Glioma tumor suppressor candidate region gene 1 (GLTSCR1) and its paralog GLTSCR1-like form SWI/SNF chromatin remodeling subcomplexes

Abstract

The mammalian SWI/SNF chromatin remodeling complex is a heterogeneous collection of related protein complexes required for gene regulation and genome integrity. It contains a central ATPase (BRM or BRG1) and various combinations of 10-14 accessory subunits (BAFs for BRM/BRG1 Associated Factors). Two distinct complexes differing in size, BAF and the slightly larger polybromo-BAF (PBAF), share many of the same core subunits but are differentiated primarily by having either AT-rich interaction domain 1A/B (ARID1A/B in BAF) or ARID2 (in PBAF). Using density gradient centrifugation and immunoprecipitation, we have identified and characterized a third and smaller SWI/SNF subcomplex. We termed this complex GBAF because it incorporates two mutually exclusive paralogs, GLTSCR1 (glioma tumor suppressor candidate region gene 1) or GLTSCR1L (GLTSCR1-like), instead of an ARID protein. In addition to GLTSCR1 or GLTSCR1L, the GBAF complex contains BRD9 (bromodomain-containing 9) and the BAF subunits BAF155, BAF60, SS18, BAF53a, and BRG1/BRM. We observed that GBAF does not contain the core BAF subunits BAF45, BAF47, or BAF57. Even without these subunits, GBAF displayed in vitro ATPase activity and bulk chromatin affinity comparable to those of BAF. GBAF associated with BRD4, but, unlike BRD4, the GBAF component GLTSCR1 was not required for the viability of the LNCaP prostate cancer cell line. In contrast, GLTSCR1 or GLTSCR1L knockouts in the metastatic prostate cancer cell line PC3 resulted in a loss in proliferation and colony-forming ability. Taken together, our results provide evidence for a compositionally novel SWI/SNF subcomplex with cell type-specific functions.

Keywords: BRD4; BRD9; BRG1; CRISPR/Cas; GLTSCR1; SWI/SNF; chromatin remodeling; prostate cancer; protein complex; protein-protein interaction.

Figure 1.

GLTSCR1 is a dedicated subunit of the SWI/SNF chromatin remodeling complex. A, current illustration of mammalian SWI/SNF complex composition. B, mass spectrometry (Spec) analysis of BRG1 IP from two human renal cancer cell lines identifies peptides from GLTSCR1. C, GLTSCR1 specific antibody identified using Gltscr1 knockout mESCs derived using three different sgRNA constructs. D, immunoprecipitation with antibodies against BRG1 confirms GLTSCR1 association. Urea denaturation with 0.5 and 2.5 m urea prior to BRG1 immunoprecipitation indicates the strong association of GLTSCR1 to BRG1, comparable to the strength of association of BRG1 to core BAF subunits ARID1A and BAF60A.

Figure 2.

GLTSCR1 is in a novel SWI/SNF subcomplex GBAF. A, Glycerol gradients from renal cancer cell line Caki1 and prostate cancer cell line PC3 indicate that GLTSCR1 does not co-sediment with BAF subunit ARID1A or PBAF subunits PBRM1 (for Caki1) or BRD7 (for PC3). B, IP experiments of BAF subunits from PC3 lysates identify GLTSCR1 association with BRG1 and BRM. C, BAF subunit and GLTSCR1 IP experiments from HEK293T lysates identify GLTSCR1 association with BAF155 and BAF53a but not BAF47, BAF57, or BAF45D. D, BAF subunit and GLTSCR1 IP experiments from HEK293T lysates identify GLTSCR1 association with SS18 and BRD9 but not BCL11A. E and F, glycerol gradient analysis (E) and BAF subunit IP experiments (F) from HEK293T lysates identify GLTSCR1 association with BRG1 and SS18 but not BAF170 and BAF47 and validate BRD9 as a subunit found in GBAF, but not BAF/PBAF. G, schematic representation of GBAF, BAF, and PBAF composition. Yellow subunits are unique to GBAF, blue subunits are unique to BAF, red subunits are unique to PBAF, green subunits are shared by GBAF and BAF, purple subunits are shared by BAF and PBAF, and gray subunits are shared by all three complexes. Subcomplex GBAF consists of BAF60A, BRG1, BAF155, BRD9, BAF53A, and SS18. H, GBAF possesses ATPase activity. ATPase activity assay was performed with BRG1 and GLTSCR1 immunoprecipitations providing similar levels of BRG1. ATPase activities normalized to respective IgG isotype controls yielded comparable fold changes (3.03 ± 0.23, for BRG1 IP; 3.24 ± 0.87, for GLTSCR1 IP). Error bars, means ± S.D. (n = 3). *, p < 0.05; ***, p < 0.001. I, sequential salt extraction analysis and immunoblot quantitation indicates that GLTSCR1 interacts with bulk chromatin at a similar strength as ARID1A (representative of BAF) and PBRM1 (representative of PBAF).

Figure 3.

