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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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The H19 DMD is required to prevent interactions between the maternal endodermal enhancers and the Igf2 promoters. The 3C analysis was carried out on samples from a H19Δ3.8 kb-5′H19 × B6(CAST7) cross. (A) Top, schematic of the H19Δ3.8 kb-5′H19 allele (see Fig. 2 for details), with 3.8 kb upstream of the H19 promoter removed. Bottom, representative gel image of 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal (M) alleles; 3C, non-digested PCR product; -L, no ligase control; +, positive control. When the H19Δ3.8 kb-5′H19 allele is inherited maternally, the endodermal enhancers on the maternal chromosome interact with the whole H19 region. (B) Top, schematic of the Igf2 locus (see Fig. 3 for details). Bottom, representative gel image of 3C PCR products digested with HaeIII, which distinguishes paternal (P) from maternal (M) alleles.
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DDN200F4: The H19 DMD is required to prevent interactions between the maternal endodermal enhancers and the Igf2 promoters. The 3C analysis was carried out on samples from a H19Δ3.8 kb-5′H19 × B6(CAST7) cross. (A) Top, schematic of the H19Δ3.8 kb-5′H19 allele (see Fig. 2 for details), with 3.8 kb upstream of the H19 promoter removed. Bottom, representative gel image of 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal (M) alleles; 3C, non-digested PCR product; -L, no ligase control; +, positive control. When the H19Δ3.8 kb-5′H19 allele is inherited maternally, the endodermal enhancers on the maternal chromosome interact with the whole H19 region. (B) Top, schematic of the Igf2 locus (see Fig. 3 for details). Bottom, representative gel image of 3C PCR products digested with HaeIII, which distinguishes paternal (P) from maternal (M) alleles.

Mentions: To prove that the restricted interaction of the endodermal enhancers on the maternal chromosome is dependent on the presence of the insulator, we used an allele that removed the DMD. When the H19Δ3.8 kb-5′H19 mutation is inherited maternally, the normally silent Igf2 gene is activated, resulting in biallelic expression (14). In contrast to behavior of the wild-type maternal chromosome, the analysis of the maternally inherited H19Δ3.8 kb-5′H19 allele showed contact between the endodermal enhancers and the regions upstream of the DMD (Fig. 4A, see maternal and paternal signals in panels 1–4).


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)

The H19 DMD is required to prevent interactions between the maternal endodermal enhancers and the Igf2 promoters. The 3C analysis was carried out on samples from a H19Δ3.8 kb-5′H19 × B6(CAST7) cross. (A) Top, schematic of the H19Δ3.8 kb-5′H19 allele (see Fig. 2 for details), with 3.8 kb upstream of the H19 promoter removed. Bottom, representative gel image of 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal (M) alleles; 3C, non-digested PCR product; -L, no ligase control; +, positive control. When the H19Δ3.8 kb-5′H19 allele is inherited maternally, the endodermal enhancers on the maternal chromosome interact with the whole H19 region. (B) Top, schematic of the Igf2 locus (see Fig. 3 for details). Bottom, representative gel image of 3C PCR products digested with HaeIII, which distinguishes paternal (P) from maternal (M) alleles.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2536502&req=5

DDN200F4: The H19 DMD is required to prevent interactions between the maternal endodermal enhancers and the Igf2 promoters. The 3C analysis was carried out on samples from a H19Δ3.8 kb-5′H19 × B6(CAST7) cross. (A) Top, schematic of the H19Δ3.8 kb-5′H19 allele (see Fig. 2 for details), with 3.8 kb upstream of the H19 promoter removed. Bottom, representative gel image of 3C PCR products: D, digested with NlaIII, which distinguishes paternal (P) from maternal (M) alleles; 3C, non-digested PCR product; -L, no ligase control; +, positive control. When the H19Δ3.8 kb-5′H19 allele is inherited maternally, the endodermal enhancers on the maternal chromosome interact with the whole H19 region. (B) Top, schematic of the Igf2 locus (see Fig. 3 for details). Bottom, representative gel image of 3C PCR products digested with HaeIII, which distinguishes paternal (P) from maternal (M) alleles.
Mentions: To prove that the restricted interaction of the endodermal enhancers on the maternal chromosome is dependent on the presence of the insulator, we used an allele that removed the DMD. When the H19Δ3.8 kb-5′H19 mutation is inherited maternally, the normally silent Igf2 gene is activated, resulting in biallelic expression (14). In contrast to behavior of the wild-type maternal chromosome, the analysis of the maternally inherited H19Δ3.8 kb-5′H19 allele showed contact between the endodermal enhancers and the regions upstream of the DMD (Fig. 4A, see maternal and paternal signals in panels 1–4).

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