A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.     

Many facades of CTCF unified by its coding for three-dimensional genome architecture

CCCTC-binding factor (CTCF) is a multifunctional zinc finger protein that is conserved in metazoan species. CTCF is consistently found to play an important role in many diverse biological processes. CTCF/cohesin-mediated active chromatin ‘loop extrusion’ architects three-dimensional (3D) genome folding. The 3D architectural role of CTCF underlies its multifarious functions, including developmental regulation of gene expression, protocadherin (Pcdh) promoter choice in the nervous system, immunoglobulin (Ig) and T-cell receptor (Tcr) V(D)J recombination in the immune system, homeobox (Hox) gene control during limb development, as well as many other aspects of biology. Here, we review the pleiotropic functions of CTCF from the perspective of its essential role in 3D genome architecture and topological promoter/enhancer selection. We envision the 3D genome as an enormous complex architecture, with tens of thousands of CTCF sites as connecting nodes and CTCF proteins as mysterious bonds that glue together genomic building parts with distinct articulation joints. In particular, we focus on the internal mechanisms by which CTCF controls higher order chromatin structures that manifest its many façades of physiological and pathological functions. We also discuss the dichotomic role of CTCF sites as intriguing 3D genome nodes for seemingly contradictory ‘looping bridges’ and ‘topological insulators’ to frame a beautiful magnificent house for a cell's nuclear home.     

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF,WAPL, and PDS5 proteins

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome‐wide function in mediating long‐range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.     

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

On the choreography of genome folding: A grand pas de deux of cohesin and CTCF

Cohesin and CTCF are key to the 3D folding of interphase chromosomes. Cohesin forms chromatin loops via loop extrusion, a process that involves the formation and enlargement of DNA loops. The architectural protein CTCF controls this process by acting as an anchor for chromatin looping. How CTCF controls cohesin has long been a mystery. Recent work shows that CTCF dictates chromatin looping via a direct interaction of its N-terminus with cohesin. CTCF's ability to regulate chromatin looping turns out to also be partially dependent on several RNA-binding domains. In this review, we discuss recent insights and consider how cohesin and CTCF together may orchestrate the folding of the genome into chromosomal loops.