GBAF contains GLTSCR1 or paralog GLTSCR1L (BICRAL). A, glycerol gradient analysis in mESCs showing that GBAF-associated BRG1 and BAF60A were preserved in fractions 11–13 in the absence of GLTSCR1, suggesting that GBAF was not completely disrupted by Gltscr1 knockout. B, pairwise alignment of amino acid sequences of GLTSCR1 and its paralog GLTSCR1L (BICRAL) show homology in the N-terminal region and strong homology at region identified at a conserved GLTSCR1 domain. C, verification of inducible expression of BICRAL-FLAG in HEK293T cells with both FLAG and endogenous BICRAL antibodies. D, immunoprecipitation analysis showed that similar to GLTSCR1, exogenous BICRAL interacts with BRD9, BAF53A, and BRG1. In addition, endogenous GLTSCR1 and BICRAL do not immunoprecipitate one another. BICRAL overexpression results in reduced GLTSCR1 protein levels. E, endogenous BICRAL does not associate with GLTSCR1, further validating that GLTSCR1 and BICRAL are mutually exclusive in GBAF context. BICRAL is detected in total BRG1 IP but not in GLTSCR1 IP, although both contain comparable levels of BRG1. Note that the same Western blotting is used in Fig. S1B to compare BRG1 levels for ATPase assay.

Figure 4.

GLTSCR1 and BICRAL are mutually exclusive subunits of GBAF that can alter SWI/SNF complex stoichiometry. A, glycerol gradient analysis in BICRAL-FLAG–overexpressing HEK293T cells indicates that BICRAL is incorporated into GBAF. Overexpression of BICRAL-FLAG increases the GBAF-associated BRG1 levels (fractions 11–14), suggesting formation of new GBAF upon BICRAL overexpression. Reduced GBAF-associated GLTSCR1 levels also validate decreased GLTSCR1 protein expression upon BICRAL overexpression. B, immunoprecipitation analysis showing that BICRAL-FLAG overexpression reduced BRG1-associated GLTSCR1 levels and enhanced BRD9 protein levels and its association with BRG1. BICRAL-FLAG overexpression also reduced BRG1-associated BAF47 and BAF57, suggesting competition between GBAF and BAF for BRG1. C, RT-qPCR showing that expression of BRG1, GLTSCR1, or BRD9 did not alter upon BICRAL overexpression. Error bars, means ± S.D. (n = 3). D, CRISPR/Cas9-mediated knockout of GLTSCR1 with or without CRISPR/Cas9-mediated knockout of BICRAL reduced the BRG1-associated BRD9 levels, as an indicator of loss of GBAF.

Figure 5.

GLTSCR1 associates with BRD4 but is not required for BRD4-mediated MYC transcription in LNCaP cells. A, immunoprecipitation of BRD4 enriches GLTSCR1, BRD9, and BAF155 but not BAF/PBAF subunit BAF47. AP, lysates treated with alkaline phosphatase; PI, lysates treated with phosphatase inhibitors. B, proliferation measurement after 6 days of growth of LNCaP cells with GLTSCR1 knockout using Alamar Blue. Error bars, means ± S.D. for n = 6 replicates. C, GLTSCR1 knockout sensitized LNCaP to BET inhibitor JQ1. Cell numbers are approximated using Alamar Blue fluorescence. IC₅₀ values are derived from curve fit calculations using GraphPad Prism and presented as means ± S.D. for n = 4 replicates. **, p < 0.01. D, MYC expression is up-regulated in GLTSCR1 knockout LNCaP cells, which reverted back to basal levels upon 50 nm JQ1 treatment. Error bars, mean ± S.D. (n = 3 replicates). *, p < 0.05.

Figure 6.

GLTSCR1 and BICRAL are expressed in most cell lines but are uniquely required for the viability of prostate cancer cell line PC3. A, immunoblot analysis of GLTSCR1 and BICRAL expression across a panel of cell lines. B, proliferation measurement after 6 days of growth of the non-transformed mouse cell lines mESCs and NMuMG with Gltscr1 knockout using Alamar Blue. C, proliferation measurement after 6 days of growth of the transformed human astrocyte cell line SVGp12 and glioblastoma cell line T98G with GLTSCR1 knockout using Alamar Blue. D, proliferation measurement after 6 days of growth of HEK293T cells with GLTSCR1 and BICRAL knockout using Alamar Blue. E, validation of knockouts using multiple guide RNAs. F, left panel, Alamar Blue assay demonstrated that loss of GLTSCR1 and BICRAL reduced the growth of PC3 cells 6 day after plating. Fluorescence values graphed (excitation, 560 nm; emission, 590 nm) represent the metric for cell number. Error bars, means ± S.D. (n = 3 biological replicates). **, p < 0.01; ***, p < 0.001 compared with control cells. Right panel, loss of GLTSCR1 reduced the clonogenic growth of prostate cell line PC3. G, PC3 cells did not display sensitivity to BRD9 inhibitor BI-7273 (IC₅₀ of 275 nm) up to 10 μm treatment for 4 days. Cell number was approximated using Alamar Blue fluorescence (n = 3 biological replicates).