restriction enzymes.

DNA fragments was probed with a 2.4-kb ICR fragment.

CTCF binding is not necessary for preventing ICR from being methylated during oogenesis

and early stage of embryo development

Fig3. Bisulfite sequencing of regions of maternally transmitted mutant ICRs.

Methods:

Bisulfite sequencing

A commonly used technique for detecting the methylation pattern of target sequence.

Mutation of ICR, which give rise to low CTCF binding, leads to expression of Igf2 on the

mutant marternal allele

Fig4. Allelic Igf2 mRNA expression in neonatal mice.

Methods:

RNase protection assay

Mutation of ICR, which give rise to low CTCF binding, results in the reduction of maternal

H19 expression

Fig5. Effect of the mutant ICR on maternal H19 expression and promoter methylation

Overall ConclusionsOverall Conclusions

• CTCF binding is necessary to CTCF binding is necessary to maintainmaintain, but not , but not establishestablish, the differential methylation of the ICR., the differential methylation of the ICR.

• CTCF binding is necessary for full expression of CTCF binding is necessary for full expression of maternal maternal H19H19..

• mutant ICR lacks enhancer-blocking activity, mutant ICR lacks enhancer-blocking activity, as the expression of as the expression of Igf2Igf2 is activated on is activated on mutant maternal chromosomes.mutant maternal chromosomes.

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

• CTCF binding may CTCF binding may maintainmaintain the unmethylated the unmethylated state of the maternal ICR throughout state of the maternal ICR throughout development. development.

• The The Insulin-like growth factor 2Insulin-like growth factor 2 ( (Igf2Igf2) and ) and H19H19 genes are genes are imprinted, resulting in silencing of the maternal and paternal imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of imprinted-control region two kilobases upstream of H19H19. On the . On the paternal chromosome this element is methylated and required paternal chromosome this element is methylated and required for the silencing of for the silencing of H19H19. On the maternal chromosome the . On the maternal chromosome the region is unmethylated and required for silencing of the region is unmethylated and required for silencing of the Igf2Igf2 gene 90 kilobases upstream. We have proposed that the gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of boundary that blocks the interaction of Igf2Igf2 with enhancers with enhancers that lie 3' of that lie 3' of H19H19. This enhancer-blocking activity would then be . This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing lost when the region was methylated, thereby allowing expression of expression of Igf2Igf2 paternally. Here we show, using transgenic paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human control regions from mouse and human H19H19 exhibit enhancer- exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.boundary in vertebrates.

• Chromatin boundaries create independent regions of Chromatin boundaries create independent regions of gene expression by preventing the expansion of gene expression by preventing the expansion of heterochromatin and by blocking enhancers from heterochromatin and by blocking enhancers from activating inappropriate promoters. An element with activating inappropriate promoters. An element with enhancer-blocking activity must reside between a enhancer-blocking activity must reside between a promoter and an enhancer to exert its repressive effect. promoter and an enhancer to exert its repressive effect.

• the ICR contains two maternal-specific nuclease the ICR contains two maternal-specific nuclease hypersensitive regions: HS1, which maps between -4.1 hypersensitive regions: HS1, which maps between -4.1 and -3.6 kilobases (kb), and HS2, between -2.7 to -and -3.6 kilobases (kb), and HS2, between -2.7 to -2.1 kb upstream of 2.1 kb upstream of H19H19. Both are candidate sites for . Both are candidate sites for boundary protein binding.boundary protein binding.

• the the H19H19 ICR has enhancer-blocking activity and is not a ICR has enhancer-blocking activity and is not a silencer. silencer.

• both hypersensitive regions of the ICR are both hypersensitive regions of the ICR are required for full enhancer-blocking activity required for full enhancer-blocking activity in vivoin vivo. In contrast, the sequence between . In contrast, the sequence between HS1 and HS2 (IVS) had minimal blocking HS1 and HS2 (IVS) had minimal blocking activity. activity.

• A CpG-rich 45-bp sequence in the human B A CpG-rich 45-bp sequence in the human B repeats was found to be 60% identical to repeats was found to be 60% identical to two sites in HS1 and three sites in HS2, as two sites in HS1 and three sites in HS2, as reported. CTCF, a zinc-finger protein reported. CTCF, a zinc-finger protein implicated in both transcriptional implicated in both transcriptional activation and repression, is required for activation and repression, is required for the enhancer-blocking function of several the enhancer-blocking function of several vertebrate boundary or insulator elements. vertebrate boundary or insulator elements.

