Proteomic and bioinformatic analysis of mammalian SWI/SNF complexes identifies extensive roles in human malignancy

Abstract

Subunits of mammalian SWI/SNF (mSWI/SNF or BAF) complexes have recently been implicated as tumor suppressors in human malignancies. To understand the full extent of their involvement, we conducted a proteomic analysis of endogenous mSWI/SNF complexes, which identified several new dedicated, stable subunits not found in yeast SWI/SNF complexes, including BCL7A, BCL7B and BCL7C, BCL11A and BCL11B, BRD9 and SS18. Incorporating these new members, we determined mSWI/SNF subunit mutation frequency in exome and whole-genome sequencing studies of primary human tumors. Notably, mSWI/SNF subunits are mutated in 19.6% of all human tumors reported in 44 studies. Our analysis suggests that specific subunits protect against cancer in specific tissues. In addition, mutations affecting more than one subunit, defined here as compound heterozygosity, are prevalent in certain cancers. Our studies demonstrate that mSWI/SNF is the most frequently mutated chromatin-regulatory complex (CRC) in human cancer, exhibiting a broad mutation pattern, similar to that of TP53. Thus, proper functioning of polymorphic BAF complexes may constitute a major mechanism of tumor suppression.

Figure 1. Identification of novel, dedicated subunits of mSWI/SNF-like BAF complexes

(a) Composition of purified SWI/SNF complexes as determined by mass spectrometric analysis. Confidence (protein probability statistic) is 1.0 for all peptides. Protein identification was completed using SEQUEST (University of Washington) as described. n.d.=not detected. (b) Glycerol gradient sedimentation analysis of CCRF-CEM human T cell nuclear extracts demonstrates that Bcl7 and Bcl11 family proteins, as well as Brd9, co-sediment with mSWI/SNF complex core subunits. Fractions of 0.5ml of the 10ml 10–30% glycerol gradient were collected and subjected to western blot analysis for various mSWI/SNF proteins. Red arrows indicate fractions with prominent BRG1 peaks. (c) Partial urea denaturation ranging from 0.25M to 5M urea prior to anti-Brg1 immunoprecipitation reveals that Bcl7 and Bcl11 family proteins, and Brd9, are mSWI/SNF complex components and must be denatured to dissociate from mSWI/SNF complexes. The co-precipitated proteins were analyzed by immunoblot with antibodies specific for established and putative subunits of mSWI/SNF complexes. (d) Reciprocal IP studies using novel anti-Bcl7a/b/c antibodies and anti-Bcl11a/b, Brd 9 also reveal BAF complex subunits. (e) Updated model of mSWI/SNF (BAF) complexes incorporating novel subunits.

Figure 2. mSWI/SNF complex subunits are mutated in human cancers with high frequency

(a) The frequency of patients harboring a mutation in an mSWI/SNF subunit is presented according to a graded color scale. Studies on the same tissue/cancer type were summed to obtain the individual frequency of each protein. Novel subunits are highlighted in red. Bottom: the total number of patients affected by any mSWI/SNF subunit mutation was summed to calculate the overall frequency of patients with mutations in the mSWI/SNF complex. (b) A volcano plot representing the Mutation rate/BMR and p value for the most frequently mutated mSWI/SNF subunit in each of the exome sequencing studies evaluated. (c) A list of the Mutation rate/BMR and p value for all the studies in which the novel subunits BCL7A, BCL11A, and BCL11B are mutated above the BMR. (d) Translocations to mSWI/SNF subunits often represent sole genomic abnormalities (Mittelman Database).

Figure 3. mSWI/SNF complexes are more frequently mutated in human cancer than other chromatin modifying complexes

The Mutation rate/BMR for 37 exome sequencing studies for the mSWI/SNF, TIP60, SRCAP, INO80, ISWI, NURD, PRC1, and PRC2 complexes is graphed. The study number refers to the publications listed in Supplementary Table 2. Statistically significant mutation rates are indicated with asterisks. The percent bar graphs under each complex name indicate the percent of studies for which that complex was the most highly mutated complex of the eight examined.

Figure 4. mSWI/SNF subunits are more frequently mutated than EP300, MLL, and HDAC family proteins

The ratio of the mutation frequency of EP300, the highest mutated MLL family member, or the highest mutated HDAC member, was plotted relative to the most frequently mutated mSWI/SNF subunit for each study. Ratios of <1 indicate that the peak mutation frequency of mSWI/SNF genes is higher than the chromatin modifier; values of this ratio >1 indicate that mutation frequency of the chromatin modifier is higher. Statistically significant ratios favoring mSWI/SNF (ratios of less than 1) are indicated.

Figure 5. Mutations to mSWI/SNF subunits occur in a broad spectrum of cancer types

The ratio of the mutation frequency of the (a) tumor suppressors TP53, PTEN, and CDKN2A, and (b) oncogenes PIK3CA, KRAS, and CTNNB1 was plotted relative to the most frequently mutated mSWI/SNF subunit. Ratios of <1 indicate that the peak mutation frequency of mSWI/SNF genes is higher than the tumor suppressor/oncogene; values of this ratio >1 indicate that mutation frequency of the tumor suppressor/oncogene is higher. Statistically significant ratios are indicated.

Figure 6. Co-occurrence of mSWI/SNF mutations in human cancers

(a) The patient profiles of all the patients harboring a mutation or deletion in more than 1 mSWI/SNF subunit are represented from the studies that provided this information (study number is listed after cancer type). Hypermutated patients are marked with a # sign. The number of patients with <1 mutation in mSWI/SNF subunits, 1 mutation, the total number of cases, and the frequency of compound heterozygous patients is displayed. (b) The cooccurrence of mSWI/SNF mutations and TP53 mutations for all the studies that had more than 1 tumor with a TP53 mutation are displayed in a Euler diagram against the total number of cases.