Structural Insights into BAF47 and BAF155 Complex Formation

https://doi.org/10.1016/j.jmb.2017.04.008Get rights and content

Highlights

  • BAF155 SWIRM domain directly interacts with the BAF47 RPT1 domain.

  • BAF47 RPT1 domain has a novel structural fold.

  • BAF155 SWIRM domain mediates protein–protein interactions, which is different from other canonical SWIRM domains with DNA binding activity.

Abstract

Mammalian BAF complexes are a subfamily of SWI/SNF ATP-dependent chromatin remodelers that dynamically modulate chromatin structure to regulate fundamental cellular processes including gene transcription, cell cycle control, and DNA damage response. So far, many distinct BAF complexes have been identified with polymorphic assemblies of up to 15 subunits from 29 genes. The evolutionarily conserved BRG1/BRM, BAF47, and BAF155/BAF170 form a stable complex that carries out essential chromatin remodeling activity and therefore have been regarded as the core components of BAF complex. Here, we first confirmed that SWIRM domain of BAF155 is responsible for its interaction with BAF47 and then narrowed down the SWIRM-binding region in BAF47 to the Repeat 1 (RPT1) domain. We further presented the high-resolution crystal structure of SWIRM/RPT1 complex. Extensive mutagenesis experiments together with isothermal titration calorimetry and NMR titrations were performed to corroborate the interactions observed in crystal structure. Overall, we demonstrated that BAF155 SWIRM is a modular domain involved in BAF47 interaction, which is functionally distinct from other characterized SWIRM domains that possess DNA binding activity.

Introduction

SWI/SNF (referred to as BRG1/BRM-associated factors in mammalian) complex, as the first identified chromatin remodeling complex, can utilize the energy of ATP hydrolysis to alter chromatin structure, thereby spatiotemporally controlling genomic DNA accessibility to nuclear machinery. So far, 15 BAF subunits have been identified in mammals, and variant subunits can assemble into different combinations that contain either BRG1 or BRM as the catalytic ATPase. It has been well documented that distinct combinatorial assembly of BAF complex produces functional diversity [1], [2], [3]. BAF complex regulates the transcription of a large number of target genes and thus plays an essential role in many developmental processes including cardiac development and nervous system development, maintaining pluoripotency in embryonic stem cells (ESCs) and promoting somatic cell reprogramming [4], [5]. Pluripotency interactome analyses have identified that esBAF complex functionally associates with Oct4 and Nanog, which greatly facilitates somatic cell reprogramming and helps the induced pluripotent stem cells maintain pluripotency [6], [7]. Overexpression of BAF155 and BRG1 in mouse embryonic fibroblasts significantly enhances Oct4-, Sox2-, Klf4-, and c-Myc-mediated reprogramming [8]. In addition, BAF complexes have also been implicated in DNA damage response [9]. BAF complex was shown to accumulate at DNA damage sites induced by laser microirradiation and promote γH2AX induction [10], [11]. Another study has demonstrated that BRIT1, an early DNA damage response factor, can help recruit BAF complex to DNA double-strand break sites to relax the nearby chromatin structure, which eventually facilitates other DNA repair factors' access to double-strand break sites [12].

Recent cancer genome sequencing studies have revealed that components of human BAF complex are among the most frequently mutated genes in a variety of tumors [13], [14]. BAF47 mutations occur almost 100% in rhabdoid tumors, and PBAF subunit BAF180 is mutated in 41% of renal cell carcinomas. More broadly, mutations in genes encoding BAF subunits have been identified in over 20% of all human cancers [13], [14], [15]. However, the molecular mechanism underlying the contributions of high frequency of BAF subunit mutations to human cancers remains largely unknown.

Although the subunit composition of mammalian BAF complex is very diverse, most BAF complexes contain a subset of core subunits including BRG1/BRM, BAF155/170, and BAF47. In vitro biochemical analysis has shown that adding BAF47, BAF155, and BAF170 to BRG1 stimulates its ATPase activity to a level comparable to the intact BAF complex [16]; also, mice lacking BAF47 or BAF155 or BRG1 die at early embryonic stage [17], [18], [19], [20], underscoring the essential roles of these core subunits in diverse BAF complexes and in early embryogenesis. Although BAF complex has been extensively studied over the past decades, the molecular mechanism by which these BAF essential subunits interact to form the core regulatory machinery is currently lacking.

