Insulators and promoters: closer than we think

Abstract

Insulators prevent promiscuous gene regulation by restricting the action of enhancers and silencers. Recent studies have revealed a number of similarities between insulators and promoters, including binding of specific transcription factors, chromatin-modification signatures and localization to specific subnuclear positions. We propose that enhancer-blockers and silencing barrier-insulators might have evolved as specialized derivatives of promoters and that the two types of element use related mechanisms to mediate their distinct functions. These insights can help to reconcile different models of insulator action.

Figure 1. Chromatin domains and regulatory elements

The relationships among silencers, enhancers, promoters and insulators are shown. Light blue circles represent nucleosomes and yellow ovals represent silencing proteins, such as heterochromatin protein 1 (HP1) and Sir3. Silencer elements (S) are sites of initiation of heterochromatin, which spreads and encompasses promoters (P2 in the diagram), silencing transcription. The I1 insulator functions to restrict the spread of heterochromatin. An enhancer (E1) that is present in an active chromatin domain flanked by insulators (I1 and I2) and that is bound by a transcription factor (TF) is able to communicate with a promoter (P1) in the same domain, whereas another enhancer (E2) is unable to communicate with promoter P1 because of an intervening insulator (I2).

Figure 2. Establishment of silencing and barrier activity

a,b|Silencing elements (S) recruit specific transcription factors (TF), which in turn recruit chromatin remodellers (CR) and histone-modifying enzymes (HM) that cooperate to modify chromatin and create binding sites in nucleosomes for repressor proteins. c,d|The binding and spreading of the repressor proteins (R) along the chromatin fibre results in the formation of a silenced heterochromatic domain. Barrier elements (B) bind a distinct set of transcription factors that recruit enzyme complexes, which modify nucleosomes with ‘active’ histone marks and evict nucleosomes. This creates a discontinuity in the chromatin fibre and thereby restricts the spread of repressor proteins.

Figure 3. Interactions among enhancers, promoters and enhancer-blocking insulators

Potential interactions between regulatory elements are shown. a|A pair of enhancer-blocking insulators (I) interact pairwise with each other, placing the enhancer (E) in a loop with a promoter (P2) to enable transcription activation, while isolating a second promoter (P1) in a separate loop. b|An enhancer-blocker functions by directly sequestering an enhancer, therefore disrupting its ability to interact with a promoter. c|The enhancer-blocker can also interact directly with a promoter, preventing it from interacting with the enhancer, which can freely interact with a second promoter.

Figure 4. Vertebrate loci at which insulator function is well-studied

a|The chicken HS4 insulator. A 16-kb heterochromatin domain is located between the folate receptor 1 (FOLR1) gene and the β-globin locus. The HS4 insulator separates the heterochromatin from the active β-globin gene domain. The insulator contains binding sites (FI to FV) for the proteins vascular endothelial zinc finger 1 (VEZF1), CCCTC-binding factor (CTCF) and upstream stimulatory factor 1 (USF1). CTCF is necessary for enhancer-blocking activity and tethering the element to the nucleolus, whereas VEZF1 and USF1 are required for barrier activity. HSA and 3′ HS are DNase I hypersensitive sites with enhancer-blocking activity. b|Enhancer-blocking activity at the imprinted insulin-like growth factor 2 (IGF2)–H19 locus. On the maternal allele, CTCF binds the imprinting control region (ICR) and interacts with the IGF2 promoter, blocking activation by the upstream enhancers, which are then able to interact with the H19 gene. On the paternal allele, the CTCF-binding sites in the ICR are methylated and CTCF is unable to bind the ICR. The enhancers can then activate the IGF2 gene while repressors bind H19. Part a is modified, with permission, from REF. © (2002) National Academy of Sciences USA.

Figure 5. Insulators and three-dimensional organization in the nucleus

a|Clustering of transcription factor IIIC (TFIIIC) in Schizosaccharomyces pombe. b|Clustering of Suppressor of hairy wing (SU(HW)) in Drosophila melanogaster. c|The interaction of CCCTC-binding factor (CTCF) insulator sites with the nucleolus in human cells. The arrows point to the HS4 insulator interacting with the nucleolus, as visualized by fluorescence in situ hybridization (FISH). d|Model of interactions among insulators, enhancers, promoters and subnuclear structures in the nucleus. Insulators interact with each other and with other regulatory elements to partition the chromosome into structural and functional domains. Image a is reproduced, with permission, from REF. © (2006) Elsevier. Image b is reproduced, with permission, from REF. © (2000) Elsevier. Image c is reproduced, with permission, from REF. © (2004) Elsevier. Image d is adapted, with permission, from REF. © (2001) Annual Reviews.