Buried in PEAT—discovery of a new silencing complex with opposing activities

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146404/1/embj2018100573.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146404/2/embj2018100573_am.pd

RNA polymerase V targets transcriptional silencing components to promoters of protein‐coding genes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/1/tpj12034-sup-0010-TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/2/tpj12034-sup-0006-FigureS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/3/tpj12034-sup-0007-FigureS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/4/tpj12034-sup-0003-FigureS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/5/tpj12034-sup-0008-FigureS6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/6/tpj12034-sup-0005-FigureS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/7/tpj12034-sup-0004-FigureS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/8/tpj12034-sup-0009-FigureS7.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/9/tpj12034.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/96338/10/tpj12034-sup-0002-MethodsS1.pd

Spatial and Functional Relationships Among Pol V-Associated Loci, Pol IV-Dependent siRNAs, and Cytosine Methylation in the \u3cem\u3eArabidopsis\u3c/em\u3e Epigenome

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%–60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites

Suppression of Histone H1 Genes in Arabidopsis Results in Heritable Developmental Defects and Stochastic Changes in DNA Methylation

Histone H1 is an abundant component of eukaryotic chromatin that is thought to stabilize higher-order chromatin structures. However, the complete knock-out of H1 genes in several lower eukaryotes has no discernible effect on their appearance or viability. In higher eukaryotes, the presence of many mutually compensating isoforms of this protein has made assessment of the global function of H1 more difficult. We have used double-stranded RNA (dsRNA) silencing to suppress all the H1 genes of Arabidopsis thaliana. Plants with a >90% reduction in H1 expression exhibited a spectrum of aberrant developmental phenotypes, some of them resembling those observed in DNA hypomethylation mutants. In subsequent generations these defects segregated independently of the anti-H1 dsRNA construct. Downregulation of H1 genes did not cause substantial genome-wide DNA hypo- or hypermethylation. However, it was correlated with minor but statistically significant changes in the methylation patterns of repetitive and single-copy sequences, occurring in a stochastic manner. These findings reveal an important and previously unrecognized link between linker histones and specific patterns of DNA methylation

Silencing: new faces of Morpheus' molecule

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102123/1/embj2009388.pd

RNA ‐directed DNA methylation requires stepwise binding of silencing factors to long non‐coding RNA

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108033/1/tpj12563.pd

Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes

Nuclear transcription is not restricted to genes but occurs throughout the intergenic and noncoding space of eukaryotic genomes. The functional significance of this widespread noncoding transcription is mostly unknown. We show that Arabidopsis RNA polymerase IVb/Pol V, a multisubunit nuclear enzyme required for siRNA-mediated gene silencing of transposons and other repeats, transcribes intergenic and noncoding sequences, thereby facilitating heterochromatin formation and silencing of overlapping and adjacent genes. Pol IVb/Pol V transcription requires the chromatin-remodeling protein DRD1 but is independent of siRNA biogenesis. However, Pol IVb/Pol V transcription and siRNA production are both required to silence transposons, suggesting that Pol IVb/Pol V generates RNAs or chromatin structures that serve as scaffolds for siRNA-mediated heterochromatinforming complexes. Pol IVb/Pol V function provides a solution to a paradox of epigenetic control: the need for transcription in order to transcriptionally silence the same region

The "drought-inducible" histone H1s of tobacco play no role in male sterility linked to alterations in H1 variants

Tobacco ( Nicotiana tabacum L.) has two major H1 variants (H1A and H1B), which account for over 80% of chromatin linker histones, and four minor variants: H1C, H1D, H1E and H1F. We have shown previously [M. Prymakowska-Bosak et al. (1999) Plant Cell 11:2317-2329] that reversal of the natural proportion of major to minor H1 variants in transgenic tobacco plants results in a characteristic male-sterility phenotype identical to that occurring in many plant species subjected to water deficit at the time of male meiosis. It has been proposed by others that the drought-induced arrest of male gametophyte development is linked to decreased sugar delivery to reproductive tissues. Within the family of angiosperm H1s there is a well-defined class of minor H1 variants named "drought inducible" because some of its members have been shown to be induced by water deficit. We have identified and cloned the tobacco H1C gene, which, based on sequence similarity, represents a "drought-inducible" minor H1 variant. Analysis of the un-translated mRNA and promoter regions of H1C suggests a regulation by sucrose concentration. Antisense silencing of H1C and its close homologue H1D in plants that do not express H1A and H1B does not affect the characteristic H1A(-)/ H1B(-) male-sterility phenotype. Silencing of H1C and H1D also has no effect on growth and development of plants. Our findings demonstrate that H1C and H1D are dispensable for normal growth and development of tobacco, and that the compensatory up-regulation of "drought-inducible" H1s observed in H1A(-)/ H1B(-) plants is not the direct cause of male sterility linked to alterations in H1 variants

High- resolution map of plastid- encoded RNA polymerase binding patterns demonstrates a major role of transcription in chloroplast gene expression

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/174809/1/tpj15882.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174809/2/tpj15882-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174809/3/tpj15882_am.pd