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FLOWERING LOCUS M (FLM) is a well-known MADS-box transcription factor that is required for preventing early flowering under low temperatures in Arabidopsis thaliana. Alternative splicing of FLM is involved in the regulation of temperature-responsive flowering. However, how the basic transcript level of FLM is regulated is largely unknown. Here, we conducted forward genetic screening and identified a previously uncharacterized flowering repressor gene, UBA2c. Genetic analyses indicated that UBA2c represses flowering at least by promoting FLM transcription. We further demonstrated that UBA2c directly binds to FLM chromatin and facilitates FLM transcription by inhibiting histone H3K27 trimethylation, a histone marker related to transcriptional repression. UBA2c encodes a protein containing two putative RNA recognition motifs (RRMs) and one prion-like domain (PrLD). We found that UBA2c forms speckles in the nucleus and that both the RRMs and PrLD are required not only for forming the nuclear speckles but also for the biological function of UBA2c. These results identify a previously unknown flowering repressor and provide insights into the regulation of flowering time.

Although previous studies have identified several autonomous pathway components that are required for the promotion of flowering, little is known about how these components cooperate. Here, we identified an autonomous pathway complex (AuPC) containing both known components (FLD, LD and SDG26) and previously unknown components (EFL2, EFL4 and APRF1). Loss-of-function mutations of all of these components result in increased FLC expression and delayed flowering. The delayed-flowering phenotype is independent of photoperiod and can be overcome by vernalization, confirming that the complex specifically functions in the autonomous pathway. Chromatin immunoprecipitation combined with sequencing indicated that, in the AuPC mutants, the histone modifications (H3Ac, H3K4me3 and H3K36me3) associated with transcriptional activation are increased, and the histone modification (H3K27me3) associated with transcriptional repression is reduced, suggesting that the AuPC suppresses FLC expression at least partially by regulating these histone modifications. Moreover, we found that the AuPC component SDG26 associates with FLC chromatin via a previously uncharacterized DNA-binding domain and regulates FLC expression and flowering time independently of its histone methyltransferase activity. Together, these results provide a framework for understanding the molecular mechanism by which the autonomous pathway regulates flowering time.

In eukaryotes, MEDIATOR is a conserved multi-subunit complex that links transcription factors and RNA polymerase II and that thereby facilitates transcriptional initiation. Although the composition of MEDIATOR has been well studied in yeast and mammals, relatively little is known about the composition of MEDIATOR in plants. By affinity purification followed by mass spectrometry, we identified 28 conserved MEDIATOR subunits in Arabidopsis thaliana, including putative MEDIATOR subunits that were not previously validated. Our results indicated that MED34, MED35, MED36, and MED37 are not Arabidopsis MEDIATOR subunits, as previously proposed. Our results also revealed that two homologous CBP/p300 histone acetyltransferases, HAC1 and HAC5 (HAC1/5) are in fact plant-specific MEDIATOR subunits. The MEDIATOR subunits MED8 and MED25 (MED8/25) are partially responsible for the association of MEDIATOR with HAC1/5, MED8/25 and HAC1/5 co-regulate gene expression and thereby affect flowering time and floral development. Our in vitro observations indicated that MED8 and HAC1 form liquid-like droplets by phase separation, and our in vivo observations indicated that these droplets co-localize in the nuclear bodies at a subset of nuclei. The formation of liquid-like droplets is required for MED8 to interact with RNA polymerase II. In summary, we have identified all of the components of Arabidopsis MEDIATOR and revealed the mechanism underlying the link of histone acetylation and transcriptional regulation.

Trimethylated histone H3 lysine 27 (H3K27me3) is a repressive histone marker that regulates a variety of developmental processes, including those that determine flowering time. However, relatively little is known about the mechanism of how H3K27me3 is recognized to regulate transcription. Here, we identified BAH domain-containing transcriptional regulator 1 (BDT1) as an H3K27me3 reader. BDT1 is responsible for preventing flowering by suppressing the expression of flowering genes. Mutation of the H3K27me3 recognition sites in the BAH domain disrupted the binding of BDT1 to H3K27me3, leading to de-repression of H3K27me3-enriched flowering genes and an early-flowering phenotype. We also found that BDT1 interacts with a family of PHD finger-containing proteins, which we named PHD1-6, and with CPL2, a Pol II carboxyl terminal domain (CTD) phosphatase responsible for transcriptional repression. Pull-down assays showed that the PHD finger-containing proteins can enhance the binding of BDT1 to the H3K27me3 peptide. Mutations in all of the PHD genes caused increased expression of flowering genes and an early-flowering phenotype. This study suggests that the binding of BDT1 to the H3K27me3 peptide, which is enhanced by PHD proteins, is critical for preventing early flowering.

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.

Adenosine triphosphate-dependent chromatin remodeling complexes are important for the regulation of transcription, DNA replication, and genome stability in eukaryotes. Although genetic studies have illustrated various biological functions of core and accessory subunits of chromatin-remodeling complexes in plants, the identification and characterization of chromatin-remodeling complexes in plants is lagging behind that in yeast and animals. Recent studies determined whether and how the Arabidopsis SWI/SNF, ISWI, INO80, SWR1, and CHD chromatin remodelers function in multi-subunit complexes in Arabidopsis. Both conserved and plant-specific subunits of chromatin-remodeling complexes have been identified and characterized. These findings provide a basis for further studies of the molecular mechanisms by which the chromatin-remodeling complexes function in plants.

