Author ORCID Identifier
Date of Graduation
Genes and Development
Doctor of Philosophy (PhD)
Michelle C Barton, PhD
Guillermina Lozano, PhD
Traver Hart, PhD
Nicholas Navin, PhD
Alastair Thompson, MBChD, MD, FRCS(Ed)
John Weinstein, MD, PhD
Wendy Woodward, MD, PhD
TRIM24 is an oncogenic chromatin reader that is frequently overexpressed in human tumors and associated with poor prognosis. However, TRIM24 is rarely mutated, duplicated, or rearranged in cancer. This raises questions about how TRIM24 is regulated and whether changes in its regulation are responsible for its activity in cancer.
To investigate this possibility, I performed a genomewide CRISPR/Cas9 screen library using fluorescence activated cell sorting (FACS) to identify regulators of TRIM24. The screen was enabled by two innovations. I engineered cells with an in-frame knock-in of mClover3 to the endogenous copy of TRIM24 to allow fluorescent monitoring of TRIM24 expression by flow cytometry. To overcome the limits of current scoring algorithms for CRISPR screens when applied to data from screens performed by FACS, I also developed and credentialed an algorithm specifically for analysis of FACS screens.
The screen nominated 220 candidate negative regulators that revealed a network of cellular processes and known repressor complexes upstream of TRIM24 by STRING analysis. Primary validation of the screen using single gene perturbation experiments demonstrated 93% specificity and 89% sensitivity for candidates nominated at an FDR<10%. Targeted CRISPR knockouts of required components of the three repressor complexes in the network caused TRIM24 overexpression, confirming their negative regulation of TRIM24. Interestingly, human genetic disorders leading to loss of function for the complexes validated in this study phenotypically converge on pathologies in neurodevelopment.
The results of this study expand our understanding of TRIM24 regulation, nominates previously unexplored contexts for this gene in biology and disease, and will serve as a resource for researchers investigating TRIM24. The algorithm presented here will also serve as resource for various genomic screens using FACS to enrich cells.
TRIM24, Gene Regulation, CRISPR/Cas9, Genomewide Screen, Fluorescence Activated Cell Sorting, Analysis of CRISPR Screen
Available for download on Wednesday, August 02, 2023