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 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.
Project description:For cells to initiate and sustain a differentiated state, it is necessary that a “memory” of this state is transmitted through mitosis to the daughter cells. Mammalian SWItch/ Sucrose Non- Fermentable (SWI/SNF) complexes, also called Brg1/ Brg- associated factors (BAF), control cell identity by modulating chromatin architecture to regulate gene expression, but whether they participate in cell fate memory is unclear. Here, we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but bound on promoters during mitosis and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets, and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 plays a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.
Project description:The 12-subunit Swi/Snf chromatin remodeling complex is conserved from yeast to humans. It functions to alter nucleosome positions by either sliding nucleosomes on DNA or evicting histones. Interestingly, 20% of all human cancers carry mutations in subunits of the Swi/Snf complex. Many of these mutations cause protein instability and loss, resulting in partial Swi/Snf complexes. Although several studies have shown that histone acetylation and activator-dependent recruitment of Swi/Snf regulate its function, it is less well understood how subunits regulate stability and function of the complex. Using functional proteomic and genomic approaches, we have assembled the network architecture of yeast Swi/Snf. In addition, we find that subunits of the Swi/Snf complex regulate occupancy of the catalytic subunit Snf2, thereby modulating gene transcription. Our findings have direct bearing on how cancer-causing mutations in orthologous subunits of human Swi/Snf may lead to aberrant regulation of gene expression by this complex.
Project description:The 12-subunit Swi/Snf chromatin remodeling complex is conserved from yeast to humans. It functions to alter nucleosome positions by either sliding nucleosomes on DNA or evicting histones. Interestingly, 20% of all human cancers carry mutations in subunits of the Swi/Snf complex. Many of these mutations cause protein instability and loss, resulting in partial Swi/Snf complexes. Although several studies have shown that histone acetylation and activator-dependent recruitment of Swi/Snf regulate its function, it is less well understood how subunits regulate stability and function of the complex. Using functional proteomic and genomic approaches, we have assembled the network architecture of yeast Swi/Snf. In addition, we find that subunits of the Swi/Snf complex regulate occupancy of the catalytic subunit Snf2, thereby modulating gene transcription. Our findings have direct bearing on how cancer-causing mutations in orthologous subunits of human Swi/Snf may lead to aberrant regulation of gene expression by this complex.
Project description:A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5M-bM-^@M-^Y ends, RNA Polymerases II and III and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins and DNA replication origins). To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members as well as with cellular constituents such as nuclear matrix proteins, key transcription factors and centromere components implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated. ChIP-Seq analysis of the SWI/SNF subunits Ini1, Brg1, BAF155 and BAF170 in HeLa S3 cells
Project description:We exogenously expressed the SWI/SNF ATPase catalytic subunits BRG1 and BRM in the lacking cell line C33A, and compared the differences with mock transfected cell at gene expression and alternative splicing level.