Association of RNA polymerase V (Pol V) with chromatin is a critical step for RNA- directed DNA methylation (RdDM) in plants. Although the methylated DNA-binding proteins SUVH2 and SUVH9 and the chromatin remodeler-containing complex DRD1-DMS3-RDM1 are known to be required for the association of Pol V with chromatin, the molecular mechanisms underlying the association of Pol V with different chromatin environments remain largely unknown. Here we found that SUVH9 interacts with FVE, a homolog of the mammalian retinoblastoma-associated protein, which has been previously identified as a shared subunit of the histone deacetylase complex and the polycomb-type histone H3K27 trimethyltransferase complex. We demonstrated that FVE facilitates the association of Pol V with chromatin and thus contributes to DNA methylation at a substantial subset of RdDM target loci. Compared with FVE-independent RdDM target loci, FVE-dependent RdDM target loci are more abundant in gene-rich chromosome arms than in pericentromeric heterochromatin regions. This study contributes to our understanding of how the association of Pol V with chromatin is regulated in different chromatin environments.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an evolutionarily conserved histone acetyltransferase complex that has a critical role in histone acetylation, gene expression, and various developmental processes in eukaryotes. However, little is known about the composition and function of the SAGA complex in plants. In this study, we found that the SAGA complex in Arabidopsis thaliana contains not only conserved subunits but also four plant-specific subunits: three functionally redundant paralogs, SCS1, SCS2A, and SCS2B (SCS1/2A/2B), and a TAF-like subunit, TAFL. Mutations in SCS1/2A/2B lead to defective phenotypes similar to those caused by mutations in the genes encoding conserved SAGA subunits HAG1 and ADA2B, including delayed juvenile-to-adult phase transition, late flowering, and increased trichome density. Furthermore, we demonstrated that SCS1/2A/2B are required for the function of the SAGA complex in histone acetylation, thereby promoting the transcription of development-related genes. These results together suggest that SCS1/2A/2B are core subunits of the SAGA complex in Arabidopsis. Compared with SAGA complexes in other eukaryotes, the SAGA complexes in plants have evolved unique features that are necessary for normal growth and development.

Small interfering RNAs (siRNAs) are responsible for establishing and maintaining DNA methylation through the RNA-directed DNA methylation (RdDM) pathway in plants. Although siRNA biogenesis is well known, it is relatively unclear about how the process is regulated. By a forward genetic screen in Arabidopsis thaliana, we identified a mutant defective in NOT1 and demonstrated that NOT1 is required for transcriptional silencing at RdDM target genomic loci. We demonstrated that NOT1 is required for Pol IV-dependent siRNA accumulation and DNA methylation at a subset of RdDM target genomic loci. Furthermore, we revealed that NOT1 is a constituent of a multi-subunit CCR4-NOT deadenylase complex by immunoprecipitation combined with mass spectrometry and demonstrated that the CCR4-NOT components can function as a whole to mediate chromatin silencing. Therefore, our work establishes that the CCR4-NOT complex regulates the biogenesis of Pol IV-dependent siRNAs, and hence facilitates DNA methylation and transcriptional silencing in Arabidopsis.

Deposition of H2A.Z in chromatin is known to be mediated by a conserved SWR1 chromatin-remodeling complex in eukaryotes. However, little is known about whether and how the SWR1 complex cooperates with other chromatin regulators. Using immunoprecipitation followed by mass spectrometry, we found all known components of the Arabidopsis thaliana SWR1 complex and additionally identified the following three classes of previously uncharacterized plant-specific SWR1 components: MBD9, a methyl-CpG-binding domain-containing protein; CHR11 and CHR17 (CHR11/17), ISWI chromatin remodelers responsible for nucleosome sliding; and TRA1a and TRA1b, accessory subunits of the conserved NuA4 histone acetyltransferase complex. MBD9 directly interacts with CHR11/17 and the SWR1 catalytic subunit PIE1, and is responsible for the association of CHR11/17 with the SWR1 complex. MBD9, TRA1a, and TRA1b function as canonical components of the SWR1 complex to mediate H2A.Z deposition. CHR11/17 are not only responsible for nucleosome sliding but also involved in H2A.Z deposition. These results indicate that the association of the SWR1 complex with CHR11/17 may facilitate the coupling of H2A.Z deposition with nucleosome sliding, thereby co-regulating gene expression, development, and flowering time.

Involvement of long non-coding RNAs (lncRNAs) in the regulation of gene expression in cis has been well studied in eukaryotes but relatively little is known whether and how lncRNAs affect gene expression in tans. In Arabidopsis thaliana, COLDAIR, a previously reported lncRNA, is produced from the first intron of FLOWERING LOCUS C (FLC), which encodes a repressor of flowering time. Our results indicated that the exogenously overexpressed COLDAIR enhances the expression of FLC in trans, resulting in a late-flowering phenotype. In 35S-COLDAIR lines, the enhanced expression of FLC is correlated with the down-regulation of the repressive histone mark H3K27me3 and with the up-regulation of the active histone mark H3K4me3 at the FLC chromatin. Furthermore, we demonstrated that overexpression of intronic lncRNAs from several other H3K27me3-enriched MADS-box genes also activates the expression of their host genes. This study suggests that the involvement of overexpressed intronic lncRNAs in gene activation may be conserved in H3K27me3-enriched genes in eukaryotes.

Heterochromatin is a tightly packed form of chromatin that is associated with DNA methylation and histone 3 lysine 9 methylation (H3K9me). Here, we identify an H3K9me2-binding protein, Agenet domain (AGD)-containing p1 (AGDP1), in Arabidopsis thaliana. Here we find that AGDP1 can specifically recognize the H3K9me2 mark by its three pairs of tandem AGDs. We determine the crystal structure of the Agenet domain 1 and 2 cassette (AGD12) of Raphanus sativus AGDP1 in complex with an H3K9me2 peptide. In the complex, the histone peptide adopts a unique helical conformation. AGD12 specifically recognizes the H3K4me0 and H3K9me2 marks by hydrogen bonding and hydrophobic interactions. In addition, we find that AGDP1 is required for transcriptional silencing, non-CG DNA methylation, and H3K9 dimethylation at some loci. ChIP-seq data show that AGDP1 preferentially occupies long transposons and is associated with heterochromatin marks. Our findings suggest that, as a heterochromatin-binding protein, AGDP1 links H3K9me2 to DNA methylation in heterochromatin regions., DNA methylation and H3K9 dimethylation are two linked epigenetic marks of silenced chromatin in plants that depend on the activity of CMT3/2 and SUVH4/5/6. Here the authors identify AGDP1 as an H3K9me2-binding protein required for heterochromatic non-CG DNA methylation, H3K9 dimethylation, and transcriptional silencing.

