Second-gen protein degradation technology revealed new avenues for CTCF research
January 31, 2023 | Biology
To intensively study a protein and how it influences the cell, it is degraded or removed from the model system so that researchers can observe the functional changes that can subsequently occur. Degradation enables the creation of a very clean background where researchers can introduce a mutant. One system for degrading proteins is the auxin-inducible degron 1 (AID1) system. However, it has limitations including a high dosage dependency on auxin, which causes cellular toxicity that affects the result.
Developed by Masato Kanemaki, Ph.D., at the National Institute of Genetics, auxin-inducible degron 2 (AID2) was applied by the scientists. This system is superior for loss-of-function studies and it addressed the limitations of the AID1 system. This allowed researchers to introduce mutations that could be tracked with their model.
Combining the AID2 system with SLAM-seq and sgRNA screening in studying how the degradation of CTCF modifies transcription, the swapping system revealed the role of the zinc finger (ZF) domain. The ZF domain is the region within CTCF with the most functional relevance, involving ZF1 and ZF10. The removal of these two from the model system uncovered the genomic regions that independently need these ZFs in binding DNA and regulating transcription.
With this superior system, scientists were able to perform further functional studies to understand the effects of such mutations. They are able to infer the gene regulatory network which gives way for far more extensive downstream analysis in understanding how regulation works.