Dataset Information

BRAHMA-containing chromatin remodelling complex prevailing in Arabidopsis vegetative organs is a homolog of mammalian non-canonical SWI/SNF complexes and requires BRD subunits for assembly and stability

ABSTRACT: The SWI/SNF family of chromatin remodeling complexes is evolutionarily conserved and present in yeast, animals and plants. While the biological functions of plant SWI/SNF complexes have been studied in detail, their composition is still elusive. To clarify this picture we used protein extracts from Arabidopsis plants in vegetative phase of growth to perform a series of immunoprecipitation followed by mass spectrometry experiments, using GFP-tagged BRM ATPase as a bait. The analysis of MS data showed that the dominant form of SWI/SNF complex present in these extracts has a specific subunit composition including ARP4 and 7, SWI3C, SWP73B and BRIP2, as well as three bromodomain containing subunits, BRD1, 2 and 13. This subunit composition and the lack of the core SWI/SNF subunit BSH (SNF5/INI1) both strongly resemble the characteristics of the specific subclass of mammalian SWI/SNF complexes referred to as non-canonical BAFs, indicating that homologues of these complexes also exist in plants. We next found that depletion of the all three BRDs severely affected the assembly of this form of BRM-associated SWI/SNF complex. However, while BRD1 and BRD2 were found sufficient to allow complex formation, BRD13 was only required under BRD1/2 deficiency. The analyses of IP/MS results using BRM-GFP in different brd mutant backgrounds as well as BRD1-GFP indicate that SWI/SNF assemblies containing only one BRD isoform, BRD1 or BRD2, do exist. Furthermore, our data indicate that BRD1/2 may be necessary for the incorporation of BRIP2 subunit into the complex. Together, our results shed new light on the structural and functional diversification of SWI/SNF complexes in Arabidopsis.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Body

SUBMITTER: Maciej Kotliński

LAB HEAD: Rafał Archacki

PROVIDER: PXD030928 | Pride | 2023-04-03

REPOSITORIES: Pride

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Publications

BRM Complex in Arabidopsis Adopts ncBAF-like Composition and Requires BRD Subunits for Assembly and Stability.

Stachula Paulina P Kapela Katarzyna K Malecka Ewelina E Jaronczyk Kamila K Patryn Jacek J Siwirykow Nina N Bucholc Maria M Marczak Malgorzata M Kotlinski Maciej M Archacki Rafal R

International journal of molecular sciences 20230215 4

ATP-dependent SWI/SNF chromatin remodelling complexes are conserved multi-subunit assemblies that control genome activity. Functions of SWI/SNF complexes in plant development and growth have been well established, but the architecture of particular assemblies is unclear. In this study, we elucidate the organization of Arabidopsis SWI/SNF complexes formed around a BRM catalytic subunit, and define the requirement of bromodomain-containing proteins BRD1/2/13 for the formation and stability of the ...[more]

PMID: 36835328

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Project description:Switch defective/sucrose non-fermentable chromatin remodeling complexes are multi-subunit machines that play vital roles in regulating chromatin structure and gene expression. However, how SWI/SNF complexes recognize target loci is still not fully understood. Here, we show that Arabidopsis bromodomain-containing homologous proteins, BRD1, BRD2 and BRD13, are core subunits of SWI/SNF complexes that are required for SWI/SNF genomic targeting. The three BRDs directly interact with multiple SWI/SNF subunits, including the BRAHMA (BRM) catalytic subunit. Phenotypic and transcriptome analysis of the brd1 brd2 brd13 triple mutants showed that the BRDs act in large redundancy to control developmental processes and gene expression that are also regulated by BRM. BRDs extensively co-localize with BRM on chromatin. brd1 brd2 brd13 mutation results in the reduced BRM protein levels and genome-wide targeting on chromatin. Finally, we demonstrate that the bromodomain of BRD2 is essential for genomic targeting of BRD2, highlighting the role of this reader domain in the recruitment of BRM-containing SWI/SNF complexes to target sites in plants. SWI/SNF chromatin remodeling complexes are evolutionarily conserved and is confirmed to use the energy derived from hydrolysis of ATP to alter the density or the position of nucleosomes on the DNA or the composition of histone octamer. Bromodomain, an acetylated histone interaction module, was found in chromatin remodeling factors. During the 29 Arabidopsis bromodomain-containing protein, like GCN5 and GTE4/6, their function has been reported. Here, we reported three BRM-interacting bromodomain-containing protein, BRD1, BRD2 and BRD13 are new core subunits of Arabidopsis SWI/SNF complexes. brd1/2/13 displayed a similar phenotype like brm, such as down-ward curled leaves, reduced fertility, shorter silique and root. Moreover, brm brd1/2/13 shows more serious phenotype just like brm-1. Y2H, Co-IP, RNA-seq and ChIP-seq assay reveal that BRDs interact with BRM at both protein and chromatin level. Future more, BRDs are required for BRM genome-wide occupancy and BRM might bind to chromatin via BRDs bromodomain by interacting with their BBC domain.

Project description:The two catalytic subunits of the SWI/SNF chromatin remodeling complex - Brg1 and Brm - have been often implicated as essential components of common biological processes, suggesting a functional redundancy between these two proteins. For instance, earlier works indicated that both proteins are required for the activation of the myogenic program. However, their mutually exclusive pattern of incorporation in SWI/SNF complexes with variable composition and functional heterogeneity predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions to coordinate gene expression in multistep programs, such as skeletal myogenesis. We detected a distinct expression profile of Brg1 and Brm during the myogenic program, with Brm being upregulated in differentiating myocytes. Genetic knockdown of either Brg1 or Brm in skeletal myoblasts, followed by gene expression analysis, revealed discrete functions during myogenic differentiation. While Brm is required for the exit from the cell cycle at the early stages of differentiation, by repressing CyclinD1 transcription, Brg1 is required for the activation of muscle gene transcription. Interestingly, at later stages of differentiation Brg1 and Brm cooperate to the activation of a cluster of common target genes. Thus, Brg1 and Brm appear to coordinate activation and repression of distinct subsets of genes during initial phase myogenesis, and converge to cooperatively activate the expression of muscle genes at later stages. C2C12 myoblasts treated with siBRM or siBRG1 or siSCR in growth medium (GM) and differentiated by medium replacemnte (DM ). Celles were collected at 18h and 48h from the onset of DM for RNA extraction and microarray analysis. Arrays were used to generate 6 data sets in duplicate.To address the question of which genes were regulated by BRM and/or BRG1 the fold changes in gene expression were calculated between Scrambled siRNA and the BRM or BRG1 siRNA. The experiments were repeated at 2 different time points (18 hours and 48 hours) .

Dataset Information

BRAHMA-containing chromatin remodelling complex prevailing in Arabidopsis vegetative organs is a homolog of mammalian non-canonical SWI/SNF complexes and requires BRD subunits for assembly and stability

Publications

BRM Complex in Arabidopsis Adopts ncBAF-like Composition and Requires BRD Subunits for Assembly and Stability.

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