Defining the relative and combined contribution of CTCF and CTCFL to genomic regulation

Abstract

Background Ubiquitously expressed CTCF is involved in numerous cellular functions, such as organizing chromatin into TAD structures. In contrast, its paralog, CTCFL, is normally only present in the testis. However, it is also aberrantly expressed in many cancers. While it is known that shared and unique zinc finger sequences in CTCF and CTCFL enable CTCFL to bind competitively to a subset of CTCF binding sites as well as its own unique locations, the impact of CTCFL on chromosome organization and gene expression has not been comprehensively analyzed in the context of CTCF function. Using an inducible complementation system, we analyze the impact of expressing CTCFL and CTCF-CTCFL chimeric proteins in the presence or absence of endogenous CTCF to clarify the relative and combined contribution of CTCF and CTCFL to chromosome organization and transcription. Results We demonstrate that the N terminus of CTCF interacts with cohesin which explains the requirement for convergent CTCF binding sites in loop formation. By analyzing CTCF and CTCFL binding in tandem, we identify phenotypically distinct sites with respect to motifs, targeting to promoter/intronic intergenic regions and chromatin folding. Finally, we reveal that the N, C, and zinc finger terminal domains play unique roles in targeting each paralog to distinct binding sites to regulate transcription, chromatin looping, and insulation. Conclusion This study clarifies the unique and combined contribution of CTCF and CTCFL to chromosome organization and transcription, with direct implications for understanding how their co-expression deregulates transcription in cancer.

CTCF as a boundary factor for cohesin-mediated loop extrusion: evidence for a multi-step mechanism

“…First, the simplest model invokes direct protein-protein interaction between CTCF and cohesin ( Figure 3d , 2). This model is supported by the observed CTCF-cohesin co-immunoprecipitation [ 36 , 40 , 41 , 70 , 104 , 106 ] and Li et al . recently elucidated the structural basis for this interaction [ 40 ] by solving the crystal structure of a short CTCF peptide (N-terminal amino acids 222–231) bound to the RAD21-STAG2 interface.…”

“…Though analyses of CTCF paralog CTCFL are informative here: CTCFL has a nearly identical 11ZF domain and motif preference to CTCF. While CTCFL cannot rescue CTCF-mediated genome organization [ 41 ], a CTCF-CTCFL chimera with the N-terminus and ZF1 + 2 from CTCF and ZF3-11 and C-terminus from CTCFL was able to largely rescue cohesin ChIP-Seq binding [ 39 ], though loop-resolution Hi-C analyses of this chimera were not performed. Beyond CTCF-DNA interactions, this roadblock model may also include CTCF-RNA interactions [ 36 , 37 , 39 ].…”

“…Originally, a small region C-terminal to CTCF’s 11 Zinc Finger domain was reported to be necessary and sufficient for binding the STAG2 subunit of cohesin and this was assumed to be the only direct interaction between CTCF and the cohesin complex [ 103 ]. However, subsequent studies found that ΔRBR i -CTCF, whose deletion encompasses this region, co-immunoprecipitated cohesin equally well as wild-type CTCF [ 36 , 41 , 104 ], indicating that this region is not required for the biochemical CTCF-cohesin interaction, though it does contribute to loop formation [ 36 ]. Instead, despite different experimental systems and different read-outs, the recent papers all reached remarkable agreement that it is the CTCF-N-terminus as well as the first two Zinc Fingers that play key roles in the CTCF-cohesin interaction, though notably, neither region is autonomously sufficient [ 39 – 42 , 105 ].…”

“…Instead, despite different experimental systems and different read-outs, the recent papers all reached remarkable agreement that it is the CTCF-N-terminus as well as the first two Zinc Fingers that play key roles in the CTCF-cohesin interaction, though notably, neither region is autonomously sufficient [ 39 – 42 , 105 ]. Notably, placing the CTCF N-terminus on the C-terminal side does not fully recapitulate function [ 41 , 42 ]. Here, we discuss three mechanistic models, that are not mutually exclusive, for the interaction between CTCF and cohesin: direct protein-protein interaction, CTCF inactivating cohesin’s ATPase function, and CTCF stabilizing cohesin on DNA by antagonizing WAPL-mediated cohesin release ( Figure 3d ).…”

“…AID-tagged CTCF [ 28 , 29 ]) [ 37 , 41 , 42 ] or alternatively in a CTCF-mutant background [ 39 ] (though this is challenging to generalize and leads to co-expression of two versions of CTCF). The inducible complementation approach [ 37 , 41 , 42 ] is more general and 1) allows studying both lethal and non-lethal mutants; 2) allows some distinction between direct and secondary effects; 3) allows higher throughput. However, disadvantages include: 1) some AID-clones tend to have significant residual wt-protein; 2) it can difficult to achieve physiological expression.…”