BAF47 is a well-known bona fide tumor suppressor that is mutated in almost all aggressive rhabdoid tumors predominantly occurring to infants and young children [21], [22]. Several recent studies have shown that BAF47 (SNF5 in yeast) plays a critical role in maintaining BAF complex integrity [23], [24], [25]. Loss of BAF47 resulted in substantially decreased levels of the BAF complex as observed in a number of rhabdoid cell lines. On the other hand, re-expression of BAF47 in BAF47-deficient cells dramatically increased the incorporation of BAF subunits including Brg1, BAF155/BAF170, and BAF250a into BAF complexes. Interestingly, a previous study has revealed that SS18-SSX evicts SS18 and BAF47 to form an altered BAF complex in human synovial sarcoma, but the process can be reversible, and wild-type BAF complex can be reassembled after increasing the protein level of wild-type SS18 [26]. In addition, BAF47 has been reported to directly bind to HIV-1 integrase to regulate the viral integration [27]. Compared to BAF47, BAF155 and its paralog BAF170 are less frequently mutated in cancers. BAF155 shares a high degree of sequence similarity with BAF170 but has a non-redundant role at the different stages of development. In mouse embryonic stem cells, the ESC-specific BAF complex is characterized by the presence of BRG1, BAF155, and BAF60a and the absence of BRM, BAF170, and BAF60c [7], [28]. In contrast, both BAF155 and BAF170 are present in human embryonic stem cells, and knockdown of BAF170 led to the loss of pluoripotency [29]. Besides, BAF155 and/or BAF170, acting as scaffold proteins, have been demonstrated to maintain the integrity of BAF complex by protecting other BAF subunits including BAF47 and BRG1 from proteasomal degradation [30], [31]. In this study, we first confirmed that BAF155 and BAF47 form a protein complex. We further identified that BAF155 SWIRM domain interacts directly with BAF47 Repeat 1 (RPT1) domain. The crystal structure of BAF155 SWIRM domain bound to BAF47 RPT1 reveals extensive interactions in which electrostatic interactions dominate, whereas hydrophobic interactions make an additive contribution. Mutagenesis together with isothermal titration calorimetry (ITC) and NMR binding studies validated the importance of these interactions.

Section snippets

BAF155 SWIRM domain binds directly to BAF47 RPT1 domain

Phelan et al. have previously demonstated that BAF155 and BAF47 can be co-purified from human cells, suggesting that these two proteins physically interact with each other [16]. In another study, Sohn et al. have reported that SWIRM domain of BAF155 is required for the interaction with BAF47 by using yeast two-hybrid assay [30]. To further clarify this observation, we performed co-immunoprecipitation assay upon overexpression of Myc-tagged BAF47 with Flag-tagged full-length BAF155 or

Discussion

Mammalian BAF complex belongs to SWI/SNF chromatin remodeling family that was originally identified in yeast [38]. Although SWI/SNF complex has lost and gained a number of subunits during evolution, BAF47 and BAF155/BAF170 are evolutionarily conserved and homologous proteins can be found in yeast, flies, plants, and mammals [39]. In addition, BAF47 and BAF155 or BAF170 are present in almost all identified tissue- or cell-type-specific BAF complexes. It has previously been demonstrated that

Plasmid construction, protein expression, and purification

The human BAF155 SWIRM domain (residue 438–540) was cloned into pGEX-6p1 plasmid as an N-terminal GST-tagged protein, and BAF47_N (residue 3–111), BAF47_C (residue 169–385), BAF47 RPT1 (residue 169–252), and BAF47_C2 (residue 255–385) were cloned into pET28a or home-engineered pRSF-SUMO vectors. Fusion proteins were expressed in Escherichia coli strain Rosetta DE3 (Novagen) and purified via either GSTrap HP column or nickel-NTA affinity column, followed by size-exclusion and/or ion exchange

Acknowledgments

We wish to acknowledge the use of the Shanghai synchrotron radiation (beamline BL17U) for X-ray data collection and Bruker Avance 600 MHz NMR spectrometer with cryoprobe installed in the Chemistry Department of HKU for NMR data collection. We thank Dr. HZ Sun for facilitating NMR data collection. This study is supported by grants from Hong Kong Research Grants Council (17127715 and 775712) to C. Qian.

Author contributions: C. Qian conceived the project. L. Yan conducted most of the experiments.

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