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Three-dimensional conformation at the H19/Igf2 locus supports a model of enhancer tracking.

Engel N, Raval AK, Thorvaldsen JL, Bartolomei SM - Hum. Mol. Genet. (2008)

Bottom Line: Furthermore, we compared wild-type chromosomes to several mutations that affect the insulator.Based on our results, we propose that physical proximity of cis-acting DNA elements is vital for their activity in vivo.We suggest that enhancers track along the chromosome until they find a suitable promoter sequence to interact with and that insulator elements block further tracking of enhancers.

View Article: PubMed Central - PubMed

Affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA. noraengel@temple.edu

ABSTRACT
Insight into how the mammalian genome is structured in vivo is key to understanding transcriptional regulation. This is especially true in complex domains in which genes are coordinately regulated by long-range interactions between cis-regulatory elements. The regulation of the H19/Igf2 imprinted region depends on the presence of several cis-acting sequences, including a methylation-sensitive insulator between Igf2 and H19 and shared enhancers downstream of H19. Each parental allele has a distinct expression pattern. We used chromosome conformation capture to assay the native three-dimensional organization of the H19/Igf2 locus on each parental copy. Furthermore, we compared wild-type chromosomes to several mutations that affect the insulator. Our results show that promoters and enhancers reproducibly co-localize at transcriptionally active genes, i.e. the endodermal enhancers contact the maternal H19 and the paternal Igf2 genes. The active insulator blocks traffic of the enhancers along the chromosome, restricting them to the H19 promoter. Conversely, the methylated inactive insulator allows the enhancers to contact the upstream regions, including Igf2. Mutations that either remove or inhibit insulator activity allow unrestricted access of the enhancers to the whole region. A mutation that allows establishment of an enhancer-blocker on the normally inactive paternal copy diminishes the contact of the enhancer with the Igf2 gene. Based on our results, we propose that physical proximity of cis-acting DNA elements is vital for their activity in vivo. We suggest that enhancers track along the chromosome until they find a suitable promoter sequence to interact with and that insulator elements block further tracking of enhancers.

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Analysis of long-range interactions at the H19 locus. (A) Schematic of the H19 locus, including the position of the coding sequence (black box), the differentially methylated domain (hatched box, DMD) and the endodermal enhancers (circles, EE). Vertical bars indicate PsuI restriction sites, numbers beneath the sites are the distance relative to the H19 transcriptional start site, and the resulting digestion fragments are numbered (1–9). Arrowheads represent the location of PCR primers for 3C analysis. The white arrowhead is the reverse primer for all reactions, which test for ligation of fragments 1–9 to the EE fragment. An asterisk indicates a restriction polymorphism that distinguishes C57BL/6J and B6(CAST7) alleles. Samples were from wild-type progeny of C57BL/6J and B6(CAST7) mice (B × C, C × B), with the female indicated first. (B) Representative gel image of 3C analysis at the H19 locus. Shown are the 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal alleles (M); 3C, non-digested PCR product; -L, no ligase control; +, positive control. The endodermal enhancers on the maternal chromosome associate with fragments in the H19 region up to the DMD, beyond which only fragments from the paternal chromosome are observed.
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DDN200F2: Analysis of long-range interactions at the H19 locus. (A) Schematic of the H19 locus, including the position of the coding sequence (black box), the differentially methylated domain (hatched box, DMD) and the endodermal enhancers (circles, EE). Vertical bars indicate PsuI restriction sites, numbers beneath the sites are the distance relative to the H19 transcriptional start site, and the resulting digestion fragments are numbered (1–9). Arrowheads represent the location of PCR primers for 3C analysis. The white arrowhead is the reverse primer for all reactions, which test for ligation of fragments 1–9 to the EE fragment. An asterisk indicates a restriction polymorphism that distinguishes C57BL/6J and B6(CAST7) alleles. Samples were from wild-type progeny of C57BL/6J and B6(CAST7) mice (B × C, C × B), with the female indicated first. (B) Representative gel image of 3C analysis at the H19 locus. Shown are the 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal alleles (M); 3C, non-digested PCR product; -L, no ligase control; +, positive control. The endodermal enhancers on the maternal chromosome associate with fragments in the H19 region up to the DMD, beyond which only fragments from the paternal chromosome are observed.

Mentions: In the wild-type mouse, H19 is expressed from the maternal allele, whereas Igf2 is active on the paternal chromosome. Our results from the 3C assay were identical for reciprocal crosses of C57BL/6J × B6(CAST7) and indicated that the maternal enhancers contacted the entire H19 region, including the promoter, correlating with the active transcriptional status of the maternal H19 gene (Fig. 2, see maternal bands in panels 5–7). However, on the maternal chromosome, the enhancers were not found beyond the DMD. This suggests that the presence of an insulator assembled in this region on the maternal copy physically bars the enhancers from moving upstream.


