All eukaryotes use three essential DNA-dependent RNA polymerases to decode the genetic information in chromosomal DNA, namely RNA Polymerases I, II and III. Remarkably, plants have evolved two additional RNA polymerases, abbreviated as Pol IV and Pol V. These novel RNA polymerases play non- redundant roles in RNA-directed DNA methylation, silencing of transposable elements, large-scale heterochromatin organization, long-distance spreading of silencing signals, and proper temporal and morphological development. Affinity purification and mass spec analyses revealed that Pol IV and Pol V are specialized forms of RNA Polymerase II, with half of their twelve subunits encoded by the same genes. Three subunits differ between Pol IV and Pol V and presumably account for their unique functions. Pol IV and Pol V are best understood with respect to their roles in the Arabidopsis siRNA-directed DNA methylation pathway. Pol IV acts early in the pathway, generating transcripts that are required for the biogenesis of 24 nt short interfering RNAs (siRNAs) that are loaded into ARGONAUTE 4 (AGO4). Independent of siRNA biogenesis, Pol V generates noncoding transcripts at target loci. siRNA-AGO4 complexes bind to these Pol V transcripts, facilitating recruitment of AGO4 to the adjacent chromatin. In subsequent steps that are not understood, the de novo DNA methyltransferase, DRM2 and histone modifying activities are recruited to target loci, generating heterochromatin that is refractive to transcription by conventional polymerases such as Pol II and Pol III. Major questions in need of answers include: what are the templates used by Pol IV and Pol V?;how are Pol IV and Pol V recruited to these templates?;are Pol IV and Pol V transcription units specified by conventional promoters or by chromatin structures?;how are Pol IV and Pol V coordinated with other proteins of the gene silencing machinery;and how do the unique subunits of Pol II, Pol IV and Pol V confer the unique functions of these novel polymerases? Using genetics and genomics as well as cell biological and biochemical approaches, our specific aims are designed to find answers to these questions. In diverse eukaryotes, including humans, flies, worms and fission yeast, siRNAs and noncoding RNAs essential processes through chromatin modifications. Examples include transposon silencing, centromere maintenance, X-chromosome inactivation and imprinting of maternal or paternal alleles. DNA methylation and chromatin modifications are also implicated in Rett, ICF, Prader-Willi, Beckwith-Wiedemann and Fragile X syndromes, as well as numerous forms of cancer. By understanding how noncoding RNAs and siRNAs specify sites of DNA methylation and gene silencing, our study will contribute to the long-term goal of understanding these processes with respect to human disease.

Public Health Relevance

Large non-coding RNAs and small RNAs are critical for X-chromosome inactivation and dosage control, imprinting of genes expressed from only one parent, defense against retrotransposons and viruses, heterochromatin formation at centromeres and genome stability. Alterations in DNA methylation and heterochromatin are involved in multiple human diseases and genetic disorders, including cancer, and targeted gene silencing using small RNA interference (RNAi) technologies are being used in medical and agricultural biotechnology applications. Our studies of RNA polymerases IV and V are significant with respect to all of these areas, especially the roles of intergenic noncoding RNAs and siRNAs in chromatin-mediated gene silencing.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Indiana University Bloomington
Schools of Arts and Sciences
United States
Zip Code
Blevins, Todd; Wang, Jing; Pflieger, David et al. (2017) Hybrid incompatibility caused by an epiallele. Proc Natl Acad Sci U S A 114:3702-3707
Wang, Jing; Blevins, Todd; Podicheti, Ram et al. (2017) Mutation of Arabidopsis SMC4 identifies condensin as a corepressor of pericentromeric transposons and conditionally expressed genes. Genes Dev 31:1601-1614
Marasco, Michelle; Li, Weiyi; Lynch, Michael et al. (2017) Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination. Nucleic Acids Res 45:11315-11326
Wendte, Jered M; Haag, Jeremy R; Singh, Jasleen et al. (2017) Functional Dissection of the Pol V Largest Subunit CTD in RNA-Directed DNA Methylation. Cell Rep 19:2796-2808
Wendte, Jered M; Pikaard, Craig S (2017) The RNAs of RNA-directed DNA methylation. Biochim Biophys Acta Gene Regul Mech 1860:140-148
Wendte, Jered M; Pikaard, Craig S (2016) Targeting Argonaute to chromatin. Genes Dev 30:2649-2650
Ream, Thomas S; Haag, Jeremy R; Pontvianne, Frederic et al. (2015) Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III-specific forms of the AC40 subunit and alternative forms of the C53 subunit. Nucleic Acids Res 43:4163-78
Blevins, Todd; Podicheti, Ram; Mishra, Vibhor et al. (2015) Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. Elife 4:e09591
Blevins, Todd; Pontvianne, Frédéric; Cocklin, Ross et al. (2014) A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54:30-42
Pikaard, Craig S; Mittelsten Scheid, Ortrun (2014) Epigenetic regulation in plants. Cold Spring Harb Perspect Biol 6:a019315

Showing the most recent 10 out of 41 publications