High affinity binding of H3K14ac through collaboration of bromodomains 2, 4 and 5 is critical for the molecular and tumor suppressor functions of PBRM1

• Published in: Molecular oncology Vol. 13; no. 4; pp. 811 - 828
• Main Authors: Liao, Lili, Alicea‐Velázquez, Nilda L., Langbein, Lauren, Niu, Xiaohua, Cai, Weijia, Cho, Eun‐Ah, Zhang, Meiling, Greer, Celeste B., Yan, Qin, Cosgrove, Michael S., Yang, Haifeng
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.04.2019; John Wiley and Sons Inc; Wiley
• Subjects: Acetylation; Amino Acid Sequence; Animals; bromodomain; Cell Line; Chromatin; Ethylenediaminetetraacetic acid; Gene expression; Gene Expression Regulation, Neoplastic; Gene mutations; H3K14ac; Histones - metabolism; kidney cancer; Lysine; Lysine - metabolism; Mice, Nude; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; PBRM1; Point Mutation - genetics; Promoter Regions, Genetic - genetics; Protein Binding; Protein Domains; Structure-Activity Relationship; synergy; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Tumor Suppressor Proteins - chemistry; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; Tumors; kidney cancer; bromodomain; PBRM1; H3K14ac; synergy
• ISSN: 1574-7891; 1878-0261; 1878-0261
• DOI: 10.1002/1878-0261.12434

Polybromo‐1 (PBRM1) is an important tumor suppressor in kidney cancer. It contains six tandem bromodomains (BDs), which are specialized structures that recognize acetyl‐lysine residues. While BD2 has been found to bind acetylated histone H3 lysine 14 (H3K14ac), it is not known whether other BDs collaborate with BD2 to generate strong binding to H3K14ac, and the importance of H3K14ac recognition for the molecular and tumor suppressor function of PBRM1 is also unknown. We discovered that full‐length PBRM1, but not its individual BDs, strongly binds H3K14ac. BDs 2, 4, and 5 were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement of the interactions between purified BD proteins and H3K14ac or nonacetylated peptides confirmed the tight and specific association of the former. Interestingly, while the structural integrity of BD4 was found to be required for H3K14ac recognition, the conserved acetyl‐lysine binding site of BD4 was not. Furthermore, simultaneous point mutations in BDs 2, 4, and 5 prevented recognition of H3K14ac, altered promoter binding and gene expression, and caused PBRM1 to relocalize to the cytoplasm. In contrast, tumor‐derived point mutations in BD2 alone lowered PBRM1's affinity to H3K14ac and also disrupted promoter binding and gene expression without altering cellular localization. Finally, overexpression of PBRM1 variants containing point mutations in BDs 2, 4, and 5 or BD2 alone failed to suppress tumor growth in a xenograft model. Taken together, our study demonstrates that BDs 2, 4, and 5 of PBRM1 collaborate to generate high affinity to H3K14ac and tether PBRM1 to chromatin. Mutations in BD2 alone weaken these interactions, and this is sufficient to abolish its molecular and tumor suppressor functions. Bromodomains (BDs) 2, 4, and 5 of polybromo‐1 (PBRM1) were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement confirmed this. Simultaneous point mutations in these bromodomains prevented recognition of H3K14ac, altered promoter binding and gene expression, and relocalized PBRM1 to the cytoplasm. In contrast, tumor‐derived point mutations in BD2 alone disrupted the molecular and tumor suppressor functions of PBRM1.