The DNA methyltransferases MET1 and CMT3 are known to be responsible for maintenance of DNA methylation at symmetric CG and CHG sites, respectively, in Arabidopsis thaliana. However, it is unknown how the expression of methyltransferase genes is regulated in different cell states and whether change in expression affects DNA methylation at the whole-genome level. Using a reverse genetic screen, we identified TCX5, a tesmin/TSO1-like CXC domain-containing protein, and demonstrated that it is a transcriptional repressor of genes required for maintenance of DNA methylation, which include MET1, CMT3, DDM1, KYP and VIMs. TCX5 functions redundantly with its paralogue TCX6 in repressing the expression of these genes. In the tcx5 tcx6 double mutant, expression of these genes is markedly increased, thereby leading to markedly increased DNA methylation at CHG sites and, to a lesser extent, at CG sites at the whole-genome level. Furthermore, our whole-genome DNA methylation analysis indicated that the CG and CHG methylation level is lower in differentiated quiescent cells than in dividing cells in the wild type but is comparable in the tcx5/6 mutant, suggesting that TCX5/6 are required for maintenance of the difference in DNA methylation between the two cell types. We identified TCX5/6-containing multi-subunit complexes, which are known as DREAM in other eukaryotes, and demonstrated that the Arabidopsis DREAM components function as a whole to preclude DNA hypermethylation. Given that the DREAM complexes are conserved from plants to animals, the preclusion of DNA hypermethylation by DREAM complexes may represent a conserved mechanism in eukaryotes.

Although the mechanism of DNA methylation-mediated gene silencing is extensively studied, relatively little is known about how promoter methylated genes are protected from transcriptional silencing. SUVH1, an Arabidopsis Su(var)3-9 homolog, was previously shown to be required for the expression of a few promoter methylated genes. By chromatin immunoprecipitation combined with sequencing, we demonstrate that SUVH1 binds to methylated genomic loci targeted by RNA-directed DNA methylation. SUVH1 and its homolog SUVH3 function partially redundantly and interact with three DNAJ domain-containing homologs, SDJ1, SDJ2, and SDJ3, thus forming a complex which we named SUVH-SDJ. The SUVH-SDJ complex components are co-localized in a large number of methylated promoters and are required for the expression of a subset of promoter methylated genes. We demonstrate that the SUVH-SDJ complex components have transcriptional activation activity. SUVH1 and SUVH3 function synergistically with SDJ1, SDJ2, and SDJ3 and are required for plant viability. This study reveals how the SUVH-SDJ complex protects promoter methylated genes from transcriptional silencing and suggests that the transcriptional activation of promoter methylated genes mediated by the SUVH-SDJ complex may play a critical role in plant growth and development.

Transposable elements and other repetitive DNA sequences are usually subject to DNA methylation and transcriptional silencing. However, anti-silencing mechanisms that promote transcription in these regions are not well understood. Here, we describe an anti-silencing factor, Bromodomain and ATPase domain-containing protein 1 (BRAT1), which we identified by a genetic screen in Arabidopsis thaliana. BRAT1 interacts with an ATPase domain-containing protein, BRP1 (BRAT1 Partner 1), and both prevent transcriptional silencing at methylated genomic regions. Although BRAT1 mediates DNA demethylation at a small set of loci targeted by the 5-methylcytosine DNA glycosylase ROS1, the involvement of BRAT1 in anti-silencing is largely independent of DNA demethylation. We also demonstrate that the bromodomain of BRAT1 binds to acetylated histone, which may facilitate the prevention of transcriptional silencing. Thus, BRAT1 represents a potential link between histone acetylation and transcriptional anti-silencing at methylated genomic regions, which may be conserved in eukaryotes., Transposons and repetitive sequences are typically subject to transcription silencing. Here, Zhang et al. find that the bromodomain-containing protein BRAT1 forms a complex with BRP1, recognizes histone acetylation and acts to prevent transcriptional silencing in Arabidopsis.

DNA methylation directed by 24-nucleotide (nt) small interfering RNAs (siRNAs) plays critical roles in gene regulation and transposon silencing in Arabidopsis. Twenty-four-nt siRNAs are known to be processed from double-stranded RNAs by Dicer-like 3 (DCL3) and loaded into the effector Argonaute 4 (AGO4). Here we report a distinct class of siRNAs independent of DCLs (sidRNAs). sidRNAs are present as ladders of ~20 to 60 nt in length, often having same 5’ ends but differing in 3’ ends by 1-nt steps. We further show that sidRNAs are associated with AGO4 and capable of directing DNA methylation. Finally we show that sidRNA production depends on distributive 3’-5’ exonucleases. Our findings suggest an alternative route for siRNA biogenesis: precursor transcripts are bound by AGO4 and subsequently subjected to 3’-5’ exonucleolytic trimming for maturation. We propose that sidRNAs generated through this route are the initial triggers of de novo DNA methylation.

