Abstract
Proteins with domains that recognize and bind post-translational modifications (PTMs) of histones are collectively termed epigenetic readers. Numerous interactions between specific reader protein domains and histone PTMs and their regulatory outcomes have been reported, but little is known about how reader proteins may in turn be modulated by these interactions. Tripartite motif-containing protein 24 (TRIM24) is a histone reader aberrantly expressed in multiple cancers. Here, our investigation revealed functional cross-talk between histone acetylation and TRIM24 SUMOylation. Binding of TRIM24 to chromatin via its tandem PHD-bromodomain, which recognizes unmethylated lysine 4 and acetylated lysine 23 of histone H3 (H3K4me0/K23ac), led to TRIM24 SUMOylation at lysine residues 723 and 741. Inactivation of the bromodomain, either by mutation or with a small-molecule inhibitor, IACS-9571, abolished TRIM24 SUMOylation. Conversely, inhibition of histone deacetylation markedly increased TRIM24's interaction with chromatin and its SUMOylation. Of note, gene expression profiling of MCF7 cells expressing WT versus SUMO-deficient TRIM24 identified cell adhesion as the major pathway regulated by the cross-talk between chromatin acetylation and TRIM24 SUMOylation. In conclusion, our findings establish a new link between histone H3 acetylation and SUMOylation of the reader protein TRIM24, a functional connection that may bear on TRIM24's oncogenic function and may inform future studies of PTM cross-talk between histones and epigenetic regulators.
Highlights
Proteins with domains that recognize and bind post-translational modifications (PTMs) of histones are collectively termed epigenetic readers
We addressed this question by focusing on Tripartite motif– containing protein 24 (TRIM24), known as transcription intermediary factor 1␣ (TIF1␣), a plant homeodomain (PHD)6/bromodomain acetyllysine reader protein [7]
We find that TRIM24 association with chromatin is a prerequisite for TRIM24 SUMOylation by SUMO1 and SUMO2/3 proteins, and this association and SUMOylation are dependent on the TRIM24 PHD-bromodomain
Summary
Post-translational modification by SUMO isoforms results in slowly migrating, higher-molecular weight forms of substrate proteins when separated by SDS-PAGE. FLAG-TRIM24 WT and mutant proteins were expressed in HEK293T cells, immunoprecipitated using FLAG antibody, and subjected to in vitro SUMOylation (Fig. 2C). Immunoblot analysis of whole-cell lysates from HEK293T cells expressing FLAG-TRIM24 WT and single SUMO site mutants of TRIM24 showed that TSA-mediated up-regulation of TRIM24 SUMOylation occurred at both Lys723 and Lys741 (Fig. 3B). To annotate and assess the biological roles of the specific subset of TRIM24-regulated genes dependent on the SUMOylation state of TRIM24, we used Database for Annotation, Visualization and Integrated Discovery (DAVID) to determine enrichment of biological process and cellular component gene ontology terms [28, 29] This analysis showed an enrichment of cell adhesion, plasma membrane, and extracellular region components among all genes differentially expressed, whether up- or down-regulated, in TRIM24 WT versus DM-expressing MCF7 cells Compared with TRIM24 WT-expressing cells, TRIM24 DM-expressing cells showed reduced cell adhesion to several ECM proteins (collagen I, collagen II, fibronectin, laminin, and tenascin), supporting a role for SUMO modification of TRIM24 in regulation of pathways associated with cell adhesion
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