We gather together: insulators and genome organization

Abstract

When placed between an enhancer and promoter, certain DNA sequence elements inhibit enhancer-stimulated gene expression. The best characterized of these enhancer-blocking insulators, gypsy in Drosophila and the CTCF-binding element in vertebrates and flies, stabilize contacts between distant genomic regulatory sites leading to the formation of loop domains. Current results show that CTCF mediates long-range contacts in the mouse beta-globin locus and at the Igf2/H19-imprinted locus. Recently described active chromatin hubs and transcription factories also involve long-range interactions; it is likely that CTCF interferes with their formation when acting as an insulator. The properties of CTCF, and its newly described genomic distribution, suggest that it may play an important role in large-scale nuclear architecture, perhaps mediated by the co-factors with which it interacts in vivo.

Figure 1. Loop domains and enhancer blocking insulators

A. Action of CTCF at the mouse Igf2/H19 locus. On the maternally transmitted allele, CTCF binds to sites in the imprinted control region (ICR) and prevents downstream enhancers from activating Igf2 expression. The ICR of the paternal allele is methylated, CTCF does not bind, and the enhancer is no longer blocked [–6]. B. Loop formation stabilized by proteins and RNA bound to gypsy sites in Drosophila [20] (Figure taken from [20]; see [22] for review of a more complete version of this complex). For clarity, one set of interactions, leading to the formation of a single loop, is shown. Clusters of such sites form insulator bodies in vivo. (a) The proteins recruited to the site include the DNA-binding protein Su(Hw), CP190, and Mod(mdg4)2.2, as well as Topors, a ubiquitin ligase, which associates with the nuclear lamina and the insulator complex and is required for insulator function. (b,c) Loop formation and insulator activity also involve RNA and are dependent on members of the RNAi processing pathway. (d) Loop formation is interfered with by the RNA-binding protein, Rm62. The protein CP190 is additionally involved in insulator function at sites that do not contain Su(Hw). C. Contacts between distant CTCF-binding sites (red circles), which are also nuclease hypersensitive sites (HS), in embryonic day 12.5 mouse erythroid progenitor cells [9,15]. HS-62 is ~ 62kb upstream, and HS5 ~ 30kb upstream, of the globin gene Ey. 3′HS1 is downstream of the globin gene array. Other hypersensitive sites (yellow) mark locus control regions; not all such sites are shown. The globin genes (blue squares) are not expressed in these cells. Mutating the CTCF site at 3′HS1 destroys the interactions between it and the other two CTCF sites. At a later stage of erythroid development additional contacts are made between locus control region and other regulatory sites to form the ACH; this structure is independent of CTCF binding [15]. D. Loop domain models for the action of the ICR at the mouse Igf2/H19 locus, showing contacts involving the ICR, detected by 3C and other methods, that differ on the maternal and paternal alleles [15,18]. It is proposed that the conformation of the paternal allele allows contacts between Igf2 promoters and the enhancer that are blocked on the maternal allele. (a,b) The ICR on the maternal allele (a) makes contact with an imprinted site (DMR1) located upstream of the Igf2 promoters. On the paternal allele (b) the ICR contacts another such site, DMR2, downstream of Igf2. This should make the Igf2 promoters accessible to the enhancers located downstream of H19. For a more elaborate model based on additional data, see [16]. (c,d) A second set of 3C experiments [18] shows that on the maternal allele (c) one of the downstream enhancers (mesodermal or endodermal specific, depending on cell type) contacts the ICR, effectively inhibiting contact between enhancer and Igf2 promoter. Other interactions, not shown, occur between the ICR and the Igf2 promoter. On the paternal allele (d), with CTCF not bound to the ICR, the enhancer makes contact with the appropriate subset of Igf2 promoters. Differences between the results shown in (a,b) and (c,d) may reflect different choices of anchor and target sites for the 3C analysis, and may not be mutually exclusive. Some of these interactions may be tissue-specific; which interactions are most important for establishing allele-specific expression is not yet clear.

