Ros1 Mutant Research Articles

Transgenerational increases in DNA methylation in Arabidopsis plants defective in active DNA demethylation

Spontaneous gain or loss of DNA methylation occurs in plant and animal genomes, and DNA methylation changes can lead to meiotically stable epialleles that generate heritable phenotypic diversity. However, it is unclear whether transgenerational epigenetic stability may be regulated by any cellular factors. Here, we examined spontaneously occurring variations in DNA methylation in wild-type and ros1 mutant Arabidopsis plants that were propagated for ten generations from single-seed descent. We found that the ros1 mutant, which is defective in active DNA demethylation, showed an increased transgenerational epimutation rate. The ros1 mutation led to more spontaneously gained methylation than lost methylation at individual cytosines, compared to the wild type which had similar numbers of spontaneously gained and lost methylation cytosines. Consistently, transgenerational differentially methylated regions were also biased toward hypermethylation in the ros1 mutant. Our results reveal a genetic contribution of the ROS1 DNA demethylase to transgenerational epigenetic stability and suggest that ROS1 may have an unexpected surveillance function in preventing transgenerational DNA methylation increases.

Raman imaging monitors the time-resolved response of A. thaliana to the artificial inhibition of PSII

The short-term (0–96 h) response of A. thaliana to the oxidative stress induced by PSII inhibitor metribuzin was examined using Raman spectroscopy. Whole leaves of wildtype (WT, Col-0) and ros1 mutant were scanned and changes in carotenoids were examined. Strong differences in Raman intensity distributions between WT and ros1 were observed. A stronger decrease of carotenoid v1(C=C) band intensity across the leaf was observed in ros1 after 48 h of exposure to metribuzin. It can be assumed that higher sensitivity to oxidative stress in ros1 mutant results in significantly faster degradation of carotenoids.

Fine mapping and characterization of RLL6 locus required for anti-silencing of a transgene and DNA demethylation in Arabidopsis thaliana.

DNA methylation patterns in plants are dynamically shaped by the antagonistic actions of DNA methylation and demethylation pathways. Although the DNA methylation pathway has been well studied, the DNA demethylation pathway, however, are not fully understood so far. To gain deeper insights into the mechanisms of DNA demethylation pathway, we conducted a genetic screening for proteins that were involved in preventing epigenetic gene silencing, and then the ones, which were also implicated in DNA demethylation pathway, were used for further studies. Eventually, a mutant with low luciferase luminescence (low LUC luminescence) was recovered, and named reduced LUC luminescence 6–1 (rll6-1). Map-based cloning revealed that rll6-1 mutation was located on chromosome 4, and there were a total of 10 candidate genes residing within such a region. Analyses of genome-wide methylation patterns of rll6-1 mutant showed that mutation of RLL6 locus led to 3,863 hyper-DMRs (DMRs for differentially methylated regions) throughout five Arabidopsis chromosomes, and elevated DNA methylation level of 2 × 35S promoter, which was similar to that found in the ros1 (repressor of silencing 1) mutant. Further analysis demonstrated that there were 1,456 common hyper-DMRs shared by rll6-1 and ros1-7 mutants, suggesting that both proteins acted together in a synergistic manner to remove DNA methylation. Further investigations demonstrated that mutation of RLL6 locus did not affect the expression of the four genes of the DNA glycosylase/lyase family. Thus, our results demonstrate that RLL6 locus-encoded protein not only participates in transcriptional anti-silencing of a transgene, but is also involved in DNA demethylation pathway.

Non-destructive insights into photosynthetic and photoprotective mechanisms in Arabidopsis thaliana grown under two light regimes

