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

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

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

From left to right: Beisi Xu, Ph.D., Chunliang Li, Ph.D.; Judith Hyle; Mohamed Nadhir Djekidel, Ph.D. Credit: St. Jude Children’s Research Hospital

CTCF is a critical protein known to play several roles in key 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 revealed the superiority of the approach in addition to providing functional information on how CTCF regulates transcription. The study, published today in genome biologypaves the way for clearer and 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 the instructions housed in DNA and ultimately translate that code into 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 transcriptional machinery is a new frontier in the search for vulnerabilities that can be exploited therapeutically.

While the biology of CTCF has been extensively studied, it is still unclear how the different domains (parts) of CTCF function in relation to the regulation of transcription.

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 information about how the protein influences a cell. One system for degrading proteins is the auxin-inducible grade 1 (AID1) system. However, this system imposes limitations to the precise investigation of CTCF function, such as high auxin dose dependence, leading to cell toxicity confounding the results.

St. Jude scientists applied the second-generation, auxin-inducible grade 2 (AID2) system 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, overcomes the limitations of the AID1 system, and eliminates 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 co-corresponding author Chunliang Li, Ph.D., St. Jude Department of Tumor Cell Biology. “Using this system, we identified the rules governing the regulation of CTCF-dependent transcription.”

“When the CTCF protein is missing, we and others have observed that very 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 disconnect between protein depletion and transcription must follow a mechanism. We identified part of the mechanism. protein not only relies on DNA binding through 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 alone when CTCF binds to these specific sequences”.

‘Swap system’ sheds light on the role of zinc finger domains

The researchers combined the AID2 system with state-of-the-art 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 fast, so we call it a fast swap system,” Li said. “This is the first time that a clean, rapid exchange system has been used to study individual CTCF mutants.”

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

“CTCF itself is a multifunctional protein,” said co-author Judith Hyle, of the St. Jude Department of Tumor Cell Biology. “It has several functions in a cell, from maintaining chromatin architecture to regulating transcription, either as a transcription activator or repressor. Our interest is how CTCF participates in transcriptional regulation, and with this new system we were able to degrade CTCF much more rapidly and zero in on the specific targets of CTCF We were able to assign some function to these poorly understood peripheral zinc fingers, demonstrating that certain regions within the genome required or depended on these zinc finger linkages for transcriptional regulation. That was the first time it had been seen or confirmed in a cellular system.”

An open door for future research.

The superior system allowed the researchers to introduce mutations that could be tracked through their model. Next, the scientists performed functional studies to understand the consequences of such mutations in relation to CTCF binding and transcriptional regulation.

Speaking about the new approach, co-author Mohamed Nadhir Djekidel, Ph.D., St. Jude Center for Applied Bioinformatics, said: “Because clean data on mutants can be obtained when the endogenous protein is degraded, you can actually inferring the gene regulatory network, and that opens the door to different 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 verification for other researchers in the field of transcriptional regulation research. The work has also revealed new avenues for investigation of this key protein.

The study’s corresponding co-author is Beisi Xu, Ph.D., St. Jude Center for Applied Bioinformatics. Other authors include Justin Williams, Shaela Wright, and Ying Shao of St. Jude.

More information:
Judith Hyle et al. genome biology (2023).

Provided by St. Jude Children’s Research Hospital

Citation: Pioneering Approach Advances Study of CTCF Protein in Transcription Biology (Jan 25, 2023) Retrieved Jan 25, 2023 from https://phys.org/news/2023-01-approach-advances -ctcf-protein-transcription.html

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