Investigating CLSY proteins and Pol-IV complex interaction in controlling DNA methylation patterns in Arabidopsis thaliana
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE278181 | GEO | 2024/10/01
REPOSITORIES: GEO
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Project description:Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a “one CLSY per Pol IV” model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.
2024-08-22 | GSE242191 | GEO
Project description:RNA polymerase IV (Pol IV) transcribes transposable elements (TEs) into precursors for small interfering RNAs (siRNAs) that guide DNA methylation to silence these potentially dangerious mobile elements in plants. Pol IV is recruited to TEs via SNF2-like CLASSY (CLSY) proteins. How Pol IV evolved to partner with the CLSYs is unknown. Using phylogenetics, we identified a conserved CYC-YPMF motif that is specific to Pol IV’s largest subunit. Modeling predicts that this motif coordinates a zinc ion and is exposed near Pol IV’s surface in a domain contacting downstream DNA. Via immunoprecipitation and mass spectrometry we found that this motif is essential for the association of all four CLSYs with Pol IV and hereby designate it as a CLSY-docking motif. Consistent with these biochemical results, mutations in the CLSY-docking motif or in neighboring sites of the Pol IV clamp domain, phenocopy pol iv null mutants and display a near-total loss of siRNA biogenesis, resulting in defects in DNA methylation and TE silencing. Together, our findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing Pol IV to target, transcribe and limit the expression of TE regions, thereby protecting genome integrity.
2024-11-28 | PXD047743 | Pride
Project description:Maize RNA Polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, repression of transposable elements (TEs), and for the regulation of specific alleles associated with TEs. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based transcriptional regulation. Surprisingly, although TE-like sequences comprise >85% of the maize genome, most TEs are not transcribed at the seedling stage, even in rpd1 mutants. Profile comparisons identify the global set of genes and TEs whose transcription is altered in the absence of RPD1, in some cases in antisense orientation. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for certain regions of the maize genome. Nuclei isolated from 10 wild-type and 10 rpd1 mutant seedlings were pooled and used to make two global run-on sequencing libraries.
2015-02-04 | E-GEOD-54166 | biostudies-arrayexpress
Project description:Maize RNA Polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, repression of transposable elements (TEs), and for the regulation of specific alleles associated with TEs. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based transcriptional regulation. Surprisingly, although TE-like sequences comprise >85% of the maize genome, most TEs are not transcribed at the seedling stage, even in rpd1 mutants. Profile comparisons identify the global set of genes and TEs whose transcription is altered in the absence of RPD1, in some cases in antisense orientation. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for certain regions of the maize genome.
2015-02-04 | GSE54166 | GEO
Project description:Intronic transposon elements (TEs) comprise a large proportion in eukaryotic genomes, but how they regulate the host genes remains to be explored. Our forward genetic screen disclosed the plant specific RNA polymerases IV and V in suppressing intronic TE-mediated cryptic transcription initiation of a chimeric transcripts at FLC (FLCTE). Initiation of FLCTE transcription is blocked by the locally formed intronic heterochromatin, and RNA Pol V direct associates and establishes intronic heterochromatin, thus inhibits the entry of RNA Pol II Ser5p and the occupancy of H3K4 methylation for promoting initiation. Genome-wide Pol II Ser5p native elongation transcription sequencing revealed that this is a common mechanism among intronic heterochromatin-containing genes. This study sheds light on deeply understanding the function of intronic heterochromatin on host genes expression in eukaryotic genome.
2019-11-01 | GSE125096 | GEO
Project description:In Arabidopsis thaliana, DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA (sRNA) transcripts. These transcripts are processed by DICER-LIKE3 into 24-nt small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nt epigenetically activated siRNAs (easiRNAs), which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernable pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV in the Brassicaceae species Capsella rubella, caused post-meiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were 2 depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nt and 24-nt siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella rubella compared to Arabidopsis thaliana, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.
2020-01-23 | GSE136847 | GEO
Project description:Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.
2018-04-14 | GSE99692 | GEO
Project description:Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.
2018-04-14 | GSE99691 | GEO
Project description:Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.
2018-04-14 | GSE99689 | GEO
Project description:Abstract: Cytosine DNA methylation plays crucial roles in gene regulation, transposon silencing, and diverse developmental processes. While methylation patterns are known to differ between cell-types, tissues, and disease states, how these differences arise remains poorly understood. In plants, DNA methylation is established via the RNA-directed DNA methylation pathway (RdDM), wherein 24-nucleotide small interfering RNAs (siRNAs) guide methylation at cognate genomic loci1. RNA POLYMERASE-IV (Pol-IV), a plant-specific polymerase, initiates the biogenesis of these methylation-targeting RNAs, thus understanding how Pol-IV is regulated is critical in determining how specific patterns of DNA methylation are generated. Here we show roles for four Pol-IV-associated factors, CLASSY (CLSY) 1-42,3, in both locus-specific and global regulation of Pol-IV function. Individually, each CLSY protein controls siRNA production and Pol-IV chromatin association at unique set of loci. This translates into locus-specific DNA methylation losses and the release of silencing. In addition to locus-specific effects, several layers of redundancy were identified: The clsy1,2 and clsy3,4 mutants act synergistically, regulating thousands more siRNA loci than the single mutants. Furthermore, the clsy1,2- and clsy3,4-dependent loci are mutually exclusive and geographically distinct, revealing a striking division of labor amongst the CLSY family. Finally, the clsy quadruple mutant causes global siRNA losses, demonstrating that Pol-IV is completely dependent on the CLSY family. Investigation into the mechanisms underlying the CLSY specificity revealed connections between clsy1,2- and clsy3,4-dependent loci and either SAWADEE HOMEODOMAIN HOMOLOG 1 or DNA METHYLTRANSFERASE 1, demonstrating a reliance on different chromatin modifications, H3K9 or CG DNA methylation, respectively4,5. Together, these findings not only shed light on Pol-IV function, but also reveal an additional layer of complexity to the RdDM pathway that enables the locus-specific control of DNA methylation patterns. Given the parallels between methylation systems in plants and mammals1, these findings will be informative for analogous processes in a broad range of organisms.
2018-04-14 | GSE99693 | GEO