Probing insights into understanding photosynthetic processes via non-invasive means has an added advantage when used in phenotyping or precision agriculture. We employed Raman spectroscopy and fluorescence-based methods to investigate both the changes in the photosynthetic processes and the underlying protective mechanisms on Arabidopsis thaliana wild-type (WT), and ros1, which is a mutant of a repressor of transcriptional gene silencing, both grown under low light (LL: 100 μmol m−2s−1) and high light (HL: 400 μmol m−2s−1) regimes. Raman imaging detected a lower carotenoid intensity after two weeks in those plants grown under HL, compared to those grown under the LL regime; we interpret this as the result of oxidative damage of β-carotene molecules. Further, the data revealed a significant depletion in carotenoids with enhanced phenolics around the midrib and tip of the WT leaves, but not in the ros1. On the contrary, small necrotic zones appeared after two weeks of HL in the ros1 mutant, pointing to the starting oxidative damage. The lower maximum quantum yield of the photochemistry (Fv/Fm) in the WT as well as in the ros1 mutant grown in HL (compared to those in the LL two weeks post-exposure), indicates the HL partially inactivated photosystems. Chlorophyll a fluorescence imaging further showed high non-photochemical quenching (NPQ) in the plants grown under the HL regime for both the WT and the ros1 mutant, but the spatial heterogeneity of NPQ images was much higher in the HL-grown ros1 mutant. Fluorescence screening methods revealed significantly high values of chlorophyll proxies in the WT as well as in the ros1 mutant two weeks after in the HL compared to those under LL. The data generally revealed an increased accumulation of phenolics under HL in both the WT and ros1 mutant plants, but the proxies of anthocyanin and flavonols were significantly lower in the ros1 mutant than in the WT. The comparatively low accumulation of anthocyanin in the ros1 mutant compared to the WT supports the Raman data. We conclude that integrated use of these techniques can be efficiently applied for a better understanding of insights into photosynthetic mechanisms.

Impact of DNA Demethylases on the DNA Methylation and Transcription of Arabidopsis NLR Genes.

Active DNA demethylation is an important epigenetic process that plays a key role in maintaining normal gene expression. In plants, active DNA demethylation is mediated by DNA demethylases, including ROS1, DME, DML2, and DML3. In this study, the available bisulfite sequencing and mRNA sequencing data from ros1 and rdd mutants were analyzed to reveal how the active DNA demethylation process shapes the DNA methylation patterns of Arabidopsis nucleotide-binding leucine-rich repeat (NLR) genes, a class of important plant disease resistance genes. We demonstrate that the CG methylation levels of three NLR genes (AT5G49140, AT5G35450, and AT5G36930) are increased in the ros1 mutants relative to the wild-type plants, whereas the CG methylation level of AT2G17050 is decreased. We also observed increased CG methylation levels of AT4G11170 and AT5G47260 and decreased CG methylation levels of AT5G38350 in rdd mutants. We further found that the expression of three NLR genes (AT1G12280, AT1G61180, and AT4G19520) was activated in both ros1 and rdd mutants, whereas the expression of another three NLR genes (AT1G58602, AT1G59620, and AT1G62630) was repressed in these two mutants. Quantitative reverse transcriptase–polymerase chain reaction detection showed that the expression levels of AT1G58602.1, AT4G19520.3, AT4G19520.4, and AT4G19520.5 were decreased in the ros1 mutant; AT3G50950.1 and AT3G50950.2 in the rdd mutant were also decreased in expression compared to Col-0, whereas AT1G57630.1, AT1G58602.2, and AT5G45510.1 were upregulated in the rdd mutant relative to Col-0. These results indicate that some NLR genes are regulated by DNA demethylases. Our study demonstrates that each DNA demethylase (ROS1, DML2, and DML3) exerts a specific effect on the DNA methylation of the NLR genes, and active DNA demethylation is part of the regulation of DNA methylation and transcriptional activity of some Arabidopsis NLR genes.

The viral suppressor HCPro decreases DNA methylation and activates auxin biosynthesis genes

Auxin has profound effects on plant growth and development. In addition to participating in plant growth and development, the auxin signaling pathway is involved in plant defense against pathogens. In this study, we investigated the molecular mechanism by which helper-component protease (HCPro) encoded by the Tobacco vein banding mosaic virus (TVBMV) activates auxin biosynthesis genes (YUCs) and interferes with the auxin signaling pathway. Our results demonstrated that the viral suppressor HCPro decreased the DNA methylation of dispersed repeats (DRs) within the promoters of YUC1, YUC5 and YUC10 and transcriptional activated these YUC genes targeted by RNA-directed DNA methylation (RdDM), leading to an increase in auxin accumulation in plants. Furthermore, we found that the induction of these YUCs by HCPro was attenuated in ros1 mutant plants, suggesting that HCPro-mediated transcriptional activation of the genes was partly dependent on ROS1-mediated DNA demethylation.

Detection of Circulating Tumor Cell Molecular Subtype in Pulmonary Vein Predicting Prognosis of Stage I-III Non-small Cell Lung Cancer Patients.

