Groundbreaking approach advances research into CTCF protein in transcription biology

CTCF is an important protein known to play diverse roles in important biological processes such as transcription. Scientists at St. Jude Children’s Research Hospital have used next-generation protein degradation technology to study CTCF. Their work demonstrated the superiority of the approach and provided functional insight into the regulation of transcription by CTCF. The study published today in genomic biologypaves the way for clearer, more nuanced studies of CTCF.

Transcription is an essential biological process in which DNA is copied into RNA. The process is the first step required in a cell to take on the instructions contained in DNA and eventually translate that code into the amino acid or polypeptide building blocks that become active proteins. Dysregulated transcription plays a role in many types of pediatric cancer. Finding ways to modify or target aspects of the transcription machinery is a new frontier in the search for vulnerabilities that can be exploited therapeutically.

While the biology of CTCF has been extensively studied, the function of the different domains (parts) of CTCF in relation to transcriptional regulation remains unclear.

One of the most valuable ways to study a protein is to degrade (remove) it from a model system. In the absence of the protein, researchers can study the functional changes that occur and gain insight into how the protein affects a cell. One system for degrading proteins is the auxin-inducible degron 1 (AID1) system. However, this system limits the detailed study of the function of CTCF, such as B. the high dose-dependency of auxin causing cellular toxicity that confounds the results.

Scientists at St. Jude applied the second-generation system, auxin-inducible degron 2 (AID2), to CTCF (the system was developed by Masato Kanemaki, Ph.D., at the National Institute of Genetics). This system is excellent for loss-of-function studies, overcoming the limitations of the AID1 system and eliminating the off-target effects seen with previous approaches.

“We disrupted the understanding of the effects of CTCF using a degradation model, the AID2 system,” said co-author Chunliang Li, Ph.D., St. Jude Department of Tumor Cell Biology. “Using this system, we have identified the rules governing CTCF-dependent transcription regulation.”

“When the CTCF protein is gone, we and others have observed that very few genes change transcriptionally,” Li said. “We know that when we remove most of the CTCF protein, the effects on transcription are minimal remove in cells. The separation between protein degradation and transcription must therefore follow a mechanism. We have identified part of the mechanism. The protein not only relies on binding to DNA through recognition of the CTCF-DNA binding motif, but also relies on distinct domains to bind to specific sequences flanking the motif. For a subset of genes, transcription is only regulated when CTCF binds to those specific sequences.

“Exchange system” sheds light on the role of zinc finger domains

Researchers combined the AID2 system with cutting-edge techniques such as SLAM-seq and sgRNA screening to study how CTCF degradation alters transcription.

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

Through their work, scientists identified the zinc finger (ZF) domain as the region within CTCF of greatest functional relevance, including ZF1 and ZF10. Removal of ZF1 and ZF10 from the model system revealed genomic regions that these ZFs independently require to bind DNA and regulate transcription.

“CTCF itself is a multifunctional protein,” said co-first author Judith Hyle, St. Jude Department of Tumor Cell Biology. “It has diverse roles in a cell, from maintaining chromatin architecture to regulating transcription, either as an activator or repressor of transcription. We are interested in how CTCF is involved in transcription regulation and with this new system we were able to degrade CTCF much faster. We were able to assign some functions to these peripheral zinc fingers that are not yet well understood, showing that certain regions within the genome required or were dependent on these zinc finger bonds for transcription regulation. This was the first time it had been seen or confirmed in a cellular system.”

An open door for further research

The superior system allowed the researchers to introduce mutations that could be tracked by their model. Scientists then performed functional studies to understand the consequences of such mutations in terms of CTCF binding and transcriptional regulation.

About the new approach, co-first author Mohamed Nadhir Djekidel, Ph.D., St. Jude Center for Applied Bioinformatics, said: “Because you can get clean data about the mutants when endogenous protein is degraded, you can actually infer the gene regulatory network, and that opens the door for various downstream analyzes to understand how regulation works.”

The study demonstrates the superiority of the AID2 system for degrading proteins and demonstrates the importance of studying CTCF in a clear system. This is important confirmation for other researchers in the field of transcription regulation research. The work has also opened up new avenues for studying this key protein.

Co-author of the study is Beisi Xu, Ph.D., St. Jude Center for Applied Bioinformatics. Other authors include Justin Williams, Shaela Wright and Ying Shao of St. Jude.