EAGER: Tools4Cells: Tools for locus-specific and interaction-specific protein depletion

The proposed research will develop a new experimental technique, to selectively remove a specific protein in a cell, but only when this protein is attached to a specific, unique spot on a chromosome. Many genetic and biochemical methods of protein depletion are known, but all of them deplete protein throughout the cell. If one wishes to study how a DNA-binding protein does its job in a cell, these methods likewise will eliminate the protein whether or not it is bound anywhere on DNA.

In contrast, the proposed technology will eliminate only those protein molecules that are bound to a specific region of interest. The project will provide training for up to four graduate students in the EPSCOR state of Kansas.

The proposed novel method will involve tagging a protein of interest with an auxin-inducible degron (AID), which has been used before to rapidly deplete nearly all the protein target throughout any cell when the plant hormone auxin is added extraneously. If a protein has multiple functions within a cell, however, this depletion can have multiple direct and secondary effects.

The proposed High Risk/High Reward research will conbine several genomic technologies in a novel way, to develop, first, a method to selectively deplete the chromatin-bound Rhino-Deadlock-Cutoff (RDC) complex at specific locus that flanks a transposable element insertion in Drosophila. Next, the method will be applied to human cells and extended to deplete specifically a complex of two interacted proteins, CTCF and cohesin, without affecting each of the proteins alone.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.