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. 2017 Feb 17;292(7):2601-2610.
doi: 10.1074/jbc.M116.746875. Epub 2017 Jan 4.

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

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Free PMC article

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

Elizabeth G Porter et al. J Biol Chem. .
Free PMC article

Abstract

The architecture of chromatin is governed, in part, by ATP-dependent chromatin remodelers. These multiprotein complexes contain targeting domains that recognize post-translational 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 (PBAF) chromatin remodeler, contains six tandem BDs and is frequently mutated in clear cell renal cell carcinoma (ccRCC). Mutations in the PBRM1 gene often lead to the 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 the loss of function mutation in PBRM1). Four of the six BDs abrogated PBRM1 tumor suppressor function, gene regulation, and chromatin affinity with the degree of importance correlating strongly to the 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.

Keywords: Polybromo-1; acetylation; bromodomain; bromodomains; cancer; cell growth; chromatin remodeling; epigenetics; histone acetylation; renal cancer.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

FIGURE 1.
FIGURE 1.
The BAF and PBAF complexes coexist in all cell types. Although BAF and PBAF share many subunits (green), BAF exclusively contains ARID1A/B, BAF45B/C/D, BRD9, SS18, and BLC7A/B/C (purple), whereas PBAF exclusively contains PBRM1, ARID2, BAF45A, and BRD7 (red). PBRM1 is the most distinguishing feature of PBAF with no homolog in BAF.
FIGURE 2.
FIGURE 2.
Missense mutations in PBRM1 identified from ccRCC patient samples indicated with black bars. Data were obtained from the COSMIC database (17).
FIGURE 3.
FIGURE 3.
Proliferation and gene expression is altered in PBRM1 BD mutant cell lines. A, thermal shift stability assays indicate that mutation of conserved asparagine 263 to alanine in recombinant BD2 abrogates binding to an H3K14Ac peptide. B, immunoblotting analysis of protein expression level of PBRM1 and BRG1 in the BD mutant (mut) cell lines from lysates (top) and PBRM1 immunoprecipitations (bottom). TBP, TATA-binding protein. C, the proliferation rate of BD mutant cell lines normalized to Caki2+Vector cells. n = 3 independent biological replicates D, transcriptional analysis by quantitative RT-PCR of PBRM1-dependent genes (IGFBP4 and CNTN6) in BD mutant cell lines normalized to Caki2+Vector cells. n = 6 independent biological replicates. A designation of * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 (paired Student's t test). ns, not significant. Error bars represent S.D.
FIGURE 4.
FIGURE 4.
Chromatin binding properties of BAF and PBAF complexes. A, analysis of binding affinity to chromatin of the BAF (ARID1A) and PBAF (PBRM1) in Caki1 cells by SSE. n = 2 independent biological replicates. B, SSE analysis of BAF and PBAF affinity in HeLa cells. C, analysis of PBAF affinity to chromatin in Caki2+Vector and Caki2+PBRM1WT cells indicated by the elution of ARID2. n = 4 independent biological replicates. D, analysis of PBAF affinity to chromatin Caki1+Vector and Caki1+shPBRM indicated by the elution of ARID2. E, comparison of PBAF elution in Caki1 cells grown under normal conditions or serum-starved conditions for 16 h. A designation of * = p < 0.05 (paired Student's t test). Error bars represent S.D.
FIGURE 5.
FIGURE 5.
Chromatin binding properties are altered in PBRM1 BD mutants. A, representative immunoblotting analysis of SSE assays designed to assess relative chromatin binding affinity of BD mutant (m) PBRM1 re-expressed in Caki2 cells. B, analysis of binding affinity to chromatin by SSE of PBRM1 in the BD mutants (mut) indicated by the percentage of PBRM1 eluted at 200 mm NaCl. n = 4 independent biological replicates. A designation of * = p < 0.05, ** = p < 0.01 (paired Student's t test). ns, not significant. Error bars represent S.D.
FIGURE 6.
FIGURE 6.
PBRM1 binding to acetylated histone peptides. A, immunoblotting analysis of PBRM1 after peptide pulldowns with H3 or singly acetylated peptide from HeLa nuclear cell lysate. B, immunoblotting analysis after peptide pulldowns with H3 or H3K14Ac peptide from Caki2+PBRM1WT and Caki2+PBRM1mBD2 nuclear lysate. Enrichment of BAF (SS18) and PBAF (PBRM1) was determined by immunoblot analysis. C, immunoblot analysis of PBRM1 after peptide pulldowns with differentially acetylated peptides from HeLa nuclear cell lysate. D, immunoblot analysis of PBRM1 after peptide pulldowns with H3 or multiply acetylated H3 peptide from the nuclear lysates of Caki2 with re-expression of wild-type and BD mutant (mut) PBRM1.
FIGURE 7.
FIGURE 7.
Quantitative ChIP in Caki2 cells. A, ChIP of PBRM1 and H3K14Ac enrichment at four sites across the CNTN6 locus in Caki2 cells. A designation of * = p < 0.05 and ** = p < 0.01 (paired Student's t test) when compared with IgG. Error bars represent S.D. B, PBRM1 ChIP at the CNTN6 locus (primer site 1) from Caki2 cells expressing wild-type or BD mutant (mut) PBRM1. A designation of * = p < 0.05 (paired Student's t test) when compared with WT PBRM1. Error bars represent S.D.
FIGURE 8.
FIGURE 8.
Proposed models for PBRM1 recognition of histone marks. BD2 binds specifically to H3K14ac, and the remaining bromodomains recognize a pattern of acetylation marks based on spatial orientation.

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