Labeling of blocked replication fork proteome by BLOCK-ID identifies the chromatin reader protein TRIM24 in promoting Alternative Lengthening of Telomeres
Introduction
Chromatin-based DNA damage response (DDR) pathways are vital for the maintenance of genome integrity, including during DNA replication. Obstacles within the genome can obstruct DNA replication, contributing to genome instability, cell fitness, and human diseases. Emerging evidence has highlighted the vital functions for chromatin reader proteins in these pathways that act by recognizing modified histone and non-histone proteins in the vicinity of DNA damage to promote recognition and resolution of the lesion. As an example, acetylated lysines are recognized by bromodomain-containing “reader” proteins and our previous work has demonstrated that over one-third of the 42 human BRD proteins are recruited to sites of DNA damage and are required for efficient DNA double-strand break (DSB) repair.
Methods
We have developed a new technique called BLOCK-ID, which allows for proteomic profiling using proximity ligation and visualization of proteins at blocked replication forks generated at LacO arrays bound to Lac repressor (LacI) fused to a tagged Bio-ID enzyme.
Results
Through BLOCK-ID, we identified and validated nearly a third of human BRD proteins associated with blocked replication forks in human cancer cells. These include the BRD protein TRIM24, which we demonstrate to be involved in replication stress response pathways. Further proteomic analyses identified TRIM24 involvement in Alternative Lengthening of Telomeres (ALT). TRIM24 deficiency resulted in telomere loss, reduced telomere sister chromatid exchanges, and reduced hallmarks of ALT including c-circles and ALT-associated PML bodies (APB). Mechanistically, TRIM24 ALT activity is governed by Post-translational modifications (PTMs), including acetylation by p300/CBP that supports TRIM24 recruitment to stressed telomeres. Forced tethering of TRIM24 to telomeres revealed PIAS1 SUMO-dependent interactions with PML, revealing a function for TRIM24 in localizing telomeres to APBs to support ALT telomere synthesis.
Conclusion
Collectively, this study delivers a new methodology for surveying replication stress proteins in human cells and identifies a PTM-driven TRIM24 pathway involved in ALT, which provides a rationale for therapeutic targeting of PTM-mediated TRIM24 pathway in ALT cancers.