Background: There was rare studies on prognosis of pulmonary venous CTC and early or advanced NSCLC patients. We want to investigate whether CTCs and the subtype of it can predict the prognosis of NSCLC patients.Patients and Methods: One hundred and fourteen patients with stage I-III NSCLC were included CanPatrol™ CTC analysis. PD-L1 expression level were detected in CTC of pulmonary vein. PD-L1, number of CTC in pulmonary, CTC's subtype, clinical characteristics, prognosis of patients were analyzed.Results: 110/114 (96.5%) patients could be found CTCs in pulmonary vein, 58/114 (50.9%) patients had CTC≥15/ml in pulmonary vein, 53/110 patients (48.2%) were defined as having MCTC subtype and 56/110 patient were found have PD-L1 (+) CTC in pulmonary vein. Multivariate analyses showed that PVCTC, MCTC, and stage were independent factors of DFS (P < 0.05). No OS difference was found between number of CTC (P = 0.33) and other CTC factors (P > 0.05), only stage was independent factor of OS (P = 0.019). There were decreases of CTC number and MCTC number in EGFR mutant subgroup (P = 0.0009 and P = 0.007). There were increases of CTC (P = 0.0217), MCTC (P = 0.0041), and PD-L1 (+) CTC (P = 0.0002) number in KRAS mutant subgroup. There was increase of MCTC (P =0.0323) number in BRAF mutant. There were fewer CTCs in pulmonary vein for patients with EGFR mutant than in patients with full wild-type gene (P = 0.0346). There were more PD-L1 positive CTCs in pulmonary vein for patients with ALK rearrangement, KRAS mutant, BRAF mutant, or ROS1 mutant than in patients with full wild-type gene (P = 0.0610, P = 0.0003, P = 0.032, and P = 0.0237). There were more mesenchymal CTCs in pulmonary vein for patients with KRAS mutant and BRAF mutant than in patients with full wild-type gene (P = 0.073 and P = 0.0381). There were fewer mesenchymal CTCs in pulmonary vein for patients with EGFR mutant than in patients with full wild-type gene (P = 0.0898).Conclusions: The patients with high number of CTCs, MCTCs, or PD-L1 (+) CTCs in pulmonary vein experienced poor prognosis of DFS. There are obvious correlations between the CTC subtype of NSCLC and the gene subgroups of tumor tissue.

Investigation on the binding mechanism of loratinib with the c-ros oncogene 1 (ROS1) receptor tyrosine kinase via molecular dynamics simulation and binding free energy calculations

The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild–type (WT) ROS1 and ROS1G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1WT and apo-ROS1G2032R systems showed similar RMSF distributions, while ROS1G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1G2032R significantly interfered the fluctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design.

Abstract 3171: Analysis of "drug addiction" mechanisms in the drug-resistant ROS1-rearranged cancer cells

Abstract ROS1-rearranged non-small-cell lung cancer (NSCLC) is observed in approximately 1% of lung cancer patients. Crizotinib, ALK/ROS1/cMET tyrosine kinase inhibitor (TKI), showed high effect against ROS1-rearranged NSCLC in the clinical trials and is approved in US and EU. However, the cancers inevitably relapse due to the acquired resistance such as ROS1-G2032R mutation. We previously reported that cabozantinib could overcome those secondary mutations mediated crizotinib-resistance. Based on this finding, we tried to establish cabozantinib-resistant ROS1 mutant cells by ENU mutagenesis screening. Surprisingly, we found out a few CD74-ROS1 mutant clones that only grew in the presence of low-dose cabozantinib. Namely, these mutant clones were “addicted” to cabozantinib. When the fluorescent labeled cabozantinib addicted ROS1 mutant cells were co-cultured with non-addicted CD74-ROS1-WT-BaF3 cell in the presence or absence of low-dose cabozantinib, the drug-addicted cells became dominant in the low-dose cabozantinib, and CD74-ROS1-WT cells became dominant in the absence of it. To analyze the molecular mechanism of “drug-addiction” in the addicted CD74-ROS1 mutant cells, we performed comprehensive analysis such as inhibitor screening, cDNA microarray, and phosphoproteome analysis. As the result, we observed that the expression and phosphorylation of CD74-ROS1 in the addicted cells were excessively upregulated within 24 hours after removal of cabozantinib or other ROS1 inhibitors, such as crizotinib or lorlatinib. We also observed that the apoptosis was induced in these cells upon ROS1 inhibitor removal, whereas ROS1 mediated growth signaling were simultaneously activated. Consistent with this, cDNA microarray analysis revealed that the expression of both cell survival and cell death related genes were changed by ROS1 inhibitor removal. Furthermore, we identified that several proteins were highly phosphorylated by excessive ROS1 activation originated from ROS1 inhibitor removal. These observations suggest that these drug-addicted cells were died by excessive oncogenic signaling, and the appropriate oncogene signaling by low-dose TKI made them survive and grow without inducing apoptosis. Thus, the excessive oncogene signaling has “double-edged sword” characteristics for cancer cell viability and those characteristics could be a new therapeutic target. Citation Format: Hayato Ogura, Jun Adachi, Takeshi Tomonaga, Naoya Fujita, Ryohei Katayama. Analysis of "drug addiction" mechanisms in the drug-resistant ROS1-rearranged cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3171. doi:10.1158/1538-7445.AM2017-3171

