The maintenance of cellular phosphate (Pi) homeostasis is of great importance in living organisms. The SPX domain-containing protein 1 (SPX1) proteins from both Arabidopsis and rice have been proposed to act as sensors of Pi status. The molecular signal indicating the cellular Pi status and regulating Pi homeostasis in plants, however, remains to be identified, as Pi itself does not bind to the SPX domain. Here, we report the identification of the inositol pyrophosphate InsP 8 as a signaling molecule that regulates Pi homeostasis in Arabidopsis. Polyacrylamide gel electrophoresis profiling of InsPs revealed that InsP 8 level positively correlates with cellular Pi concentration. We demonstrated that the homologs of diphosphoinositol pentakisphosphate kinase (PPIP5K), VIH1 and VIH2, function redundantly to synthesize InsP 8 , and that the vih1 vih2 double mutant overaccumulates Pi. SPX1 directly interacts with PHR1, the central regulator of Pi starvation responses, to inhibit its function under Pi-replete conditions. However, this interaction is compromised in the vih1 vih2 double mutant, resulting in the constitutive induction of Pi starvation-induced genes, indicating that plant cells cannot sense cellular Pi status without InsP 8 . Furthermore, we showed that InsP 8 could directly bind to the SPX domain of SPX1 and is essential for the interaction between SPX1 and PHR1. Collectively, our study suggests that InsP 8 is the intracellular Pi signaling molecule serving as the ligand of SPX1 for controlling Pi homeostasis in plants.
De novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway and active DNA demethylation play important roles in controlling genome-wide DNA methylation patterns in plants. Little is known about how cells manage the balance between DNA methylation and active demethylation activities. Here, we report the identification of a unique RdDM target sequence, where DNA methylation is required for maintaining proper active DNA demethylation of the Arabidopsis genome. In a genetic screen for cellular antisilencing factors, we isolated several REPRESSOR OF SILENCING 1 (ros1) mutant alleles, as well as many RdDM mutants, which showed drastically reduced ROS1 gene expression and, consequently, transcriptional silencing of two reporter genes. A helitron transposon element (TE) in the ROS1 gene promoter negatively controls ROS1 expression, whereas DNA methylation of an RdDM target sequence between ROS1 5′ UTR and the promoter TE region antagonizes this helitron TE in regulating ROS1 expression. This RdDM target sequence is also targeted by ROS1, and defective DNA demethylation in loss-of-function ros1 mutant alleles causes DNA hypermethylation of this sequence and concomitantly causes increased ROS1 expression. Our results suggest that this sequence in the ROS1 promoter region serves as a DNA methylation monitoring sequence (MEMS) that senses DNA methylation and active DNA demethylation activities. Therefore, the ROS1 promoter functions like a thermostat (i.e., methylstat) to sense DNA methylation levels and regulates DNA methylation by controlling ROS1 expression.NA methylation is a conserved epigenetic mark important for development and stress responses in plants and many animals (1-4). Genome-wide DNA methylation patterns are dynamically regulated by establishment, maintenance, and removal activities (4, 5). In plants, de novo DNA methylation is controlled by the RNA-directed DNA methylation (RdDM) pathway, in which complementary pairing between long noncoding RNAs and siRNAs mediates cytosine methylation in a sequence-specific manner (2, 4, 6, 7). Best characterized in Arabidopsis, RdDM involves a complex array of regulators and mainly targets heterochromatic regions that are enriched with transposon elements (TEs) and other DNA repeat sequences (8, 9). Once established, Arabidopsis DNA methylation is maintained via different mechanisms depending on the cytosine contexts [i.e., CG, CHG, CHH (H represents A, T, or C)]. CG and CHG methylation is maintained by MET1 and CMT3, respectively (2), whereas CHH methylation within pericentromeric long TEs can be catalyzed by CMT2 and CHH methylation at other loci is established de novo by DRM2 during every cell cycle (2, 10). In contrast to DNA methyltransferases that establish and/or maintain cytosine methylation, plant 5-methylcytosine DNA glycosylases initiate a base excision repair pathway that erases DNA methylation, thereby generating, together with the establishment and maintenance activities, a dynamic landscape of DNA methylation (11, 12).The Arabidopsis ...
SUMMARY DNA methylation is a conserved epigenetic mark that plays important roles in plant and vertebrate development, genome stability, and gene regulation. Canonical Methyl-CpG-Binding Domain (MBD) proteins are important interpreters of DNA methylation that recognize methylated CG sites and recruit chromatin remodelers, histone deacetylases and histone methyltransferases to repress transcription. Here, we show that Arabidopsis MBD7 and Increased DNA Methylation 3 (IDM3) are anti-silencing factors that prevent gene repression and DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1 and the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn limit DNA methylation and prevent transcriptional gene silencing.
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