Three-dimensional conformation at the H19/Igf2 locus supports a model of enhancer tracking.

Engel N, Raval AK, Thorvaldsen JL, Bartolomei SM - Hum. Mol. Genet. (2008)

Analysis of long-range interactions at the H19 locus. (A) Schematic of the H19 locus, including the position of the coding sequence (black box), the differentially methylated domain (hatched box, DMD) and the endodermal enhancers (circles, EE). Vertical bars indicate PsuI restriction sites, numbers beneath the sites are the distance relative to the H19 transcriptional start site, and the resulting digestion fragments are numbered (1–9). Arrowheads represent the location of PCR primers for 3C analysis. The white arrowhead is the reverse primer for all reactions, which test for ligation of fragments 1–9 to the EE fragment. An asterisk indicates a restriction polymorphism that distinguishes C57BL/6J and B6(CAST7) alleles. Samples were from wild-type progeny of C57BL/6J and B6(CAST7) mice (B × C, C × B), with the female indicated first. (B) Representative gel image of 3C analysis at the H19 locus. Shown are the 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal alleles (M); 3C, non-digested PCR product; -L, no ligase control; +, positive control. The endodermal enhancers on the maternal chromosome associate with fragments in the H19 region up to the DMD, beyond which only fragments from the paternal chromosome are observed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2536502&req=5

DDN200F2: Analysis of long-range interactions at the H19 locus. (A) Schematic of the H19 locus, including the position of the coding sequence (black box), the differentially methylated domain (hatched box, DMD) and the endodermal enhancers (circles, EE). Vertical bars indicate PsuI restriction sites, numbers beneath the sites are the distance relative to the H19 transcriptional start site, and the resulting digestion fragments are numbered (1–9). Arrowheads represent the location of PCR primers for 3C analysis. The white arrowhead is the reverse primer for all reactions, which test for ligation of fragments 1–9 to the EE fragment. An asterisk indicates a restriction polymorphism that distinguishes C57BL/6J and B6(CAST7) alleles. Samples were from wild-type progeny of C57BL/6J and B6(CAST7) mice (B × C, C × B), with the female indicated first. (B) Representative gel image of 3C analysis at the H19 locus. Shown are the 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal alleles (M); 3C, non-digested PCR product; -L, no ligase control; +, positive control. The endodermal enhancers on the maternal chromosome associate with fragments in the H19 region up to the DMD, beyond which only fragments from the paternal chromosome are observed.
Mentions: In the wild-type mouse, H19 is expressed from the maternal allele, whereas Igf2 is active on the paternal chromosome. Our results from the 3C assay were identical for reciprocal crosses of C57BL/6J × B6(CAST7) and indicated that the maternal enhancers contacted the entire H19 region, including the promoter, correlating with the active transcriptional status of the maternal H19 gene (Fig. 2, see maternal bands in panels 5–7). However, on the maternal chromosome, the enhancers were not found beyond the DMD. This suggests that the presence of an insulator assembled in this region on the maternal copy physically bars the enhancers from moving upstream.

Bottom Line: Furthermore, we compared wild-type chromosomes to several mutations that affect the insulator.Based on our results, we propose that physical proximity of cis-acting DNA elements is vital for their activity in vivo.We suggest that enhancers track along the chromosome until they find a suitable promoter sequence to interact with and that insulator elements block further tracking of enhancers.

View Article: PubMed Central - PubMed

Affiliation: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA. noraengel@temple.edu

ABSTRACT
Insight into how the mammalian genome is structured in vivo is key to understanding transcriptional regulation. This is especially true in complex domains in which genes are coordinately regulated by long-range interactions between cis-regulatory elements. The regulation of the H19/Igf2 imprinted region depends on the presence of several cis-acting sequences, including a methylation-sensitive insulator between Igf2 and H19 and shared enhancers downstream of H19. Each parental allele has a distinct expression pattern. We used chromosome conformation capture to assay the native three-dimensional organization of the H19/Igf2 locus on each parental copy. Furthermore, we compared wild-type chromosomes to several mutations that affect the insulator. Our results show that promoters and enhancers reproducibly co-localize at transcriptionally active genes, i.e. the endodermal enhancers contact the maternal H19 and the paternal Igf2 genes. The active insulator blocks traffic of the enhancers along the chromosome, restricting them to the H19 promoter. Conversely, the methylated inactive insulator allows the enhancers to contact the upstream regions, including Igf2. Mutations that either remove or inhibit insulator activity allow unrestricted access of the enhancers to the whole region. A mutation that allows establishment of an enhancer-blocker on the normally inactive paternal copy diminishes the contact of the enhancer with the Igf2 gene. Based on our results, we propose that physical proximity of cis-acting DNA elements is vital for their activity in vivo. We suggest that enhancers track along the chromosome until they find a suitable promoter sequence to interact with and that insulator elements block further tracking of enhancers.

Show MeSH