CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2 - PubMed (original) (raw)

CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2

Sreenivasulu Kurukuti et al. Proc Natl Acad Sci U S A. 2006.

Abstract

It is thought that the H19 imprinting control region (ICR) directs the silencing of the maternally inherited Igf2 allele through a CTCF-dependent chromatin insulator. The ICR has been shown to interact physically with a silencer region in Igf2, differentially methylated region (DMR)1, but the role of CTCF in this chromatin loop and whether it restricts the physical access of distal enhancers to Igf2 is not known. We performed systematic chromosome conformation capture analyses in the Igf2/H19 region over >160 kb, identifying sequences that interact physically with the distal enhancers and the ICR. We found that, on the paternal chromosome, enhancers interact with the Igf2 promoters but that, on the maternal allele, this is prevented by CTCF binding within the H19 ICR. CTCF binding in the maternal ICR regulates its interaction with matrix attachment region (MAR)3 and DMR1 at Igf2, thus forming a tight loop around the maternal Igf2 locus, which may contribute to its silencing. Mutation of CTCF binding sites in the H19 ICR leads to loss of CTCF binding and de novo methylation of a CTCF target site within Igf2 DMR1, showing that CTCF can coordinate regional epigenetic marks. This systematic chromosome conformation capture analysis of an imprinting cluster reveals that CTCF has a critical role in the epigenetic regulation of higher-order chromatin structure and gene silencing over considerable distances in the genome.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Parent-of-origin-specific patterns of physical proximity between the H19 endodermal enhancer and the Igf2/H19 domain in neonatal liver. H19, Igf2, and the flanking Ins2 and L23mrp genes are indicated by squares. The orientation of the primers used is represented by the direction of the arrows at the top of the diagram. EcoRI restriction enzyme sites that are present all along the locus are shown below the locus diagram, whereas arrowheads pointing up show the location of the 3C primer with reference to the corresponding EcoRI restriction site. The hot-stop PCR analysis of the proximity between en4 and the entire Igf2/H19 domain was performed by comparing relative crosslinking frequencies between the maternal en4 allele and the rest of the locus after normalization of the wild-type paternal SD7 allele frequencies to 100% (red line). Also see Fig. 9 for direct comparison of frequencies of interactions. 3C analysis and allelic bias were corrected for as described in Supporting Materials and Methods. The rightmost image exemplifies a hot-stop PCR analysis of 3C samples, which were digested with KpnI to identify the SD7 allele. The completeness of the KpnI digestion was verified by incorporating a fragment covering the KpnI site, which is specific for the SD7 allele, as an internal digestion control. See Materials and Methods and Supporting Materials and Methods for additional information.

Fig. 2.

Fig. 2.

Analysis of parent-of-origin-specific patterns of physical proximity between ICR and the Igf2/H19 domain in neonatal liver. The 3C analysis was performed by comparing relative crosslinking frequencies between the fixed ICR of the maternal allele and the rest of the locus after normalizing the maternal allele frequencies to 100% (blue line). Also see Fig. 11 for direct comparison of frequencies of interactions. Because of a lack of signal for the maternal allele in some instances, the blue line makes a dip, as indicated. 3C analysis and allelic bias were corrected for as described in Fig. 1 (see Fig. 12). The bottommost image exemplifies a hot-stop PCR analysis of 3C samples, which were digested with FauI to identify the SD7 allele. See Material and Methods for additional information.

Fig. 3.

Fig. 3.

CTCF target sites control the interaction between the H19 ICR and Igf2 DMR1 regions. (A) Schematic map of the Igf2 and H19 loci. The Igf2 DMR1 and H19 ICR domains are expanded to show the locations of 3C primers (marked with roman numerals and thick arrows to indicate their directions). The numbers indicate their distance from the HindIII sites. Primers IV/V span a polymorphic restriction site for DraI specific to the SD7 allele. (B) Three independent samples from each cross were subjected to the 3C assay. The PCR products were digested with DraI and subjected to Southern blot hybridization analysis to verify specificity of the amplified DNA fragments. The amplification of HindIII-digested and ligated yeast artificial chromosome DNA covering the entire Igf2/H19 domain was used as a positive control. (C) The intensity of the ICR–DMR1 bands of the image in B was calculated to normalize against variation in the 3C assay. The same result was obtained when the PCR products were quantified from ethidium bromide-stained agarose gels (data not shown). (D) CTCF is present in the ICR–DMR1 complex as determined by 3C analysis of CTCF–DNA complexes that had undergone ChIP. See Materials and Methods for additional information.

Fig. 4.

Fig. 4.

ICR–DMR1, CTCF, and long-range epigenetic coordination. (A) (Upper) Effects of in vitro CpG methylation on CTCF binding to positive DNA fragments 2, 3, and 7 (the last fragment covers only CpG site nr 5; see Fig. 13). Digestion of unmethylated/methylated probe with the methylation-sensitive enzyme HpaII is depicted in lane d for each panel. Three pair of panels with both unmethylated and methylated [32P]DNA probes and in vitro_-translated proteins are depicted (lanes are marked as in Fig. 13 Upper). (Lower) Shown is a map of core DMR1 fragment with CpG numbers and the positions of the overlapping DNA fragments. (B and C) CTCF interacts with the Igf2 DMR1 in vivo. ChIP analysis of neonatal liver derived from a cross between C57BL/6 and SD7 (B) or from 142* × SD7 and SD7 × 142* crosses (C). The DraI polymorphism specific for the M. spretus allele of the DMR1 in the recombinant SD7 mouse strain was exploited (see Fig. 3_A). (D) Bisulfite sequencing data of DMR1 in neonatal liver. Filled and open circles represent methylated and unmethylated CpGs, respectively. Maternal and paternal alleles were distinguished using the DraI polymorphism. (E) Summary of the overall bisulfite data or, specifically, the fifth CpG site, given in percentage of CpG methylation.

Fig. 5.

Fig. 5.

Model showing contacts established within the maternal allele at the H19 locus in neonatal liver. The model suggests a mechanism of H19 ICR function that takes into account active chromatin hubs (ACH) (40) and the repressing IDM–DMR–MAR3 complex on the maternal chromosome. This model is based on results from neonatal liver only and may not apply to other tissues. Additional work will be required (see Discussion) to determine the exact roles of DMR1 and MAR3 in liver, because complete deletion of DMR1 results in reexpression of the maternal Igf2 allele only in mesodermal tissues.

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References

    1. Bell A. C., West A. G., Felsenfeld G. Science. 2001;291:447–450. - PubMed
    1. Bell A., West A., Felsenfeld G. Cell. 1999;98:387–396. - PubMed
    1. Mukhopadhyay R., Yu W.-Q., Whitehead J., Xu J.-W., Kanduri C., Kanduri M., Ginjala V., Vostrov A., Quitschke W., Chernukhin I., et al. Genome Res. 2004;14:1594–1602. - PMC - PubMed
    1. Ohlsson R., Renkawitz R., Lobanenkov V. Trends Genet. 2001;17:520–527. - PubMed
    1. Pant V., Kurukuti S., Pugacheva E., Shamsuddin S., Mariano P., Renkawitz R., Klenova E., Lobanenkov V., Ohlsson R. Mol. Cell. Biol. 2004;24:3497–3504. - PMC - PubMed

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