The C-REPEAT-BINDING FACTOR (CBF) pathway has important roles in plant responses to cold stress. How the CBF genes themselves are activated after cold acclimation remains poorly understood. In this study, we characterized cold tolerance of null mutant of RNA-DIRECTED DNA METHYLATION 4 (RDM4), which encodes a protein that associates with RNA polymerases Pol V and Pol II, and is required for RNA-directed DNA methylation (RdDM) in Arabidopsis. The results showed that dysfunction of RDM4 reduced cold tolerance, as evidenced by decreased survival and increased electrolyte leakage. Mutation of RDM4 resulted in extensive transcriptomic reprogramming. CBFs and CBF regulon genes were down-regulated in rdm4 but not nrpe1 (the largest subunit of PolV) mutants, suggesting that the role of RDM4 in cold stress responses is independent of the RdDM pathway. Overexpression of RDM4 constitutively increased the expression of CBFs and regulon genes and decreased cold-induced membrane injury. A great proportion of genes affected by rdm4 overlapped with those affected by CBFs. Chromatin immunoprecipitation results suggested that RDM4 is important for Pol II occupancy at the promoters of CBF2 and CBF3. We present evidence of a considerable role for RDM4 in regulating gene expression at low temperature, including the CBF pathway in Arabidopsis.

Chromatin modifications are known to affect flowering time in plants, but little is known about how these modifications regulate flowering time in response to environmental signals like photoperiod. In Arabidopsis thaliana, HDC1, a conserved subunit of the RPD3-like histone deacetylase (HDAC) complex, was previously reported to regulate flowering time via the same mechanism as does the HDAC HDA6. Here, we demonstrate that HDC1, SNLs and MSI1 are shared subunits of the HDA6 and HDA19 HDAC complexes. While the late-flowering phenotype of the hda6 mutant is independent of photoperiod, the hda19, hdc1 and snl2/3/4 mutants flower later than or at a similar time to the wild-type in long-day conditions but flower earlier than the wild-type in short-day conditions. Our genome-wide analyses indicate that the effect of hdc1 on histone acetylation and transcription is comparable with that of hda19 but is different from that of hda6. Especially, we demonstrate that the HDA19 complex directly regulates the expression of two flowering repressor genes related to the gibberellin signaling pathway. Thus, the study reveals a photoperiod-dependent role of the HDA19 HDAC complex in the regulation of flowering time.

The CHD3-like chromatin remodeling protein MOM1 and the PIAS-type SUMO E3 ligase-like protein PIAL2 are known to interact with each other and mediate transcriptional silencing in Arabidopsis. However, it is poorly understood whether and how the interaction is involved in transcriptional silencing. Here, we demonstrate that, while the PIAL2 interaction domain (PIAL2-IND) is required for PIAL2 dimerization, MOM-PIAL2 interaction, and transcriptional silencing, a transgene fusing the wild-type MOM1 protein with the PIAL2 protein defective in PIAL2-IND can completely restore transcriptional silencing in the mom1/pial2 double mutant, demonstrating that the artificial fusion of MOM1 and PIAL2 mimics the in vivo interaction of these two proteins so that PIAL2-IND is no longer required for transcriptional silencing in the fusion protein. Further, our yeast two-hybrid assay identifies a previously unrecognized SUMO interaction motif (SIM) in the conserved MOM1 motif CMM3 and demonstrates that the SIM is responsible for the interaction of MOM1 with SUMO. Given that eukaryotic PIAS-type SUMO E3 ligases have a conserved role in chromatin regulation, the findings reported in this study may represent a conserved chromatin regulatory mechanism in higher eukaryotes.

In eukaryotes, heterochromatin regions are typically subjected to transcriptional silencing. DNA methylation has an important role in such silencing and has been studied extensively. However, little is known about how methylated heterochromatin regions are subjected to silencing. We conducted a genetic screen and identified an epcr ( enhancer of polycomb‐related ) mutant that releases heterochromatin silencing in Arabidopsis thaliana . We demonstrated that EPCR1 functions redundantly with its paralog EPCR2 and interacts with PWWP domain‐containing proteins (PWWPs), AT‐rich interaction domain‐containing proteins (ARIDs), and telomere repeat binding proteins (TRBs), thus forming multiple functionally redundant protein complexes named PEAT (PWWPs‐EPCRs‐ARIDs‐TRBs). The PEAT complexes mediate histone deacetylation and heterochromatin condensation and thereby facilitate heterochromatin silencing. In heterochromatin regions, the production of small interfering RNAs (siRNAs) and DNA methylation is repressed by the PEAT complexes. The study reveals how histone deacetylation, heterochromatin condensation, siRNA production, and DNA methylation interplay with each other and thereby maintain heterochromatin silencing.

The SU(VAR)3-9-like histone methyltransferases usually catalyze repressive histone H3K9 methylation and are involved in transcriptional gene silencing in eukaryotic organisms. We identified a putative SU(VAR)3-9-like histone methyltransferase SUVR2 by a forward genetic screen and demonstrated that it is involved in transcriptional gene silencing at genomic loci targeted by RNA-directed DNA methylation (RdDM). We found that SUVR2 has no histone methyltransferase activity and the conserved catalytic sites of SUVR2 are dispensable for the function of SUVR2 in transcriptional silencing. SUVR2 forms a complex with its close homolog SUVR1 and associate with three previously uncharacterized SNF2-related chromatin-remodeling proteins CHR19, CHR27, and CHR28. SUVR2 was previously thought to be a component in the RdDM pathway. We demonstrated that SUVR2 contributes to transcriptional gene silencing not only at a subset of RdDM target loci but also at many RdDM-independent target loci. Our study suggests that the involvement of SUVR2 in transcriptional gene silencing is related to nucleosome positioning mediated by its associated chromatin-remodeling proteins.

LHP1 mediates recruitment of the PRC2 histone methyltransferase complex to chromatin and thereby facilitates maintenance of H3K27me3 on FLC, a key flowering repressor gene. Here, we report that the PWWP domain proteins (PDPs) interact with FVE and MSI5 to suppress FLC expression and thereby promote flowering. We demonstrated that FVE, MSI5, and PDP3 were co-purified with LHP1. The H3K27me3 level on FLC was decreased in the pdp mutants as well as in the fve/msi5 double mutant. This study suggests that PDPs function together with FVE and MSI5 to regulate the function of the PRC2 complex on FLC.