The role of DNA (de)methylation in immune responsiveness of Arabidopsis.

SummaryDNA methylation is antagonistically controlled by DNA methyltransferases and DNA demethylases. The level of DNA methylation controls plant gene expression on a global level. We have examined impacts of global changes in DNA methylation on the Arabidopsis immune system. A range of hypo‐methylated mutants displayed enhanced resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), whereas two hyper‐methylated mutants were more susceptible to this pathogen. Subsequent characterization of the hypo‐methylated nrpe1 mutant, which is impaired in RNA‐directed DNA methylation, and the hyper‐methylated ros1 mutant, which is affected in DNA demethylation, revealed that their opposite resistance phenotypes are associated with changes in cell wall defence and salicylic acid (SA)‐dependent gene expression. Against infection by the necrotrophic pathogen Plectosphaerella cucumerina, nrpe1 showed enhanced susceptibility, which was associated with repressed sensitivity of jasmonic acid (JA)‐inducible gene expression. Conversely, ros1 displayed enhanced resistance to necrotrophic pathogens, which was not associated with increased responsiveness of JA‐inducible gene expression. Although nrpe1 and ros1 were unaffected in systemic acquired resistance to Hpa, they failed to develop transgenerational acquired resistance against this pathogen. Global transcriptome analysis of nrpe1 and ros1 at multiple time‐points after Hpa infection revealed that 49% of the pathogenesis‐related transcriptome is influenced by NRPE1‐ and ROS1‐controlled DNA methylation. Of the 166 defence‐related genes displaying augmented induction in nrpe1 and repressed induction in ros1, only 25 genes were associated with a nearby transposable element and NRPE1‐ and/or ROS1‐controlled DNA methylation. Accordingly, we propose that the majority of NRPE1‐ and ROS1‐dependent defence genes are regulated in trans by DNA methylation.

Regulatory link between DNA methylation and active demethylation in Arabidopsis

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.

The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis

Although DNA methylation is known to play an important role in the silencing of transposable elements (TEs) and introduced transgenes, the mechanisms that generate DNA methylation-independent transcriptional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation (RdDM) is required for the silencing of the RD29A-LUC transgene in the Arabidopsis ros1 mutant background with defective DNA demethylase. Loss of function of ARGONAUTE 4 (AGO4) gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the ros1/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC transgene expression in the ros1/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcriptional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.

Cabozantinib overcomes crizotinib resistance in ROS1 fusion-positive cancer.

ROS1 rearrangement leads to constitutive ROS1 activation with potent transforming activity. In an ongoing phase I trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib shows remarkable initial responses in patients with non-small cell lung cancer (NSCLC) harboring ROS1 fusions; however, cancers eventually develop crizotinib resistance due to acquired mutations such as G2032R in ROS1. Thus, understanding the crizotinib-resistance mechanisms in ROS1-rearranged NSCLC and identification of therapeutic strategies to overcome the resistance are required. The sensitivity of CD74-ROS1-transformed Ba/F3 cells to multiple ALK inhibitors was examined. Acquired ROS1 inhibitor-resistant mutations in CD74-ROS1 fusion were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3 cells. To overcome the resistance mutation, we performed high-throughput drug screening with small-molecular inhibitors and anticancer drugs used in clinical practice or being currently tested in clinical trials. The effect of the identified drug was assessed in the CD74-ROS1-mutant Ba/F3 cells and crizotinib-resistant patient-derived cancer cells (MGH047) harboring G2032R-mutated CD74-ROS1. We identified multiple novel crizotinib-resistance mutations in the ROS1 kinase domain, including the G2032R mutation. As the result of high-throughput drug screening, we found that the cMET/RET/VEGFR inhibitor cabozantinib (XL184) effectively inhibited the survival of CD74-ROS1 wild-type (WT) and resistant mutants harboring Ba/F3 and MGH047 cells. Furthermore, cabozantinib could overcome all the resistance by all newly identified secondary mutations. We developed a comprehensive model of acquired resistance to ROS1 inhibitors in NSCLC with ROS1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance.

Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation.

DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes, but there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells.

Mutation in the catalytic subunit of DNA polymerase α influences transcriptional gene silencing and homologous recombination in Arabidopsis

REPRESSOR OF SILENCING 1 (ROS1) encodes a DNA demethylase that actively removes DNA methylation. Mutation in ROS1 leads to transcriptional gene silencing of a T-DNA locus that contains two genes, RD29A-LUC and 35S-NPTII, originally expressed in the C24 wild type. These units have different silencing regulation mechanisms: the former mechanism is dependent on small interfering RNA (siRNA)-directed DNA methylation, but the latter is not. We studied the latter gene silencing mechanism by screening the suppressors of the ros1 mutant using the silenced 35S-NPTII as a selection marker gene. The polalpha/incurvata2 (icu2) gene was isolated as one ros1 suppressor because its mutation leads to the reactivation of the silenced 35S-NPTII gene. POLalpha/ICU2 encodes a catalytic subunit of DNA polymerase alpha. Mutation of POLalpha/ICU2 did not affect DNA methylation, but reduced histone H3 Lys9 dimethylation (H3K9me2) modification in the 35S promoter. The polalpha mutation also influences the development of the shoot apical meristem, and delays the G2/M phase with high expression of a G2/M marker gene CycB1;1:GUS. Furthermore, the frequency of homologous recombination is greater in the polalpha/icu2 mutant than in the C24 wild type. Our results suggest that DNA polymerase alpha is involved in mediating epigenetic states and in DNA homologous recombination in Arabidopsis.

Epigenetic Regulation, Somatic Homologous Recombination, and Abscisic Acid Signaling Are Influenced by DNA Polymerase ϵ Mutation inArabidopsis

Based on abscisic acid (ABA) inhibition of seed germination and seedling growth assays, we isolated an ABA overly sensitive mutant (abo4-1) caused by a mutation in the Arabidopsis thaliana POL2a/TILTED1(TIL1) gene encoding a catalytic subunit of DNA polymerase epsilon. The dominant, ABA-insensitive abi1-1 or abi2-1 mutations suppressed the ABA hypersensitivity of the abo4-1 mutant. The abo4/til1 mutation reactivated the expression of the silenced Athila retrotransposon transcriptional silent information (TSI) and the silenced 35S-NPTII in the ros1 mutant and increased the frequency of somatic homologous recombination (HR) approximately 60-fold. ABA upregulated the expression of TSI and increased HR in both the wild type and abo4-1. MEIOTIC RECOMBINATION11 and GAMMA RESPONSE1, both of which are required for HR and double-strand DNA break repair, are expressed at higher levels in abo4-1 and are enhanced by ABA, while KU70 was suppressed by ABA. abo4-1 mutant plants are sensitive to UV-B and methyl methanesulfonate and show constitutive expression of the G2/M-specific cyclin CycB1;1 in meristems. The abo4-1 plants were early flowering with lower expression of FLOWER LOCUS C and higher expression of FLOWER LOCUS T and changed histone modifications in the two loci. Our results suggest that ABO4/POL2a/TIL1 is involved in maintaining epigenetic states, HR, and ABA signaling in Arabidopsis.

The DNA Glycosylase/Lyase ROS1 Functions in Pruning DNA Methylation Patterns in Arabidopsis

The Arabidopsis DNA glycosylase/lyase ROS1 participates in active DNA demethylation by a base-excision pathway. ROS1 has been shown to be required for demethylating a transgene promoter. To determine the function of ROS1 in demethylating endogenous loci, we carried out bisulfite-sequencing analysis of several transposons and other genes in the ros1 mutant. In the wild-type, although CpG sites at the majority of these loci are heavily methylated, many of the CpXpG and CpXpX sites have low levels of methylation or are not at all methylated. However, these CpXpG and CpXpX sites become heavily methylated in the ros1 mutant. Associated with this increased DNA methylation, these loci show decreased expression in the ros1 mutant. Our results suggest that active DNA demethylation is important in pruning the methylation patterns of the genome, and even the normally "silent" transposons are under dynamic control by both methylation and demethylation. This dynamic control may be important in keeping the plant epigenome plastic so that it can efficiently respond to developmental and environmental cues.

