New System Illuminates How CTCF Regulates Transcription

Using AID2, an improved auxin-inducible degron technology, scientists at St. Jude Children’s Research Hospital were able to gain functional insights into how CTCF regulates transcription. The study, published recently in Genome Biology, paves the way for more clear, nuanced studies of CTCF. 

One of the most valuable ways to study a protein is to degrade, or remove, it from a model system. In the protein’s absence, researchers can study the functional changes that occur, providing insight into how the protein influences a cell. One system for degrading proteins is the auxin-inducible degron 1 (AID1) system. However, this system has limitations when investigating the function of CTCF, such as the high dosage dependency of auxin, which causes cellular toxicity that muddles results. 

The St. Jude team applied the second-generation system, auxin-inducible degron 2 (AID2) to CTCF. This system is superior for loss-of-function studies, overcoming the limitations of the AID1 system and eliminating the off-target effects seen with previous approaches. 

“We’ve cracked open the understanding of the impact of CTCF using a degradation model, the AID2 system,” said co-corresponding author Chungliang Li. “Using this system, we identified the rules that govern CTCF-dependent transcription regulation.”

“When the CTCF protein is gone, we and others have observed that very few genes transcriptionally change,” Li said. “We know when we remove most of the CTCF protein in cells, the impact on transcription is minimal. So, the disconnect between the depletion of protein and transcription must be following a mechanism. We identified part of the mechanism. The protein not only relies on binding to the DNA through the recognition of the CTCF DNA binding motif, but also relies on certain domains to bind to specific sequences flanking the motif. For a subset of genes, transcription is regulated only when CTCF binds to these specific sequences.”

The researchers combined the AID2 system with leading-edge techniques such as SLAM-seq and sgRNA screening to study how the degradation of CTCF alters transcription. 

“With degradation we can create a very clean background, and then introduce a mutant. This switch happens very fast, so we call it a fast-swapping system,” Li said. “This is the first time a clean and fast-swapping system has been used to study individual mutants of CTCF.”

Through their work the scientists identified the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZF1 and ZF10. Removing ZF1 and ZF10 from the model system revealed genomic regions that independently require these ZFs for binding DNA and regulating transcription.

“CTCF itself is a multifunctional protein,” said co-first author Judith Hyle. “It has various roles in a cell from chromatin architecture maintenance to transcription regulation, either as an activator or repressor of transcription. Our interest is how CTCF is involved in transcriptional regulation, and with this new system we were able to degrade CTCF much more rapidly, and home in on the specific targets of CTCF. We were able to assign some function to these peripheral zinc fingers that have not been well understood, showing that certain regions within the genome required or were dependent upon these zinc finger bindings for transcriptional regulation. That was the first time that had been seen or confirmed in a cellular system.”