Figure 1. Loop domains and enhancer blocking insulators

A. Action of CTCF at the mouse Igf2/H19 locus. On the maternally transmitted allele, CTCF binds to sites in the imprinted control region (ICR) and prevents downstream enhancers from activating Igf2 expression. The ICR of the paternal allele is methylated, CTCF does not bind, and the enhancer is no longer blocked [–6]. B. Loop formation stabilized by proteins and RNA bound to gypsy sites in Drosophila [20] (Figure taken from [20]; see [22] for review of a more complete version of this complex). For clarity, one set of interactions, leading to the formation of a single loop, is shown. Clusters of such sites form insulator bodies in vivo. (a) The proteins recruited to the site include the DNA-binding protein Su(Hw), CP190, and Mod(mdg4)2.2, as well as Topors, a ubiquitin ligase, which associates with the nuclear lamina and the insulator complex and is required for insulator function. (b,c) Loop formation and insulator activity also involve RNA and are dependent on members of the RNAi processing pathway. (d) Loop formation is interfered with by the RNA-binding protein, Rm62. The protein CP190 is additionally involved in insulator function at sites that do not contain Su(Hw). C. Contacts between distant CTCF-binding sites (red circles), which are also nuclease hypersensitive sites (HS), in embryonic day 12.5 mouse erythroid progenitor cells [9,15]. HS-62 is ~ 62kb upstream, and HS5 ~ 30kb upstream, of the globin gene Ey. 3′HS1 is downstream of the globin gene array. Other hypersensitive sites (yellow) mark locus control regions; not all such sites are shown. The globin genes (blue squares) are not expressed in these cells. Mutating the CTCF site at 3′HS1 destroys the interactions between it and the other two CTCF sites. At a later stage of erythroid development additional contacts are made between locus control region and other regulatory sites to form the ACH; this structure is independent of CTCF binding [15]. D. Loop domain models for the action of the ICR at the mouse Igf2/H19 locus, showing contacts involving the ICR, detected by 3C and other methods, that differ on the maternal and paternal alleles [15,18]. It is proposed that the conformation of the paternal allele allows contacts between Igf2 promoters and the enhancer that are blocked on the maternal allele. (a,b) The ICR on the maternal allele (a) makes contact with an imprinted site (DMR1) located upstream of the Igf2 promoters. On the paternal allele (b) the ICR contacts another such site, DMR2, downstream of Igf2. This should make the Igf2 promoters accessible to the enhancers located downstream of H19. For a more elaborate model based on additional data, see [16]. (c,d) A second set of 3C experiments [18] shows that on the maternal allele (c) one of the downstream enhancers (mesodermal or endodermal specific, depending on cell type) contacts the ICR, effectively inhibiting contact between enhancer and Igf2 promoter. Other interactions, not shown, occur between the ICR and the Igf2 promoter. On the paternal allele (d), with CTCF not bound to the ICR, the enhancer makes contact with the appropriate subset of Igf2 promoters. Differences between the results shown in (a,b) and (c,d) may reflect different choices of anchor and target sites for the 3C analysis, and may not be mutually exclusive. Some of these interactions may be tissue-specific; which interactions are most important for establishing allele-specific expression is not yet clear.

Figure 1. Loop domains and enhancer blocking insulators

A. Action of CTCF at the mouse Igf2/H19 locus. On the maternally transmitted allele, CTCF binds to sites in the imprinted control region (ICR) and prevents downstream enhancers from activating Igf2 expression. The ICR of the paternal allele is methylated, CTCF does not bind, and the enhancer is no longer blocked [–6]. B. Loop formation stabilized by proteins and RNA bound to gypsy sites in Drosophila [20] (Figure taken from [20]; see [22] for review of a more complete version of this complex). For clarity, one set of interactions, leading to the formation of a single loop, is shown. Clusters of such sites form insulator bodies in vivo. (a) The proteins recruited to the site include the DNA-binding protein Su(Hw), CP190, and Mod(mdg4)2.2, as well as Topors, a ubiquitin ligase, which associates with the nuclear lamina and the insulator complex and is required for insulator function. (b,c) Loop formation and insulator activity also involve RNA and are dependent on members of the RNAi processing pathway. (d) Loop formation is interfered with by the RNA-binding protein, Rm62. The protein CP190 is additionally involved in insulator function at sites that do not contain Su(Hw). C. Contacts between distant CTCF-binding sites (red circles), which are also nuclease hypersensitive sites (HS), in embryonic day 12.5 mouse erythroid progenitor cells [9,15]. HS-62 is ~ 62kb upstream, and HS5 ~ 30kb upstream, of the globin gene Ey. 3′HS1 is downstream of the globin gene array. Other hypersensitive sites (yellow) mark locus control regions; not all such sites are shown. The globin genes (blue squares) are not expressed in these cells. Mutating the CTCF site at 3′HS1 destroys the interactions between it and the other two CTCF sites. At a later stage of erythroid development additional contacts are made between locus control region and other regulatory sites to form the ACH; this structure is independent of CTCF binding [15]. D. Loop domain models for the action of the ICR at the mouse Igf2/H19 locus, showing contacts involving the ICR, detected by 3C and other methods, that differ on the maternal and paternal alleles [15,18]. It is proposed that the conformation of the paternal allele allows contacts between Igf2 promoters and the enhancer that are blocked on the maternal allele. (a,b) The ICR on the maternal allele (a) makes contact with an imprinted site (DMR1) located upstream of the Igf2 promoters. On the paternal allele (b) the ICR contacts another such site, DMR2, downstream of Igf2. This should make the Igf2 promoters accessible to the enhancers located downstream of H19. For a more elaborate model based on additional data, see [16]. (c,d) A second set of 3C experiments [18] shows that on the maternal allele (c) one of the downstream enhancers (mesodermal or endodermal specific, depending on cell type) contacts the ICR, effectively inhibiting contact between enhancer and Igf2 promoter. Other interactions, not shown, occur between the ICR and the Igf2 promoter. On the paternal allele (d), with CTCF not bound to the ICR, the enhancer makes contact with the appropriate subset of Igf2 promoters. Differences between the results shown in (a,b) and (c,d) may reflect different choices of anchor and target sites for the 3C analysis, and may not be mutually exclusive. Some of these interactions may be tissue-specific; which interactions are most important for establishing allele-specific expression is not yet clear.