The SU(VAR)-3-9-related protein family member SUVR2 has been previously identified to be involved in transcriptional gene silencing both in RNA-dependent and -independent pathways. It interacts with the chromatin-remodeling proteins CHR19, CHR27, and CHR28 (CHR19/27/28), which are also involved in transcriptional gene silencing. Here our study demonstrated that SUVR2 is almost fully mono-sumoylated in vivo. We successfully identified the exact SUVR2 sumoylation site by combining in vitro mass spectrometric analysis and in vivo immunoblotting confirmation. The luminescence imaging assay and quantitative RT-PCR results demonstrated that SUVR2 sumoylation is involved in transcriptional gene silencing. Furthermore, we found that SUVR2 sumoylation is required for the interaction of SUVR2 with CHR19/27/28, which is consistent with the fact that SUMO proteins are necessary for transcriptional gene silencing. These results suggest that SUVR2 sumoylation contributes to transcriptional gene silencing by facilitating the interaction of SUVR2 with the chromatin-remodeling proteins CHR19/27/28.

DNA methylation is a conserved epigenetic marker in plants and animals. In Arabidopsis, DNA methylation can be established through an RNA-directed DNA methylation (RdDM) pathway. By screening for suppressors of ros1, we identified STA1, a PRP6-like splicing factor, as a new RdDM regulator. Whole-genome bisulfite sequencing suggested that STA1 and the RdDM pathway share a large number of common targets in the Arabidopsis genome. Small RNA deep sequencing demonstrated that STA1 is predominantly involved in the accumulation of the siRNAs that depend on both Pol IV and Pol V. Moreover, the sta1 mutation partially reduces the levels of Pol V-dependent RNA transcripts. Immunolocalization assay indicated that STA1 signals are exclusively present in the Cajal body and overlap with AGO4 in most nuclei. STA1 signals are also partially overlap with NRPE1. Localization of STA1 to AGO4 and NRPE1 signals is probably related to the function of STA1 in the RdDM pathway. Based on these results, we propose that STA1 acts downstream of siRNA biogenesis and facilitates the production of Pol V-dependent RNA transcripts in the RdDM pathway.

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.

The SU(VAR)3-9 homolog SUVH9 and the double-stranded RNA-binding protein IDN2 were thought to be components of an RNA-directed DNA methylation (RdDM) pathway in Arabidopsis. We previously found that SUVH9 interacts with MORC6 but how the interaction contributes to transcriptional silencing remains elusive. Here, our genetic analysis indicates that SUVH2 and SUVH9 can either act in the same pathway as MORC6 or act synergistically with MORC6 to mediate transcriptional silencing. Moreover, we demonstrate that IDN2 interacts with MORC6 and mediates the silencing of a subset of MORC6 target loci. Like SUVH2, SUVH9, and IDN2, other RdDM components including Pol IV, Pol V, RDR2, and DRM2 are also required for transcriptional silencing at a subset of MORC6 target loci. MORC6 was previously shown to mediate transcriptional silencing through heterochromatin condensation. We demonstrate that the SWI/SNF chromatin-remodeling complex components SWI3B, SWI3C, and SWI3D interact with MORC6 as well as with SUVH9 and then mediate transcriptional silencing. These results suggest that the RdDM components are involved not only in DNA methylation but also in MORC6-mediated heterochromatin condensation. This study illustrates how DNA methylation is linked to heterochromatin condensation and thereby enhances transcriptional silencing at methylated genomic regions., Author Summary DNA methylation is a conserved epigenetic mark that is required for the silencing of transposons and introduced transgenes in eukaryotes. An RNA-directed DNA methylation pathway mediates de novo DNA methylation and thereby leads to transcriptional silencing in Arabidopsis. In this study, we find that two RNA-directed DNA methylation components interact with the microrchidia (MORC) protein MORC6 and lead to transcriptional silencing through a mechanism that is distinct from the RNA-directed DNA methylation pathway. MORC6 was previously thought to mediate transcriptional silencing through heterochromatin condensation. Our study suggests that the interaction of the RNA-directed DNA methylation components with MORC6 may mediate a link between DNA methylation and heterochromatin condensation.

The mechanism by which MORPHEUS' MOLECULE1 (MOM1) contributes to transcriptional gene silencing has remained elusive since the gene was first identified and characterized. Here, we report that two Arabidopsis thaliana PIAS (PROTEIN INHIBITOR OF ACTIVATED STAT)-type SUMO E3 ligase-like proteins, PIAL1 and PIAL2, function redundantly to mediate transcriptional silencing at MOM1 target loci. PIAL1 and PIAL2 physically interact with each other and with MOM1 to form a high molecular mass complex. In the absence of either PIAL2 or MOM1, the formation of the high molecular mass complex is disrupted. We identified a previously uncharacterized IND (interacting domain) in PIAL1 and PIAL2 and demonstrated that IND directly interacts with MOM1. The CMM2 (conserved MOM1 motif 2) domain of MOM1 was previously shown to be required for the dimerization of MOM1. We demonstrated that the CMM2 domain is also required for the interaction of MOM1 with PIAL1 and PIAL2. We found that although PIAL2 has SUMO E3 ligase activity, the activity is dispensable for PIAL2's function in transcriptional silencing. This study suggests that PIAL1 and PIAl2 act as components of the MOM1-containing complex to mediate transcriptional silencing at heterochromatin regions.

RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. RdDM mediates the transcriptional silencing of many endogenous genomic loci, most of which are transposon related. A forward genetics screen identified DTF1 (DNA-binding transcription factor 1) as a new component for RdDM in Arabidopsis. Loss-of-function mutations in DTF1 release the transcriptional silencing of RdDM target loci and reduce the accumulation of 24-nt small interfering RNAs (siRNAs) from some of the targets. Interestingly, in the dtf1 mutant plants, the release of transcriptional gene silencing at solo-LTR is accompanied by decreased siRNA accumulation but not by reduced DNA methylation. These results suggest that DTF1 is an atypical component of RdDM and has both DNA methylation-dependent and -independent roles in transcriptional gene silencing. We suggest that besides DNA methylation, siRNAs may cause some other uncharacterized epigenetic modifications that lead to transcriptional gene silencing.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

Nuclear factor Y (NF-Y) is a ubiquitous transcription factor composed of three distinct subunits (NF-YA, NF-YB, and NF-YC). We found that the Arabidopsis thaliana NFYA5 transcript is strongly induced by drought stress in an abscisic acid (ABA)-dependent manner. Promoter:β-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells and that part of the induction by drought was transcriptional. NFYA5 contains a target site for miR169, which targets mRNAs for cleavage or translational repression. We found that miR169 was downregulated by drought stress through an ABA-dependent pathway. Analysis of the expression of miR169 precursors showed that miR169a and miR169c were substantially downregulated by drought stress. Coexpression of miR169 and NFYA5 suggested that miR169a was more efficient than miR169c at repressing the NFYA5 mRNA level. nfya5 knockout plants and plants overexpressing miR169a showed enhanced leaf water loss and were more sensitive to drought stress than wild-type plants. By contrast, transgenic Arabidopsis plants overexpressing NFYA5 displayed reduced leaf water loss and were more resistant to drought stress than the wild type. Microarray analysis indicated that NFYA5 is crucial for the expression of a number of drought stress–responsive genes. Thus, NFYA5 is important for drought resistance, and its induction by drought stress occurs at both the transcriptional and posttranscriptional levels.

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.

Plants use 24-nucleotide small interfering RNAs (24-nt siRNAs) and long non-coding RNAs (lncRNAs) to direct de novo DNA methylation and transcriptional gene silencing. This process is called RNA-directed DNA methylation (RdDM). An important question in the RdDM model is what explains the target specificity of RNA polymerase IV (Pol IV), the enzyme that initiates siRNA production. Two recent papers addressed this question by characterizing the DTF1/SHH1 protein, which contains a homeodomain in the N-terminus and a novel histone-binding domain SAWADEE in the C terminus. Here we review the main results of the two studies and discuss several possible mechanisms that could contribute to Pol IV and Pol V recruitment.

RNA-directed DNA methylation (RdDM) is required for transcriptional silencing of transposons and other DNA repeats in Arabidopsis thaliana. Although previous research has demonstrated that the SET domain-containing SU(VAR)3–9 homologs SUVH2 and SUVH9 are involved in the RdDM pathway, the underlying mechanism remains unknown. Our results indicated that SUVH2 and/or SUVH9 not only interact with the chromatin-remodeling complex termed DDR (DMS3, DRD1, and RDM1) but also with the newly characterized complex composed of two conserved Microrchidia (MORC) family proteins, MORC1 and MORC6. The effect of suvh2suvh9 on Pol IV-dependent siRNA accumulation and DNA methylation is comparable to that of the Pol V mutant nrpe1 and the DDR complex mutant dms3, suggesting that SUVH2 and SUVH9 are functionally associated with RdDM. Our CHIP assay demonstrated that SUVH2 and SUVH9 are required for the occupancy of Pol V at RdDM loci and facilitate the production of Pol V-dependent noncoding RNAs. Moreover, SUVH2 and SUVH9 are also involved in the occupancy of DMS3 at RdDM loci. The putative catalytic active site in the SET domain of SUVH2 is dispensable for the function of SUVH2 in RdDM and H3K9 dimethylation. We propose that SUVH2 and SUVH9 bind to methylated DNA and facilitate the recruitment of Pol V to RdDM loci by associating with the DDR complex and the MORC complex., Author Summary Small RNA-induced transcriptional silencing at transposable elements and other DNA repeats is an evolutionarily conserved mechanism in plants, fungi, and animals. In Arabidopsis thaliana, an RNA-directed DNA methylation pathway is involved in transcriptional silencing. Noncoding RNAs produced by the plant-specific DNA-dependent RNA polymerase V are required for RNA-directed DNA methylation. A chromatin-remodeling complex was previously demonstrated to be required for the occupancy of DNA-dependent RNA polymerase V at RNA-directed DNA methylation loci. Our results suggest that two putative histone methyltransferases are inactive in their enzymatic activity and act as adaptor proteins to facilitate the recruitment of DNA-dependent RNA polymerase V to chromatin by associating with the chromatin-remodeling complex. In combination with previous studies, we propose that the inactive histone methyltransferases bind to methylated DNA, thereby linking DNA methylation to Pol V transcription at RNA-directed DNA methylation loci.

DNA methylation is an important epigenetic mark in many eukaryotic organisms. De novo DNA methylation in plants can be achieved by the RNA-directed DNA methylation (RdDM) pathway, where the plant-specific DNA-dependent RNA polymerase IV (Pol IV) transcribes target sequences to initiate 24-nt siRNA production and action. The putative DNA binding protein DTF1/SHH1 of Arabidopsis has been shown to associate with Pol IV and is required for 24-nt siRNA accumulation and transcriptional silencing at several RdDM target loci. However, the extent and mechanism of DTF1 function in RdDM is unclear. We show here that DTF1 is necessary for the accumulation of the majority of Pol IV-dependent 24-nt siRNAs. It is also required for a large proportion of Pol IV-dependent de novo DNA methylation. Interestingly, there is a group of RdDM target loci where 24-nt siRNA accumulation but not DNA methylation is dependent on DTF1. DTF1 interacts directly with the chromatin remodeling protein CLASSY 1 (CLSY1), and both DTF1 and CLSY1 are associated in vivo with Pol IV but not Pol V, which functions downstream in the RdDM effector complex. DTF1 and DTF2 (a DTF1-like protein) contain a SAWADEE domain, which was found to bind specifically to histone H3 containing H3K9 methylation. Taken together, our results show that DTF1 is a core component of the RdDM pathway, and suggest that DTF1 interacts with CLSY1 to assist in the recruitment of Pol IV to RdDM target loci where H3K9 methylation may be an important feature. Our results also suggest the involvement of DTF1 in an important negative feedback mechanism for DNA methylation at some RdDM target loci.

