Imitation Switch chromatin remodeling factors and their interacting RINGLET proteins act together in controlling the plant vegetative phase in Arabidopsis

During their life cycle, flowering plants must experience a transition from vegetative to reproductive growth. Here, we report that double mutations in the Arabidopsis thaliana IMITATION SWITCH (AtISWI) genes, CHROMATIN REMODELING11 (CHR11) and CHR17, and the plant‐specific DDT‐domain containing genes, RINGLET1 (RLT1) and RLT2, resulted in plants with similar developmental defects, including the dramatically accelerated vegetative‐to‐reproductive transition. We demonstrated that AtISWI physically interacts with RLTs in preventing plants from activating the vegetative‐to‐reproductive transition early by regulating several key genes that contribute to flower timing. In particular, AtISWI and RLTs repress FT, SEP1, SEP3, FUL, and SOC1, but promote FLC in the leaf. Furthermore, AtISWI and RLTs may directly repress FT and SEP3 through associating with the FT and SEP3 loci. Our study reveals that AtISWI and RLTs represent a previously unrecognized genetic pathway that is required for the maintenance of the plant vegetative phase.     

ISWI proteins participate in the genome‐wide nucleosome distribution in Arabidopsis

Chromatin is a highly organized structure with repetitive nucleosome subunits. Nucleosome distribution patterns, which contain information on epigenetic controls, are dynamically affected by ATP‐dependent chromatin remodeling factors (remodelers). However, whether plants have specific nucleosome distribution patterns and how plant remodelers contribute to the pattern formation are not clear. In this study we used the micrococcal nuclease digestion followed by deep sequencing (MNase‐seq) assay to show the genome‐wide nucleosome pattern in Arabidopsis thaliana. We demonstrated that the nucleosome distribution patterns of Arabidopsis are associated with the gene expression level, and have several specific characteristics that are different from those of animals and yeast. In addition, we found that remodelers in the A. thaliana imitation switch (AtISWI) subfamily are important for the formation of the nucleosome distribution pattern. Double mutations in the AtISWI genes, CHROMATIN REMODELING 11 (CHR11) and CHR17, resulted in the loss of the evenly spaced nucleosome pattern in gene bodies, but did not affect nucleosome density, supporting a previous idea that the primary role of ISWI is to slide nucleosomes in gene bodies for pattern formation.     

The DNA-binding domain of the Chd1 chromatin-remodelling enzyme contains SANT and SLIDE domains

The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6-9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome.     

FHA2 is a plant-specific ISWI subunit responsible for stamen development and plant fertility

Imitation Switch (ISWI) chromatin remodelers are known to function in diverse multi‐subunit complexes in yeast and animals. However, the constitution and function of ISWI complexes in Arabidopsis thaliana remain unclear. In this study, we identified forkhead‐associated domain 2 (FHA2) as a plant‐specific subunit of an ISWI chromatin‐remodeling complex in Arabidopsis. By in vivo and in vitro analyses, we demonstrated that FHA2 directly binds to RLT1 and RLT2, two redundant subunits of the ISWI complex in Arabidopsis. The stamen filament is shorter in the fha2 and rlt1/2 mutants than in the wild type, whereas their pistil lengths are comparable. The shorter filament, which is due to reduced cell size, results in insufficient pollination and reduced fertility. The rlt1/2 mutant shows an early‐flowering phenotype, whereas the phenotype is not shared by the fha2 mutant. Consistent with the functional specificity of FHA2, our RNA‐seq analysis indicated that the fha2 mutant affects a subset of RLT1/2‐regulated genes that does not include genes involved in the regulation of flowering time. This study demonstrates that FHA2 functions as a previously uncharacterized subunit of the Arabidopsis ISWI complex and is exclusively involved in regulating stamen development and plant fertility.     

Nuclear Import of Chromatin Remodeler Isw1 Is Mediated by Atypical Bipartite cNLS and Classical Import Pathway

The protein Isw1 of Saccharomyces cerevisiae is an imitation‐switch chromatin‐remodeling factor. We studied the mechanisms of its nuclear import and found that the nuclear localization signal (NLS) mediating the transport of Isw1 into the nucleus is located at the end of the C‐terminus of the protein (aa1079–1105). We show that it is an atypical bipartite signal with an unconventional linker of 19 aa (KRIR X₁₉ KKAK) and the only nuclear targeting signal within the Isw1 molecule. The efficiency of Isw1 nuclear import was found to be modulated by changes to the amino acid composition in the vicinity of the KRIR motif, but not by the linker length. Live‐cell imaging of various karyopherin mutants and in vitro binding assays of Isw1NLS to importin‐α revealed that the nuclear translocation of Isw1 is mediated by the classical import pathway. Analogous motifs to Isw1NLS are highly conserved in Isw1 homologues of other yeast species, and putative bipartite cNLS were identified in silico at the end of the C‐termini of imitation switch (ISWI) proteins from higher eukaryotes. We suggest that the C‐termini of the ISWI family proteins play an important role in their nuclear import.     

