Brm Mutants Research Articles

The SWI2/SNF2 Chromatin-Remodeling ATPase BRAHMA Regulates Chlorophyll Biosynthesis in Arabidopsis

Chlorophyll biosynthesis is critical for chloroplast development and photosynthesis in plants. Although reactions in the chlorophyll biosynthetic pathway have been largely known, little is known about the regulatory mechanisms of this pathway. In this study, we found that the dark-grown knockout and knockdown mutants as well as RNA-interference transgenic seedlings of BRAHMA (BRM), which encodes an SWI2/SNF2 chromatin-remodeling ATPase, had higher greening rates, accumulated less protochlorophyllide, and produced less reactive oxygen species than Arabidopsis wild-type plants did upon light exposure. The expression of NADPH:protochlorophyllide oxidoreductase A (PORA), PORB, and PORC, which catalyze a key step in chlorophyll biosynthesis, was increased in the brm mutants. We found that BRM physically interacted with the bHLH transcription factor PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) through its N-terminal domains. Furthermore, we demonstrated that BRM was directly recruited to the cis-regulatory regions of PORC, but not of PORA and PORB, at least partially in a PIF1-dependent manner and the level of histone H3 lysine 4 tri-methylation (H3K4me3) at PORC loci was increased in the brm mutant. Taken together, our data indicate that the chromatin-remodeling enzyme BRM modulates PORC expression through interacting with PIF1, providing a novel regulatory mechanism by which plants fine-tune chlorophyll biosynthesis during the transition from heterotrophic to autotrophic growth.

Regulation of Vegetative Phase Change by SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA.

Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

HD2C histone deacetylase and a SWI/SNF chromatin remodelling complex interact and both are involved in mediating the heat stress response in Arabidopsis.

Studies in yeast and animals have revealed that histone deacetylases (HDACs) often act as components of multiprotein complexes, including chromatin remodelling complexes (CRCs). However, interactions between HDACs and CRCs in plants have yet to be demonstrated. Here, we present evidence for the interaction between Arabidopsis HD2C deacetylase and a BRM-containing SWI/SNF CRC. Moreover, we reveal a novel function of HD2C as a regulator of the heat stress response. HD2C transcript levels were strongly induced in plants subjected to heat treatment, and the expression of selected heat-responsive genes was up-regulated in heat-stressed hd2c mutant, suggesting that HD2C acts to down-regulate heat-activated genes. In keeping with the HDAC activity of HD2C, the altered expression of HD2C-regulated genes coincided in most cases with increased histone acetylation at their loci. Microarray transcriptome analysis of hd2c and brm mutants identified a subset of commonly regulated heat-responsive genes, and the effect of the brm hd2c double mutation on the expression of these genes was non-additive. Moreover, heat-treated 3-week-old hd2c, brm and brm hd2c mutants displayed similar rates of growth retardation. Taken together, our findings suggest that HD2C and BRM act in a common genetic pathway to regulate the Arabidopsis heat stress response.

The Arabidopsis SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA Targets Directly to PINs and Is Required for Root Stem Cell Niche Maintenance.

BRAHMA (BRM), a SWI/SNF chromatin remodeling ATPase, is essential for the transcriptional reprogramming associated with development and cell differentiation in Arabidopsis thaliana. In this study, we show that loss-of-function mutations in BRM led to defective maintenance of the root stem cell niche, decreased meristematic activity, and stunted root growth. Mutations of BRM affected auxin distribution by reducing local expression of several PIN-FORMED (PIN) genes in the stem cells and impaired the expression of the stem cell transcription factor genes PLETHORA (PLT1) and PLT2. Chromatin immunoprecipitation assays showed that BRM could directly target to the chromatin of PIN1, PIN2, PIN3, PIN4, and PIN7. In addition, genetic interaction assays indicate that PLTs acted downstream of BRM, and overexpression of PLT2 partially rescued the stem cell niche defect of brm mutants. Taken together, these results support the idea that BRM acts in the PLT pathway to maintain the root stem cell niche by altering the expression of PINs.

The Arabidopsis SWI2/SNF2 chromatin Remodeler BRAHMA regulates polycomb function during vegetative development and directly activates the flowering repressor gene SVP.

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.

Evidence for chromatin-remodeling complex PBAP-controlled maintenance of the Drosophila ovarian germline stem cells.