DNA methylation in transposons and other DNA repeats is conserved in plants as well as in animals. In Arabidopsis thaliana, an RNA-directed DNA methylation (RdDM) pathway directs de novo DNA methylation. We performed a forward genetic screen for suppressors of the DNA demethylase mutant ros1 and identified a novel Zinc-finger and OCRE domain-containing Protein 1 (ZOP1) that promotes Pol IV-dependent siRNA accumulation, DNA methylation, and transcriptional silencing. Whole-genome methods disclosed the genome-wide effects of zop1 on Pol IV-dependent siRNA accumulation and DNA methylation, suggesting that ZOP1 has both RdDM-dependent and -independent roles in transcriptional silencing. We demonstrated that ZOP1 is a pre-mRNA splicing factor that associates with several typical components of the splicing machinery as well as with Pol II. Immunofluorescence assay revealed that ZOP1 overlaps with Cajal body and is partially colocalized with NRPE1 and DRM2. Moreover, we found that the other development-defective splicing mutants tested including mac3a3b, mos4, mos12 and mos14 show defects in RdDM and transcriptional silencing. We propose that the splicing machinery rather than specific splicing factors is involved in promoting RdDM and transcriptional silencing.

Cytosine DNA methylation is a stable epigenetic mark that is frequently associated with the silencing of genes and transposable elements (TEs). In Arabidopsis, the establishment of DNA methylation is through the RNA-directed DNA methylation (RdDM) pathway. Here, we report the identification and characterization of RDM16, a new factor in the RdDM pathway. Mutation of RDM16 reduced the DNA methylation levels and partially released the silencing of a reporter gene as well as some endogenous genomic loci in the DNA demethylase ros1-1 mutant background. The rdm16 mutant had morphological defects and was hypersensitive to salt stress and abscisic acid (ABA). Map-based cloning and complementation test led to the identification of RDM16, which encodes a pre-mRNA-splicing factor 3, a component of the U4/U6 snRNP. RNA-seq analysis showed that 308 intron retention events occurred in rdm16, confirming that RDM16 is involved in pre-mRNA splicing in planta. RNA-seq and mRNA expression analysis also revealed that the RDM16 mutation did not affect the pre-mRNA splicing of known RdDM genes, suggesting that RDM16 might be directly involved in RdDM. Small RNA expression analysis on loci showing RDM16-dependent DNA methylation suggested that unlike the previously reported putative splicing factor mutants, rdm16 did not affect small RNA levels; instead, the rdm16 mutation caused a decrease in the levels of Pol V transcripts. ChIP assays revealed that RDM16 was enriched at some Pol V target loci. Our results suggest that RDM16 regulates DNA methylation through influencing Pol V transcript levels. Finally, our genome-wide DNA methylation analysis indicated that RDM16 regulates the overall methylation of TEs and gene-surrounding regions, and preferentially targets Pol IV-dependent DNA methylation loci and the ROS1 target loci. Our work thus contributes to the understanding of RdDM and its interactions with active DNA demethylation., Author Summary Both plants and animals utilize cytosine DNA methylation as an important epigenetic mark to suppress transposable elements (TEs), repeat sequences and genes, which is crucial for the genome integrity and development. In plants, de novo DNA methylation can be mediated by the RNA-directed DNA methylation (RdDM) pathway. Plants have also evolved a pathway for active DNA demethylation that is initiated by the ROS1 subfamily of 5-methylcytosine DNA glycosylases, to counteract the RdDM pathway to prevent undesirable silencing. In this study, we identified RDM16, a new factor in the RdDM pathway. We show that RDM16 is a pre-mRNA splicing factor and its function in the regulation of DNA methylation and gene silencing is not through influencing siRNA levels or the expression or splicing of genes encoding known RdDM components, but likely through affecting Pol V transcripts. We also show that RDM16 preferentially affects ROS1 target loci. Together, our findings contribute to the understanding of RdDM and its interactions with ROS1-mediated DNA demethylation.

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM., Author Summary In eukaryotes, transposable elements and other DNA repeats are important parts of genomes. Suppression of these sequences is required for genome stability and integrity. DNA methylation is an important chromatin modification that prevents the expression and movement of these repeat sequences. Small interfering RNAs initiate DNA methylation and transcriptional gene silencing of the sequences. In the plant model organism Arabidopsis thaliana, DNA methylation is mediated by an RNA–directed DNA methylation (RdDM) pathway. Here we report that the double-stranded RNA-binding protein IDN2 and it paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2) cooperate and form a novel complex that is required for RNA–directed DNA methylation. Unlike IDN2, IDP1 and IDP2 have no double-stranded RNA binding ability. We propose that the IDN2-IDP1/2 is a complex that functions at a downstream step of the RdDM pathway. The findings significantly increase our understanding of the plant RdDM pathway as well as of the RNA–mediated chromatin changes in yeast and animals.

DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review.