No need for a power stroke in ISWI‐mediated nucleosome sliding

Nucleosome remodelling enzymes of the ISWI family reposition nucleosomes in eukaryotes. ISWI contains an ATPase and a HAND‐SANT‐SLIDE (HSS) domain. Conformational changes between these domains have been proposed to be critical for nucleosome repositioning by pulling flanking DNA into the nucleosome. We inserted flexible linkers at strategic sites in ISWI to disrupt this putative power stroke and assess its functional importance by quantitative biochemical assays. Notably, the flexible linkers did not disrupt catalysis. Instead of engaging in a power stroke, the HSS module might therefore assist DNA to ratchet into the nucleosome. Our results clarify the roles had by the domains and suggest that the HSS domain evolved to optimize a rudimentary remodelling engine.     

The chromatin remodeling complex imitation of switch controls stamen filament elongation by promoting jasmonic acid biosynthesis in Arabidopsis

Plant reproduction requires the coordinated development of both male and female reproductive organs.Jasmonic acid(JA) plays an essential role in stamen filament elongation. However, the mechanism by which the JA biosynthesis genes are regulated to promote stamen elongation remains unclear. Here, we show that the chromatin remodeling complex Imitation of Switch(ISWI) promotes stamen filament elongation by regulating JA biosynthesis. We show that AT-Rich Interacting Domain 5(ARID5) interacts with CHR11,CHR17, and RLT1, several known subunits of ISWI. Mutations in ARID5 and RLTs caused a reduced seed set due to greatly shortened stamen filaments. RNA-seq analyses reveal that the expression of key genes responsible for JA biosynthesis is significantly down-regulated in the arid5 and rlt mutants. Consistently, the JA levels are drastically decreased in both arid5 and rlt mutants. Chromatin immunoprecipitationquantitative PCR analyses further show that ARID5 is recruited to the chromatin of JA biosynthesis genes. Importantly, exogenous JA treatments can fully rescue the defects of stamen filament elongation in both arid5 and rlt mutants, leading to the partial recovery of fertility. Our results provide a clue how JA biosynthesisis positively regulated by the chromatin remodeling complex ISWI, thereby promoting stamen filament elongation in Arabidopsis.     

Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI

Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.     

HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.     

ISWI is an ATP-dependent nucleosome remodeling factor

The ATPase ISWI is a subunit of several distinct nucleosome remodeling complexes that increase the accessibility of DNA in chromatin. We found that the isolated ISWI protein itself was able to carry out nucleosome remodeling, nucleosome rearrangement, and chromatin assembly reactions. The ATPase activity of ISWI was stimulated by nucleosomes but not by free DNA or free histones, indicating that ISWI recognizes a specific structural feature of nucleosomes. Nucleosome remodeling, therefore, does not require a functional interaction between ISWI and the other subunits of ISWI complexes. The role of proteins associated with ISWI may be to regulate the activity of the remodeling engine or to define the physiological context within which a nucleosome remodeling reaction occurs.     

Molecular organization,biochemical function,cellular role and evolution of NfuA,an atypical Fe‐S carrier

Biosynthesis of iron–sulphur (Fe‐S) proteins is catalysed by multi‐protein systems, ISC and SUF. However, ‘non‐ISC, non‐SUF’ Fe‐S biosynthesis factors have been described, both in prokaryotes and eukaryotes. Here we report in vitro and in vivo investigations of such a ‘non‐ISC, non SUF’ component, the Nfu proteins. Phylogenomic analysis allowed us to define four subfamilies. Escherichia coli NfuA is within subfamily II. Most members of this subfamily have a Nfu domain fused to a ‘degenerate’ A‐type carrier domain (ATC*) lacking Fe‐S cluster co‐ordinating Cys ligands. The Nfu domain binds a [4Fe‐4S] cluster while the ATC* domain interacts with NuoG (a complex I subunit) and aconitase B (AcnB). In vitro, holo‐NfuA promotes maturation of AcnB. In vivo, NfuA is necessary for full activity of complex I under aerobic growth conditions, and of AcnB in the presence of superoxide. NfuA receives Fe‐S clusters from IscU/HscBA and SufBCD scaffolds and eventually transfers them to the ATCs IscA and SufA. This study provides significant information on one of the Fe‐S biogenesis factors that has been often used as a building block by ISC and/or SUF synthesizing organisms, including bacteria, plants and animals.     

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.     

Histidine protein kinases: key signal transducers outside the animal kingdom

Histidine protein kinases (HPKs) are a large family of signal-transduction enzymes that autophosphorylate on a conserved histidine residue. HPKs form two-component signaling systems together with their downstream target proteins, the response regulators, which have a conserved aspartate in a so-called 'receiver domain' that is phosphorylated by the HPK. Two-component signal transduction is prevalent in bacteria and is also widely used by eukaryotes outside the animal kingdom. The typical HPK is a transmembrane receptor with an amino-terminal extracellular sensing domain and a carboxy-terminal cytosolic signaling domain; most, if not all, HPKs function as dimers. They show little similarity to protein kinases that phosphorylate serine, threonine or tyrosine residues, but may share a distant evolutionary relationship with these enzymes. In excess of a thousand known genes encode HPKs, which are important for multiple functions in bacteria, including chemotaxis and quorum sensing, and in eukaryotes, including hormone-dependent developmental processes. The proteins divide into at least 11 subfamilies, only one of which is present in eukaryotes, suggesting that lateral gene transfer gave rise to two-component signaling in these organisms.