Guided nuclear exploration increases CTCF target search efficiency

Abstract

The enormous size of mammalian genomes means that for a DNA-binding protein, the number of non-specific, off-target sites vastly exceeds the number of specific, cognate sites. How mammalian DNA-binding proteins overcome this challenge to efficiently locate their target sites is not known. Here through live-cell single-molecule tracking, we show that CCCTC-binding factor, CTCF, is repeatedly trapped in small zones that likely correspond to CTCF clusters, in a manner that is largely dependent on an internal RNA-binding region (RBR i). We develop a new theoretical model, Anisotropic Diffusion through transient Trapping in Zones (ADTZ), to explain CTCF dynamics. Functionally, transient RBR i-mediated trapping increases the efficiency of CTCF target search by ~2.5 fold. Overall, our results suggest a "guided" mechanism where CTCF clusters concentrate diffusing CTCF proteins near cognate binding sites, thus increasing the local ON-rate. We suggest that local guiding may allow DNA-binding proteins to more efficiently locate their target sites. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

An RNA-binding region regulates CTCF clustering and chromatin looping

“…Next, we analyzed our CTCF and cohesin (Smc1a) ChIP-Seq data for wt-CTCF and RBR-CTCF mESCs in more detail (Figure 6B; Figure S5B). Consistent with FRAP experiments, which showed no change in residence time at cognate binding sites for RBR-CTCF (Hansen et al, 2018b), RBR-CTCF also still binds the majority of CTCF sites, although the number and occupancy levels were generally reduced (63% of 81,785 wt-CTCF ChIP-Seq peaks maintained in RBR-CTCF mESCs; spike-in normalized ChIP-Seq in Figure S5B). Similarly, about 60% of the cohesin binding sites detected in wt-CTCF mESCs were also occupied in RBR-CTCF mESCs ( Figure 6C, Figure S5B).…”

“…In a companion paper focused on the biophysics of nuclear sub-diffusion (Hansen et al, 2018b), we show using single-particle tracking experiments that CTCF exhibits unusual anisotropic diffusion: once CTCF has moved in one direction, it is substantially more likely to move backwards, than to continue forwards. Our theoretical work suggests that this is due to transient trapping in localized zones/domains of a characteristic size (~200 nm).…”

“…Moreover, we show that CTCF's cognate DNA-target search mechanism is RBR-guided, such that RBR-CTCF takes ~2.5-fold longer to locate a cognate DNAbinding site. We provide full details on the theory and analysis of the diffusion mechanism in our companion paper (Hansen et al, 2018b). Here we focus on the primary function of CTCF, which is to regulate 3D genome organization.…”

Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains

“…Finally, it is tempting to speculate a connection between our identified clusters of closely located CTCF binding sites on the genome and the reportedly observed 3D "clusters" (or "hubs") of CTCF protein molecules (Hansen et al, 2018b(Hansen et al, , 2018c. In particular, Hansen et al have proposed a guided mechanism where an RNA strand can bind to and gather together multiple CTCF protein molecules near cognate binding sites.…”

“…In particular, Hansen et al have proposed a guided mechanism where an RNA strand can bind to and gather together multiple CTCF protein molecules near cognate binding sites. These CTCF molecule hubs apparently enhance the search 15 for target binding sites, increase the binding rate of CTCF to its related sites (also as part of the LMC model) and are often implicated in chromatin loop formation (Hansen et al, 2018b(Hansen et al, , 2018c. It is possible that our identified CTCF site clusters act synergistically with this mechanism as nearby sites for the concentrated CTCF molecules to bind.…”