SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes mediate ATP-dependent alterations of DNA-histone contacts. The minimal functional core of conserved SWI/SNF complexes consists of a SWI2/SNF2 ATPase, SNF5, SWP73, and a pair of SWI3 subunits. Because of early duplication of the SWI3 gene family in plants, Arabidopsis thaliana encodes four SWI3-like proteins that show remarkable functional diversification. Whereas ATSWI3A and ATSWI3B form homodimers and heterodimers and interact with BSH/SNF5, ATSWI3C, and the flowering regulator FCA, ATSWI3D can only bind ATSWI3B in yeast two-hybrid assays. Mutations of ATSWI3A and ATSWI3B arrest embryo development at the globular stage. By a possible imprinting effect, the atswi3b mutations result in death for approximately half of both macrospores and microspores. Mutations in ATSWI3C cause semidwarf stature, inhibition of root elongation, leaf curling, aberrant stamen development, and reduced fertility. Plants carrying atswi3d mutations display severe dwarfism, alterations in the number and development of flower organs, and complete male and female sterility. These data indicate that, by possible contribution to the combinatorial assembly of different SWI/SNF complexes, the ATSWI3 proteins perform nonredundant regulatory functions that affect embryogenesis and both the vegetative and reproductive phases of plant development.
Switch (SWI)/Sucrose Nonfermenting (SNF)-type chromatin-remodeling complexes (CRCs) are involved in regulation of transcription, DNA replication and repair, and cell cycle. Mutations of conserved subunits of plant CRCs severely impair growth and development; however, the underlying causes of these phenotypes are largely unknown. Here, we show that inactivation of SWI3C, the core component of Arabidopsis (Arabidopsis thaliana) SWI/SNF CRCs, interferes with normal functioning of several plant hormone pathways and alters transcriptional regulation of key genes of gibberellin (GA) biosynthesis. The resulting reduction of GA 4 causes severe inhibition of hypocotyl and root elongation, which can be rescued by exogenous GA treatment. In addition, the swi3c mutation inhibits DELLA-dependent transcriptional activation of GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptor genes. Down-regulation of GID1a in parallel with the DELLA repressor gene REPRESSOR OF GA1-3 1 in swi3c indicates that lack of SWI3C also leads to defects in GA signaling. Together with the recent demonstration of function of SWI/SNF ATPase BRAHMA in the GA pathway, these results reveal a critical role of SWI/SNF CRC in the regulation of GA biosynthesis and signaling. Moreover, we demonstrate that SWI3C is capable of in vitro binding to, and shows in vivo bimolecular fluorescence complementation interaction in cell nuclei with, the DELLA proteins RGA-LIKE2 and RGA-LIKE3, which affect transcriptional activation of GID1 and GA3ox (GIBBERELLIN 3-OXIDASE) genes controlling GA perception and biosynthesis, respectively. Furthermore, we show that SWI3C also interacts with the O-GlcNAc (O-linked N-acetylglucosamine) transferase SPINDLY required for proper functioning of DELLAs and acts hypostatically to (SPINDLY) in the GA response pathway. These findings suggest that DELLA-mediated effects in GA signaling as well as their role as a hub in hormonal cross talk may be, at least in part, dependent on their direct physical interaction with complexes responsible for modulation of chromatin structure.
ATP-dependent nucleosome remodeling plays a central role in the regulation of access to chromatin DNA. Swi/Snf remodeling complexes characterized in yeast, Drosophila and mammals all contain a conserved set of core subunits composed of homologs of yeast SNF2-type DNA-dependent ATPase, SNF5 and SWI3 proteins. So far, no complete Swi/Snf-type complex has been characterized in plants. Arabidopsis contains a single SNF5-type gene, BSH, which has been shown to complement the yeast snf5 mutation. Here we describe the characterization of AtSWI3B, the smallest of the four Arabidopsis homologs of SWI3. The gene encoding AtSWI3B is expressed ubiquitously in the plant. AtSWI3B is localized to nuclei and is associated mostly with the chromatin and soluble protein fractions. When expressed in Saccharomyces cerevisiae, the cDNA encoding AtSWI3B partially complements the swi3 mutant phenotype. However, like BSH, AtSWI3B is unable to activate transcription in yeast when tethered to DNA. The analysis by yeast two-hybrid indicates that AtSWI3B is capable of forming homodimers and interacts with BSH as well as with two other members of the Arabidopsis SWI3 family: AtSWI3A and AtSWI3C. The results of phage display screen using recombinant protein, confirmed by direct yeast two-hybrid analyses, indicate that AtSWI3B interacts with FCA, a regulator of flowering time in Arabidopsis. This interaction is through the C-terminal region of FCA, located outside the conserved RNA- and protein-binding domains of this protein.
