CTCF-dependent enhancer-blocking by alternative chromatin loop formation

Abstract

The mechanism underlying enhancer-blocking by insulators is unclear. We explored the activity of human ␤-globin HS5, the orthologue of the CTCF-dependent chicken HS4 insulator. An extra copy of HS5 placed between the ␤-globin locus control region (LCR) and downstream genes on a transgene fulfills the classic predictions for an enhancer-blocker. Ectopic HS5 does not perturb the LCR but blocks gene activation by interfering with RNA pol II, activator and coactivator recruitment, and epigenetic modification at the downstream ␤-globin gene. Underlying these effects, ectopic HS5 disrupts chromatin loop formation between ␤-globin and the LCR, and instead forms a new loop with endogenous HS5 that topologically isolates the LCR. Both enhancer-blocking and insulatorloop formation depend on an intact CTCF site in ectopic HS5 and are sensitive to knock-down of the CTCF protein by siRNA. Thus, intrinsic looping activity of CTCF sites can nullify LCR function.beta-globin genes ͉ insulator ͉ locus control region ͉ epigenetics ͉ transcription regulation C hromatin insulators are thought to establish domains within which proper enhancer-gene interactions occur: these domains can be visualized in Drosophila cells where a protein complex including Su(Hw) forms loops that tether gypsy insulators to the nuclear lamina (1, 2). Insulators can also interfere with enhancer-gene interaction when placed between the two elements, but the molecular mechanisms underlying enhancerblocking are not well understood.In vertebrates, the protein CTCF is associated with enhancerblocking (3). On maternal chromosomes, the CTCF-dependent imprinting control region (ICR) in the Igf2/H19 locus is thought to form a small loop with a second site, DMR1, upstream of Igf2 that includes Igf2 and restricts access to the gene by enhancers it shares with H19 (4, 5). Because detection of CTCF at DMR1 is dependent on its interaction at ICR, it is not clear whether CTCF or other proteins interact at DMR1 and participate in the long-range interactions. Other data indicate the ICR directly contacts Igf2 and the enhancers (6), suggesting that a complex series of chromosomal interactions may be involved in enhancerblocking at this locus. CTCF also mediates the enhancerblocking activity of the chicken ␤-globin insulator, 5ЈHS4, which forms the upstream border of the globin locus (7). Although a prediction (8), enhancer-blocking through loop formation by interacting CTCF insulator sites has not been demonstrated.Locus control regions (LCRs) are complex enhancers that activate genes over long distances through their ability to establish close contacts with target promoters (9). For example, the ␤-globin LCR and active globin genes come into proximity to form a chromatin loop in erythroid cells (10, 11), an interaction that requires the erythroid factors GATA-1 and erythroid Kruppel-like factor (EKLF) (12, 13). The human ␤-globin LCR is composed of four DNase I hypersensitive sites, HS1 to HS4, far upstream of the structural genes. HS5, 3-Kb upstream of HS4, is more widely ...

Cell type specificity of chromatin organization mediated by CTCF and cohesin

“…S1). Importantly, the optimized ACME parameters identified peaks at the locus flanking β-globin HS5 and 3′HS1 sites where numerous reports have documented CTCF binding in vivo (10,13,16); these sites had not been consistently identified as peaks in earlier genome scans (17,18). In other respects, such as the degree of overlap in CTCF sites between cell types and the CTCF motif shared by the sites, there was a high level of agreement between this analysis and the earlier studies (Fig.…”

“…The 3C assay was performed as described (13). Briefly, formaldehyde-fixed nuclei were digested with EcoRI to generate conveniently sized fragments, followed by ligation with T4 DNA ligase at 16°C for 4 h. Cross-links were reversed, and DNA was extensively purified before PCR amplification.…”

“…In a β-globin transgenic mouse model, we showed that CTCFbinding HS5 has intrinsic, portable enhancer blocking activity (13). When an extra copy of HS5 was placed between the locus control region (LCR) and downstream genes, it formed a new insulator loop with endogenous HS5 that topologically isolated the LCR and nullified its activity.…”

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells

“…Enhancer-blocking insulators are defined by their ability to prevent enhancer activation of the promoter when placed between the enhancer and the promoter. A large body of evidence is consistent with the idea that insulators work by binding proteins that form DNA loops to other insulators (31)(32)(33)(34)(35). This mechanism of insulator action is rationalized by the loop domain model (36), which proposes that the formation of a DNA loop creates a separate topological domain that inhibits interaction between any site within the loop and any site outside the loop (Fig.…”

Tumour-specific methylation of PTPRG intron 1 locus in sporadic and Lynch syndrome colorectal cancer

“…Differential binding of the insulator protein CTCF could have a major influence on expression of distant genes through alternative loop formation, as has been observed in b-globin expression in mouse models. 33 A recent study has shown that loss of CTCF binding to a boundary region upstream of CDKN2A resulted in spreading of repressed chromatin and DNA methylation into the p16 promoter with sequential downregulation of p16 expression. 34 The same study described that loss of upstream CTCF binding resulted in promoter DNA methylation of RASSF1 and CDH1., 34 Contradictory to the finding that CTCF binding abrogation was shown to be causative of heterochromatin spreading and DNA methylation 34,35 is the observation that DNA methylation of CTCF binding sites is suggested to regulate CTCF binding.…”