Polybromo‐1 missense mutations found in renal cancer patients affect bromodomain stability and biological function

Abstract

Chromatin structure is important in modulating gene expression. A central component of chromatin structure is the covalent post‐translational modification (PTM) of histones. Distinct histone PTMs can determine the accessibility of chromatin and recruit different effector proteins and components of the transcriptional machinery. Histone are modified by “writer” and “eraser” enzymes that can add and remove PTMs, which are recognized by evolutionarily conserved reader domains. Bromodomain is one type of evolutionarily conserved “reader” domain composed of ~110 amino acids, including a conserved asparagine that specifically binds acetylated lysine. There are approximately 80 bromodomains in humans, primarily in chromatin‐associated proteins. Most proteins contain one or two bromodomains. The exception is Polybromo‐1, which contains six tandem bromodomains in addition to two bromo‐adjacent homolog (BAH) domains involved in protein‐protein interactions and a high mobility group (HMG) for interacting with DNA. PBRM1 is a subunit of the polybromo‐associated BAF (PBAF) chromatin remodeling complex, one of three major SWI/SNF subcomplexes found humans. These chromatin remodeling complexes alter chromatin accessibility by evicting, sliding and exchanging nucleosomes on DNA. In clear cell renal cell cancer carcinoma (ccRCC), the most common subtype of kidney cancer, PBRM1is the second most mutated gene after VHL, with PBRM1mutations found in approximately 40% of ccRCC patients. Mouse models indicate that PBRM1 deletion paired with VHL deletion in the kidney results in tumors, establishing PBRM1 as a bona fide tumor suppressor. While most PBRM1 mutations in patients are also loss of function mutations, ~15‐20% of PBRM1 mutations are missense mutations, with a large portion found at highly conserved residues in the fourth bromodomain (BD4). Using recombinant PBRM1 BD4 mutants, we have assessed how the most common missense mutations found in patients affect the stability and acetyllysine binding of PBRM1 BD4, revealing new insight into bromodomain structure and function. In addition we have expressed a subset of these PBRM1 missense mutations in renal cancer cell lines and assessed how they affect PBRM1 binding to acetyllysine substrates, and subsequent renal cancer growth.