Figure 1. Loop domains and enhancer blocking insulators

A. Action of CTCF at the mouse Igf2/H19 locus. On the maternally transmitted allele, CTCF binds to sites in the imprinted control region (ICR) and prevents downstream enhancers from activating Igf2 expression. The ICR of the paternal allele is methylated, CTCF does not bind, and the enhancer is no longer blocked [–6]. B. Loop formation stabilized by proteins and RNA bound to gypsy sites in Drosophila [20] (Figure taken from [20]; see [22] for review of a more complete version of this complex). For clarity, one set of interactions, leading to the formation of a single loop, is shown. Clusters of such sites form insulator bodies in vivo. (a) The proteins recruited to the site include the DNA-binding protein Su(Hw), CP190, and Mod(mdg4)2.2, as well as Topors, a ubiquitin ligase, which associates with the nuclear lamina and the insulator complex and is required for insulator function. (b,c) Loop formation and insulator activity also involve RNA and are dependent on members of the RNAi processing pathway. (d) Loop formation is interfered with by the RNA-binding protein, Rm62. The protein CP190 is additionally involved in insulator function at sites that do not contain Su(Hw). C. Contacts between distant CTCF-binding sites (red circles), which are also nuclease hypersensitive sites (HS), in embryonic day 12.5 mouse erythroid progenitor cells [9,15]. HS-62 is ~ 62kb upstream, and HS5 ~ 30kb upstream, of the globin gene Ey. 3′HS1 is downstream of the globin gene array. Other hypersensitive sites (yellow) mark locus control regions; not all such sites are shown. The globin genes (blue squares) are not expressed in these cells. Mutating the CTCF site at 3′HS1 destroys the interactions between it and the other two CTCF sites. At a later stage of erythroid development additional contacts are made between locus control region and other regulatory sites to form the ACH; this structure is independent of CTCF binding [15]. D. Loop domain models for the action of the ICR at the mouse Igf2/H19 locus, showing contacts involving the ICR, detected by 3C and other methods, that differ on the maternal and paternal alleles [15,18]. It is proposed that the conformation of the paternal allele allows contacts between Igf2 promoters and the enhancer that are blocked on the maternal allele. (a,b) The ICR on the maternal allele (a) makes contact with an imprinted site (DMR1) located upstream of the Igf2 promoters. On the paternal allele (b) the ICR contacts another such site, DMR2, downstream of Igf2. This should make the Igf2 promoters accessible to the enhancers located downstream of H19. For a more elaborate model based on additional data, see [16]. (c,d) A second set of 3C experiments [18] shows that on the maternal allele (c) one of the downstream enhancers (mesodermal or endodermal specific, depending on cell type) contacts the ICR, effectively inhibiting contact between enhancer and Igf2 promoter. Other interactions, not shown, occur between the ICR and the Igf2 promoter. On the paternal allele (d), with CTCF not bound to the ICR, the enhancer makes contact with the appropriate subset of Igf2 promoters. Differences between the results shown in (a,b) and (c,d) may reflect different choices of anchor and target sites for the 3C analysis, and may not be mutually exclusive. Some of these interactions may be tissue-specific; which interactions are most important for establishing allele-specific expression is not yet clear.