RNA-directed DNA methylation (RdDM) is an important epigenetic mechanism for silencing transgenes and endogenous repetitive sequences such as transposons. The RD29A promoter-driven LUCIFERASE transgene and its corresponding endogenous RD29A gene are hypermethylated and silenced in the Arabidopsis DNA demethylase mutant ros1. By screening for second-site suppressors of ros1, we identified the RDM12 locus. The rdm12 mutation releases the silencing of the RD29A-LUC transgene and the endogenous RD29A gene by reducing the promoter DNA methylation. The rdm12 mutation also reduces DNA methylation at endogenous RdDM target loci, including transposons and other repetitive sequences. In addition, the rdm12 mutation affects the levels of small interfering RNAs (siRNAs) from some of the RdDM target loci. RDM12 encodes a protein with XS and coiled-coil domains, and is similar to SGS3, which is a partner protein of RDR6 and can bind to double-stranded RNAs with a 5' overhang, and is required for several post-transcriptional gene silencing pathways. Our results show that RDM12 is a component of the RdDM pathway, and suggest that RdDM may involve double-stranded RNAs with a 5' overhang and the partnering between RDM12 and RDR2.

RNA-directed DNA methylation (RdDM) is a conserved mechanism for epigenetic silencing of transposons and other repetitive elements. We report that the rdm4 (RNA-directed DNA Methylation4) mutation not only impairs RdDM, but also causes pleiotropic developmental defects in Arabidopsis. Both RNA polymerase II (Pol II)- and Pol V-dependent transcripts are affected in the rdm4 mutant. RDM4 encodes a novel protein that is conserved from yeast to humans and interacts with Pol II and Pol V in plants. Our results suggest that RDM4 functions in epigenetic regulation and plant development by serving as a transcriptional regulator for RNA Pol V and Pol II, respectively.

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.

MMS19 is an essential component of the cytoplasmic iron-sulfur (Fe-S) cluster assembly complex in fungi and mammals; the mms19 null mutant alleles are lethal. Our study demonstrates that MMS19/MET18 in Arabidopsis thaliana interacts with the cytoplasmic Fe-S cluster assembly complex but is not an essential component of the complex. We find that MMS19 also interacts with the catalytic subunits of DNA polymerases, which have been demonstrated to be involved in transcriptional gene silencing (TGS), DNA repair, and flowering time regulation. Our results indicate that MMS19 has a similar biological function, suggesting a functional link between MMS19 and DNA polymerases. In the mms19 null mutant, the assembly of Fe-S clusters on the catalytic subunit of DNA polymerase α is reduced but not blocked, which is consistent with the viability of the mutant. Our study suggests that MMS19 assists the assembly of Fe-S clusters on DNA polymerases in the cytosol, thereby facilitating transcriptional gene silencing, DNA repair, and flowering time control.

An NAC-type transcription factor gene AtNAC2 was identified from Arabidopsis thaliana when expression patterns of the genes from a microarray analysis were examined. The AtNAC2 expression was induced by salt stress and this induction was reduced in magnitude in the transgenic Arabidopsis plants overexpressing tobacco ethylene receptor gene NTHK1. AtNAC2 is localized in the nucleus and has transcriptional activation activity. It can form a homodimer in yeast. AtNAC2 was highly expressed in roots and flowers, but less expressed in other organs examined. In addition to the salt induction, the AtNAC2 can also be induced by abscisic acid (ABA), ACC and NAA. The salt induction was enhanced in the ethylene overproducer mutant eto1-1, but suppressed in the ethylene-insensitive mutants etr1-1 and ein2-1, and in the auxin-insensitive mutant tir1-1when compared with that in wild-type plants. However, the salt induction of AtNAC2 was not significantly affected in the ABA-insensitive mutants abi2-1, abi3-1 and abi4-1. These results indicate that the salt response of AtNAC2 requires ethylene signaling and auxin signaling pathways but does not require ABI2, ABI3 and ABI4, intermediates of the ABA signaling pathway. Overexpression of AtNAC2 in transgenic Arabidopsis plants resulted in promotion of lateral root development. AtNAC2 also promoted or inhibited downstream gene expressions. These results indicate that AtNAC2 may be a transcription factor incorporating the environmental and endogenous stimuli into the process of plant lateral root development.

Cell division is a fundamental biological process sharing conserved features and controls in all eukaryotes. The cell cycle is usually divided into four phases: G 1 , S, G 2 , and M. Regulated gene expression is an important mechanism for controlling cell cycle progression and genes involved in cell division-related processes often show transcriptional regulation dependent on cell cycle position. In the present report, a novel cell cycle-related gene (AtCPR) from Arabidopsis thaliana was isolated and characterized. Sequence analysis revealed that the deduced amino acid sequence of AtCPR showed 53.2% identity with p38-2G4, a mouse G 1 -to-S cell cycle specifically modulated and proliferation-associated nuclear protein. Assay of expression of AtCPR in partially synchronized cells suggested that AtCPR mRNA was expressed in the G 1 -to-S phase. In the AtCPR transgenic plants, no apparent phenotypic change was observed. By fusing a GFP tag to the AtCPR protein, it was found that AtCPR was mainly located in the nucleus. However, AtCPR does not have any transcriptional activation ability. cDNA microarray analysis showed that a total of 17 and 30 genes were identified as up-regulated and down-regulated, respectively.

DNA methylation is a conserved epigenetic mark in plants and mammals. In Arabidopsis, DNA methylation can be triggered by small interfering RNAs (siRNAs) through an RNA-directed DNA methylation (RdDM) pathway. Here we report the identification of a new RdDM effector, RDM3/KTF1. Loss-of-function mutations in RDM3/KTF1 reduce DNA methylation and release the silencing of RdDM target loci without abolishing the siRNA triggers. KTF1 has similarity to the transcription elongation factor SPT5 and contains a C-terminal extension rich in GW/WG repeats. KTF1 colocalizes with ARGONAUTE 4 (AGO4) in punctate nuclear foci, and binds AGO4 and RNA transcripts. Our results suggest KTF1 as an adaptor protein that binds scaffold transcripts generated by Pol V and recruits AGO4 and AGO4-bound siRNAs to form an RdDM effector complex. The dual interaction of an effector protein with AGO and small RNA target transcripts may be a general feature of RNA silencing effector complexes.