From peptide screens to biological insights, FOXO4 and the PBAF complex

Bromodomains are ubiquitous among chromatin regulators, and while they are potentially exciting therapeutic targets for cancer, the specific functions of many of them remain unknown. In a high-throughput peptide screen for potential bromodomain binding partners, acetylated peptides of transcription factors FOXO1/3/4 emerged as significant binders to the polybromo protein PBRM1, a subunit of the SWI/SNF chromatin remodeler PBAF. FOXOs are of particular interest because they are well-conserved mediators of stress adaptation in cells and are regulated by several PTMs. Under oxidative stress, FOXOs are acetylated and translocate to the nucleus, where PBRM1, within PBAF, resides. We sought to characterize the interaction between acetylated FOXOs and PBRM1 and their potential cooperation in stress responses. (1) We validated the interaction by Isothermal titration calorimetry (ITC) and found acetylated FOXO peptides bind to PBRM1 bromodomains with affinities comparable to peptides including the preferred histone modification, H3K14ac. (2) Using purified, site-specifically acetylated FOXO4 proteins as bait, we purified interactors for mass spec analysis, finding that FOXO4 acetylated at K189 interacts with PBAF components. (3) We employed genomic profiling assays ATAC-seq, CUT&RUN-seq, and Bru-Seq to dissect the role of FOXO4 under endogenous conditions versus oxidative stress, with select studies also performed in FOXO4 CRISPR knockout cells. We identified peaks where FOXO4 and PBRM1 co-localize and exhibit changes in peak intensity under acute stress. We also established a transcriptional profile of cellular response to oxidative stress and compared affected genes to the genes bound by FOXO4 and PBRM1. While our findings suggest that the FOXO4-PBRM1 interaction may not be the key driver of transcription in the oxidative stress response, this framework allows us to systematically assess other potential functions of the interaction. Together, by combining biophysical, biochemical, cell growth assays, and functional genomics assays, we have defined a likely aspect of the biology of the tumor suppressor PBRM1, namely regulation of FOXO transcription factor genomic localization.