• Maternal chromosome-specific hypersensitivity to Maternal chromosome-specific hypersensitivity to nuclease digestion has been demonstrated at two nuclease digestion has been demonstrated at two regions that are ~ 2.4 kb and 3.8 kb upstream of regions that are ~ 2.4 kb and 3.8 kb upstream of the the H19H19 promoter. Also, this region displays promoter. Also, this region displays paternal chromosome-specific hypermethylation paternal chromosome-specific hypermethylation that extends from approximately 4.0 kb to 2.0 that extends from approximately 4.0 kb to 2.0 kb. Therfore, methylation of this region has been kb. Therfore, methylation of this region has been suggested to be responsible for controlling the suggested to be responsible for controlling the imprinted expression of imprinted expression of H19H19 and and Igf2Igf2. .

• A 2-kb region located 5' to the imprinted mouse A 2-kb region located 5' to the imprinted mouse H19H19 gene is hypermethylated on the inactive gene is hypermethylated on the inactive paternal allele throughout development. paternal allele throughout development.

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

• Nature. 1995 May 4;375(6526):34-9.   Nature. 1995 May 4;375(6526):34-9.   Disruption of imprinting caused by deletion of the H19 gene Disruption of imprinting caused by deletion of the H19 gene region in mice.region in mice.

Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Leighton PA, Ingram RS, Eggenschwiler J, Efstratiadis A, Tilghman SM.Tilghman SM.

The imprinted H19 gene, which encodes an untranslated RNA, lies at 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 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 insulin-2 and insulin-like growth factor 2 genes, which lie about 100 kilobases upstream of H19, can be disrupted by maternal inheritance kilobases upstream of H19, can be disrupted by maternal inheritance of a targeted deletion of the H19 gene and its flanking sequence. of a targeted deletion of the H19 gene and its flanking sequence. Animals inheriting the H19 mutation from their mothers are 27% Animals inheriting the H19 mutation from their mothers are 27% heavier than those inheriting it from their fathers. Paternal inheritance heavier than those inheriting it from their fathers. Paternal inheritance of the disruption has no effect, which presumably reflects the normally of the disruption has no effect, which presumably reflects the normally silent state of the paternal gene. The somatic overgrowth of silent state of the paternal gene. The somatic overgrowth of heterozygotes for the maternal deletion is attributed to a gain 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 function of insulin-like growth factor 2, rather than a loss of function of H19.H19.

• A role for CTCF-mediated enhancer blocking activity has been A role for CTCF-mediated enhancer blocking activity has been demonstrated most clearly at the demonstrated most clearly at the Igf2/H19Igf2/H19 locus in mouse and locus in mouse and human (human (10-1210-12). In this imprinted locus the maternally transmitted ). In this imprinted locus the maternally transmitted allele expresses H19 but not Igf2, whereas the paternally allele expresses H19 but not Igf2, whereas the paternally transmitted allele expresses Igf2 but not H19. Furthermore the transmitted allele expresses Igf2 but not H19. Furthermore the paternal allele is methylated at a site (the ICR or imprinted control paternal allele is methylated at a site (the ICR or imprinted control region) located between the two genes (Fig. region) located between the two genes (Fig. 55). Earlier work had ). Earlier work had suggested that the ICR might contain an enhancer blocking activity suggested that the ICR might contain an enhancer blocking activity that would prevent downstream endodermal enhancers from that would prevent downstream endodermal enhancers from activating activating Igf2.Igf2. Direct examination reveals that the ICR contains Direct examination reveals that the ICR contains four CTCF binding sites in the mouse ICR and seven in human. four CTCF binding sites in the mouse ICR and seven in human. Methylation of these sites abolishes CTCF binding. These results Methylation of these sites abolishes CTCF binding. These results indicate that CTCF plays an important role as an insulator protein indicate that CTCF plays an important role as an insulator protein in allele-specific regulation at this imprinted locus, and that the in allele-specific regulation at this imprinted locus, and that the insulator function can be modulated by DNA methylation, thus insulator function can be modulated by DNA methylation, thus making the CTCF sites susceptible to epigenetic regulation. Quite making the CTCF sites susceptible to epigenetic regulation. Quite recently a cluster of differentially methylated CTCF sites has been recently a cluster of differentially methylated CTCF sites has been identified at the identified at the XistXist gene promoter, and it has been suggested gene promoter, and it has been suggested that these are enhancer-blocking elements important for X that these are enhancer-blocking elements important for X chromosome inactivation (chromosome inactivation (1313). ).

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

• Strong support in favor of this role Strong support in favor of this role also comes from mutant studies in also comes from mutant studies in which DNA methyltransferase gene which DNA methyltransferase gene ((dnmtdnmt) has been deleted. In these ) has been deleted. In these mutants, mutants, H19H19 was shown to be was shown to be expressed in a biallelic manner, expressed in a biallelic manner, whereas whereas Igf2Igf2 expression was expression was completely lost (Li et al. 1993completely lost (Li et al. 1993 ).).