In the Drosophila oogenesis, germline stem cells (GSCs) continuously self-renew and differentiate into daughter cells for consecutive germline lineage commitment. This developmental process has become an in vivo working platform for studying adult stem cell fate regulation. An increasing number of studies have shown that while concerted actions of extrinsic signals from the niche and intrinsic regulatory machineries control GSC self-renewal and germline differentiation, epigenetic regulation is implicated in the process. Here, we report that Brahma (Brm), the ATPase subunit of the Drosophila SWI/SNF chromatin-remodeling complexes, is required for maintaining GSC fate. Removal or knockdown of Brm function in either germline or niche cells causes a GSC loss, but does not disrupt normal germline differentiation within the germarium evidenced at the molecular and morphological levels. There are two Drosophila SWI/SNF complexes: the Brm-associated protein (BAP) complex and the polybromo-containing BAP (PBAP) complex. More genetic studies reveal that mutations in polybromo/bap180, rather than gene encoding Osa, the BAP complex-specific subunit, elicit a defect in GSC maintenance reminiscent of the brm mutant phenotype. Further genetic interaction test suggests a functional association between brm and polybromo in controlling GSC self-renewal. Taken together, studies in this paper provide the first demonstration that Brm in the form of the PBAP complex functions in the GSC fate regulation.

The SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA Represses Abscisic Acid Responses in the Absence of the Stress Stimulus inArabidopsis

The survival of plants as sessile organisms depends on their ability to cope with environmental challenges. Of key importance in this regard is the phytohormone abscisic acid (ABA). ABA not only promotes seed dormancy but also triggers growth arrest in postgermination embryos that encounter water stress. This is accompanied by increased desiccation tolerance. Postgermination ABA responses in Arabidopsis thaliana are mediated in large part by the ABA-induced basic domain/leucine zipper transcription factor ABA INSENSITIVE5 (ABI5). Here, we show that loss of function of the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) causes ABA hypersensitivity during postgermination growth arrest. ABI5 expression was derepressed in brm mutants in the absence of exogenous ABA and accumulated to high levels upon ABA sensing. This effect was likely direct; chromatin immunoprecipitation revealed BRM binding to the ABI5 locus. Moreover, loss of BRM activity led to destabilization of a nucleosome likely to repress ABI5 transcription. Finally, the abi5 null mutant was epistatic to BRM in postgermination growth arrest. In addition, vegetative growth defects typical of brm mutants in the absence of ABA treatment could be partially overcome by reduction of ABA responses, and brm mutants displayed increased drought tolerance. We propose a role for BRM in the balance between growth or stress responses.

Brahma Is Required for Proper Expression of the Floral Repressor FLC in Arabidopsis

BackgroundBRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.Methodology/Principal FindingsHere we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.Conclusions/SignificanceBRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.

Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes

In yeast and mammals, ATP-dependent chromatin remodelling complexes of the SWI/SNF family play critical roles in the regulation of transcription, cell proliferation, differentiation 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 effects 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-specific 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 differentiation of reproductive organs suggests that its regulatory functions do not entirely overlap those of ATSWI3C. The detection of two different 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.

The putative SWI/SNF complex subunit BRAHMA activates flower homeotic genes in Arabidopsis thaliana

Arabidopsis thaliana BRAHMA (BRM, also called AtBRM) is a SNF2 family protein homolog of Brahma, the ATPase of the Drosophila SWI/SNF complex involved in chromatin remodeling during transcription. Here we show that, in contrast to its Drosophila counterpart, BRM is not an essential gene. Thus, homozygous BRM loss of function mutants are viable but exhibit numerous defects including dwarfism, altered leaf and root development and several reproduction defects. The analysis of the progeny of self-fertilized heterozygous brm plants and reciprocal crosses between heterozygous and wild type plants indicated that disruption of BRM reduced both male and female gametophyte transmission. This was consistent with the presence of aborted ovules in the self-fertilized heterozygous flowers that contained arrested embryos predominantly at the two terminal cells stage. Furthermore, brm homozygous mutants were completely sterile. Flowers of brm loss-of-function mutants have several developmental abnormalities, including homeotic transformations in the second and third floral whorls. In accordance with these results, brm mutants present reduced levels of APETALA2, APETALA3, PISTILLATA and NAC-LIKE, ACTIVATED BY AP3/PI. We have previously shown that BRM strongly interacts with AtSWI3C. Now we extend our interaction studies demonstrating that BRM interacts weakly with AtSWI3B but not with AtSWI3A or AtSWI3D. In agreement with these results, the phenotype described in this study for brm plants is very similar to that previously described for the AtSWI3C mutant plants, suggesting that both proteins participate in the same genetic pathway or form a molecular complex.

Difficulty to establish vegetative compatibility of Japanese isolates of Verticillium dahliae Kleb. using melanin-synthesis deficient mutants.

Japanese isolates of Verticillium dahliae were formerly designated as tomato pathotype and non-tomato pathotype by pathogenicity tests. We attempted to classify 18 Japanese isolates based on vegetative compatibility. Only three isolates of the tomato pathotype induced compatible alm-brm mutants. A non-secreting brm mutant was not induced from isolate of the non-tomato pathotype. Complementation was not achieved by a pair of mutants from the tomato pathotype and those from the non-tomato pathotype. From these results, Japanese isolates of V. dahliae could not be separated into any vegetative compatibility groups using melanin synthesis-deficient mutants.