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

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 entire complex. Using affinity purification followed by mass spectrometry, we identify a set of BRM-associated subunits and demonstrate that the BRM complexes strongly resemble mammalian non-canonical BAF complexes. Furthermore, we identify BDH1 and 2 proteins as components of the BRM complex and, using mutant analyses, show that BDH1/2 are important for vegetative and generative development, as well as hormonal responses. We further show that BRD1/2/13 represent unique subunits of the BRM complexes, and their depletion severely affects the integrity of the complex, resulting in the formation of residual assemblies. Finally, analyses of BRM complexes after proteasome inhibition revealed the existence of a module consisting of the ATPase, ARP, and BDH proteins, assembled with other subunits in a BRD-dependent manner. Together, our results suggest modular organization of plant SWI/SNF complexes and provide a biochemical explanation for mutant phenotypes.

Histone H1 ­ structure and function / Histon H1 ­ struktura i funkcja.

Linker histones (H1) are proteins found in the nuclei of the vast majority of Eucaryota, playing important roles in their life and development. H1 takes part in processes such as chromatin condensation, transcriptional regulation of gene expression, apoptosis induction and many more. Despite its common presence and essential function, many questions remain unanswered. Experiments conducted to date don't provide unambiguous information on such crucial issues as e.g. the way linker histones bind to nucleosome. There is also much surprising information about H1 participating in physiological processes not connected directly to its widely known function ­ e.g. providing a microtubule organization center in plants or contributing to the defense against pathogens in fish. The objective of the present work is to provide insights into many aspects of linker histone structure and function, collect and systematize current knowledge and to outline questions worth answering in the future.

Histone H1 Purification and Post-Translational Modification Profiling by High-Resolution Mass Spectrometry.

It has proven particularly difficult to purify Linker (H1) histones from the model plant Arabidopsis thaliana. This is most likely due to its low nuclear DNA content and the abundance of substances that interfere with protein isolation. These problems have hindered the use of Arabidopsis for in-depth characterization of nuclear proteins by modern techniques based on mass spectrometry (MS). Here, we describe an improved methodology for preparing pure Arabidopsis H1s and separating them by HPLC into fractions corresponding to nonallelic variants. In addition, we outline basic approaches enabling the identification of posttranslational modifications of H1 by MS and their mapping by digestion with different proteases. We also discuss the analysis and interpretation of the acquired data.

Phylogeny-Based Systematization of Arabidopsis Proteins with Histone H1 Globular Domain.

H1 (or linker) histones are basic nuclear proteins that possess an evolutionarily conserved nucleosome-binding globular domain, GH1. They perform critical functions in determining the accessibility of chromatin DNA to trans-acting factors. In most metazoan species studied so far, linker histones are highly heterogenous, with numerous nonallelic variants cooccurring in the same cells. The phylogenetic relationships among these variants as well as their structural and functional properties have been relatively well established. This contrasts markedly with the rather limited knowledge concerning the phylogeny and structural and functional roles of an unusually diverse group of GH1-containing proteins in plants. The dearth of information and the lack of a coherent phylogeny-based nomenclature of these proteins can lead to misunderstandings regarding their identity and possible relationships, thereby hampering plant chromatin research. Based on published data and our in silico and high-throughput analyses, we propose a systematization and coherent nomenclature of GH1-containing proteins of Arabidopsis (Arabidopsis thaliana [L.] Heynh) that will be useful for both the identification and structural and functional characterization of homologous proteins from other plant species.

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.

Histone H1 Variants in Arabidopsis Are Subject to Numerous Post-Translational Modifications, Both Conserved and Previously Unknown in Histones, Suggesting Complex Functions of H1 in Plants.

Linker histones (H1s) are conserved and ubiquitous structural components of eukaryotic chromatin. Multiple non-allelic variants of H1, which differ in their DNA/nucleosome binding properties, co-exist in animal and plant cells and have been implicated in the control of genetic programs during development and differentiation. Studies in mammals and Drosophila have revealed diverse post-translational modifications of H1s, most of which are of unknown function. So far, it is not known how this pattern compares with that of H1s from other major lineages of multicellular Eukaryotes. Here, we show that the two main H1variants of a model flowering plant Arabidopsis thaliana are subject to a rich and diverse array of post-translational modifications. The distribution of these modifications in the H1 molecule, especially in its globular domain (GH1), resembles that occurring in mammalian H1s, suggesting that their functional significance is likely to be conserved. While the majority of modifications detected in Arabidopsis H1s, including phosphorylation, acetylation, mono- and dimethylation, formylation, crotonylation and propionylation, have also been reported in H1s of other species, some others have not been previously identified in histones.

A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis.

Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants.

Chromatin reprogramming during the somatic-to-reproductive cell fate transition in plants.

The life cycle of flowering plants is marked by several post-embryonic developmental transitions during which novel cell fates are established. Notably, the reproductive lineages are first formed during flower development. The differentiation of spore mother cells, which are destined for meiosis, marks the somatic-to-reproductive fate transition. Meiosis entails the formation of the haploid multicellular gametophytes, from which the gametes are derived, and during which epigenetic reprogramming takes place. Here we show that in the Arabidopsis female megaspore mother cell (MMC), cell fate transition is accompanied by large-scale chromatin reprogramming that is likely to establish an epigenetic and transcriptional status distinct from that of the surrounding somatic niche. Reprogramming is characterized by chromatin decondensation, reduction in heterochromatin, depletion of linker histones, changes in core histone variants and in histone modification landscapes. From the analysis of mutants in which the gametophyte fate is either expressed ectopically or compromised, we infer that chromatin reprogramming in the MMC is likely to contribute to establishing postmeiotic competence to the development of the pluripotent gametophyte. Thus, as in primordial germ cells of animals, the somatic-to-reproductive cell fate transition in plants entails large-scale epigenetic reprogramming.

BRAHMA ATPase of the SWI/SNF chromatin remodeling complex acts as a positive regulator of gibberellin-mediated responses in arabidopsis.

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.

Different statins produce highly divergent changes in gene expression profiles of human hepatoma cells: a pilot study.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol biosynthesis pathway. Statin therapy is commonly regarded as well tolerated. However, serious adverse effects have also been reported, especially during high-dose statin therapy. The aim of our study was to investigate the effect of statins on gene expression profiles in human hepatoma HepG2 cells using Affymetrix Human Genome U133 Plus 2.0 arrays. Expression of 102, 857 and 1091 genes was changed substantially in HepG2 cells treated with simvastatin, fluvastatin and atorvastatin, respectively. Pathway and gene ontology analysis showed that many of the genes with changed expression levels were involved in a broad range of metabolic processes. The presented data clearly indicate substantial differences between the tested statins.