Individual Bromodomains of Polybromo-1 Contribute to Chromatin Association and Tumor Suppression in Clear Cell Renal Carcinoma [Gene Regulation]

The architecture of chromatin is governed, in part, by adenosine triphosphate (ATP)-dependent chromatin remodelers. These multi-protein complexes contain targeting domains that recognize posttranslational marks on histones. One such targeting domain is the bromodomain (BD), which recognizes acetyl-lysines and recruits proteins to sites of acetylation across the genome. Polybromo1 (PBRM1), a subunit of the polybromo-associated BRG1- or hBRM-associated factors (P-BAF) chromatin remodeler, contains six tandem BDs and is frequently mutated in Renal Clear Cell Carcinoma (ccRCC). Mutations in the PBRM1 gene often lead to loss of protein expression; however, missense mutations in PBRM1 have been identified and tend to cluster in the BDs, particularly BD2 and BD4, suggesting that individual BDs are critical for PBRM1 function. To study the role of these six BDs, we inactivated each of the six BDs of PBRM1 and re-expressed these mutants in Caki2 cells (ccRCC cells with loss of function mutation of PBRM1). Four of the six BDs abrogated PBRM1 tumor suppressor, gene regulation, and chromatin affinity with degree of importance correlating strongly to rate of missense mutations in patients. Furthermore, we identified BD2 as the most critical for PBRM1 and confirmed BD2 mediated association to histone H3 peptides acetylated at lysine 14 (H3K14Ac), validating the importance of this specific acetylation mark for PBRM1 binding. From these data we conclude that four of the BDs act together to target PBRM1 to sites on chromatin; when a single BD is mutated, PBRM1 no longer controls gene expression properly, leading to increased cell proliferation.

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