The protein kinase TOUSLED is required for maintenance of transcriptional gene silencing in Arabidopsis

TOUSLED-like kinases (TLKs) are highly conserved in plants and animals, but direct evidence linking TLKs and transcriptional gene silencing is lacking. We isolated two new alleles of TOUSLED (TSL). Mutations of TSL in ros1 reactivate the transcriptionally silent 35S-NPTII transgene and the transcriptionally silent endogenous loci TSI (TRANSCRIPTIONAL SILENCING INFORMATION). Chromatin immunoprecipitation (ChIP) analysis shows that histone H3Lys9 dimethylation is decreased in the reactivated transgene and endogenous TSI loci in the tsl ros1 mutant. However, there is no change in DNA methylation in the affected loci. Western blot and ChIP assay suggest that TSL might not be responsible for histone H3Ser10 phosphorylation. The tsl seedlings were more sensitive to DNA damage reagent methyl methanesulphonate and UV-B light. Our results provide direct evidence for a crucial role of the TOUSLED protein kinase in the maintenance of transcriptional gene silencing in some genomic regions in a DNA-methylation-independent manner in Arabidopsis.

ROR1/RPA2A, a Putative Replication Protein A2, Functions in Epigenetic Gene Silencing and in Regulation of Meristem Development inArabidopsis

We screened for suppressors of repressor of silencing1 (ros1) using the silenced 35S promoter-neomycin phosphotransferase II (Pro(35S):NPTII) gene as a marker and identified two allelic mutants, ror1-1 and ror1-2 (for suppressor of ros1). Map-based cloning revealed that ROR1 encodes a 31-kD protein similar to DNA replication protein A2 (RPA2A). Mutations in ROR1 reactivate the silenced Pro(35S):NPTII gene but not RD29A promoter-luciferase in the ros1 mutant. DNA methylation in rDNA, centromeric DNA, and RD29A promoter regions is not affected by ror1. However, chromatin immunoprecipitation data suggest that histone H3 acetylation is increased and histone H3K9 dimethylation is decreased in the 35S promoter in the ror1 ros1 mutant compared with ros1. These results indicate that release of silenced Pro(35S):NPTII by ror1 mutations is independent of DNA methylation. ROR1/RPA2A is strongly expressed in shoot and root meristems. Mutations in ROR1/RPA2A affect cell division in meristems but not final cell sizes. Our work suggests important roles of ROR1/RPA2A in epigenetic gene silencing and in the regulation of plant development.

Mutations in a Conserved Replication Protein Suppress Transcriptional Gene Silencing in a DNA- Methylation-Independent Manner in Arabidopsis

Mutations in the DNA glycosylase/lyase ROS1 cause transcriptional silencing of the linked RD29A-LUC and 35S-NPTII transgenes in Arabidopsis. We report here that mutations in the Arabidopsis RPA2 locus release the silencing of 35S-NPTII but not RD29A-LUC in the ros1 mutant background. The rpa2 mutation also leads to enhanced expression of some transposons. Neither DNA methylation nor siRNAs at any of the reactivated loci are blocked by rpa2. Histone H3 methylation at lysine 4 was increased and histone H3 methylation at lysine 9 was decreased at the 35S promoter in the ros1rpa2 mutant compared to the ros1 background. RPA2 encodes a nuclear protein similar to the second subunit of the replication protein A conserved from yeast to mammals. Ectopic expression of the Arabidopsis RPA2 could complement the yeast rfa2 (rpa2) mutant. These results suggest an essential role of RPA2 in the maintenance of transcriptional gene silencing at specific loci in a DNA-methylation-independent manner. In addition, we found that rpa2 mutants are hypersensitive to the genotoxic agent methyl methanesulphonate, and the RPA2 protein interacts with ROS1 in vitro and in vivo, suggesting that RPA2 also functions together with ROS1 in DNA repair.