SWI/SNF chromatin remodeling complexes perform a pivotal function in the regulation of eukaryotic gene expression. Arabidopsis (Arabidopsis thaliana) mutants in major SWI/SNF subunits display embryo-lethal or dwarf phenotypes, indicating their critical role in molecular pathways controlling development and growth. As gibberellins (GA) are major positive regulators of plant growth, we wanted to establish whether there is a link between SWI/SNF and GA signaling in Arabidopsis. This study revealed that in brm-1 plants, depleted in SWI/SNF BRAHMA (BRM) ATPase, a number of GA-related phenotypic traits are GA-sensitive and that the loss of BRM results in markedly decreased level of endogenous bioactive GA. Transcriptional profiling of brm-1 and the GA biosynthesis mutant ga1-3, as well as the ga1-3/brm-1 double mutant demonstrated that BRM affects the expression of a large set of GA-responsive genes including genes responsible for GA biosynthesis and signaling. Furthermore, we found that BRM acts as an activator and directly associates with promoters of GA3ox1, a GA biosynthetic gene, and SCL3, implicated in positive regulation of the GA pathway. Many GA-responsive gene expression alterations in the brm-1 mutant are likely due to depleted levels of active GAs. However, the analysis of genetic interactions between BRM and the DELLA GA pathway repressors, revealed that BRM also acts on GA-responsive genes independently of its effect on GA level. Given the central position occupied by SWI/SNF complexes within regulatory networks controlling fundamental biological processes, the identification of diverse functional intersections of BRM with GA-dependent processes in this study suggests a role for SWI/SNF in facilitating crosstalk between GA-mediated regulation and other cellular pathways.
(T.J.S.)Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control.
In yeast and mammals, ATP-dependent chromatin remodelling complexes of the SWI/SNF family play critical roles in the regulation of transcription, cell proliferation, diVerentiation and development. Homologues of conserved subunits of SWI/SNF-type complexes, including Snf2-type ATPases and SWI3-type proteins, participate in analogous processes in Arabidopsis. Recent studies indicate a remarkable similarity between phenotypic eVects of mutations in the SWI3 homologue ATSWI3C and bromodomain-ATPase BRM genes. To verify the extent of functional similarity between BRM and ATSWI3C, we have constructed atswi3c brm double mutants and compared their phenotypic traits to those of simultaneously grown single atswi3c and brm mutants. In addition to inheritance of characteristic developmental abnormalities shared by atswi3c and brm mutants, some additive brm-speciWc traits were also observed in the atswi3c brm double mutants. Unlike atswi3c, the brm mutation results in the enhancement of abnormal carpel development and pollen abortion leading to complete male sterility. Despite the overall similarity of brm and atswi3c phenotypes, a critical requirement for BRM in the diVerentiation of reproductive organs suggests that its regulatory functions do not entirely overlap those of ATSWI3C. The detection of two diVerent transcript isoforms indicates that BRM is regulated by alternative splicing that creates an in-frame premature translation stop codon in its SNF2-like ATPase coding domain. The analysis of Arabidopsis mutants in nonsense-mediated decay suggests an involvement of this pathway in the control of alternative BRM transcript level.
Aberrant expression of DNA polymerase β, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol β expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3′UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3′UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element—the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA–protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol β mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol β.
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