A pioneering approach advances the study of the CTCF protein in transcriptional biology

A pioneering approach advances the study of the CTCF protein in transcriptional biology

A pioneering approach advances the study of the CTCF protein in transcriptional biology

L to R: Beisi Xu, Ph.D., Chunliang Li, Ph.D.; Judith Hyle; Mohamed Nachir Djékidel, Ph.D. Credit: St. Mary’s Children’s Research Hospital. Jude

CTCF is a critical protein known to play a variety of roles in key biological processes such as transcription. Scientists at St. Jude Children’s Research Hospital used next-generation protein degradation technology to study CTCF. Their work demonstrated the superiority of the approach as well as providing functional insights into how CTCF regulates transcription. The study, published today in Genome Biologypaves the way for clearer, more modern studies of CTCF.

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

Although the biology of the CTCF has been extensively studied, it remains unclear how the different domains (parts) of the CTCF function in transcriptional regulation.

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, providing insight into how a protein affects a cell. One system for degrading proteins is the auxin-inducible degron 1 (AID1) system. However, this system places limitations on the accurate investigation of CTCF function, such as the high-dose dependence of action, leading to cellular toxicity that results in confounding results.

Scientists in St. Jude the second-generation, auxin-inducible degron 2 (AID2) system applied to CTCF (the system was developed by Masato Kanemaki, Ph.D. at the National Institute of Genetics). 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 have opened up the understanding of the impact of CTCF using a degradation model, the AID2 system,” said corresponding author Chunliang Li, Ph.D., Department of Tumor Cell Biology St. “Using this system, we identified the rules that govern CTCF-dependent transcriptional regulation.”

“When the CTCF protein is gone, we and others have noticed that few genes change transcriptionally,” Li said. “We know that when we remove most of the CTCF protein in cells, the impact on transcription is minimal. Therefore, the disconnection between protein depletion and transcription must follow a mechanism. We have identified part of the mechanism The protein not only relies on binding to the DNA by recognizing the CTCF DNA binding motif, but also relies on certain domains to bind to specific sequences adjacent to the motif. For a subset of genes, the transcription only when CTCF binds to these specific sequences.”

‘Exchange system’ provides insight into the role of zinc finger domains

The 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 change happens very quickly, so we call it a fast-switching system,” Li said. “This is the first time that a clean and rapid exchange system has been used to study CTCF single mutants.”

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

“CTCF itself is a multifunctional protein,” said co-first author Judith Hyle, St. John’s Department of Tumor Cell Biology. Jude. “It has different roles in a cell, from the maintenance of chromatin architecture to the regulation of transcription, 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 CTCF degrade much faster. , and home to the specific targets of the CTCF We were able to assign some function to these poorly understood peripheral zinc fingers, showing that certain regions within the genome required or depended on the connections this zinc fingers for transcriptional control. That was the first time it was 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 through their model. The scientists then performed functional studies to understand the consequences of such mutations for CTCF binding and transcriptional regulation.

Speaking about the new approach, co-first author Mohamed Nadhir Djekidel, Ph.D., of the St. Jude of Applied Bioinformatics, “Because you can get clean data about the mutants when an endogenous protein is degraded, you can draw the conclusion. 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 shows the importance of studying CTCF in a clear system. This is an important validation for other researchers in the field of transcriptional regulation research. The work also revealed new ways to research this key protein.

Beisi Xu, Ph.D., St. Jude for Applied Bioinformatics is the corresponding author of the study. Additional authors are Justin Williams, Shaela Wright and Ying Shao of St. Jude.

More information:
Judith Hyle et al. Genome Biology (2023).

Provided by St. Mary’s Children’s Research Hospital. Jude

Quote: Advances approach to study CTCF protein in transcription biology (2023, January 25) Retrieved January 25, 2023 from https://phys.org/news/2023-01-approach-advances-ctcf-protein-transcription .html

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