Biology subjects

Pikaard, C. S.

Publications and source records attributed to Pikaard, C. S..

3 recordsLinked to original sources

bioRxiv2017

siRNA biogenesis, DNA methylation and target locus silencing are distinct and separable Pol IV sub-functions differentially dependent on the largest subunit's CTD

Plant nuclear multisubunit RNA polymerase IV plays a key role in the RNA-directed DNA methylation (RdDM) pathway for transcriptional silencing of transposons, viruses and specific genes by synthesizing precursors of 24 nt siRNAs that guide the process. The Pol IV largest subunit, NRPD1 is derived from the Pol II largest subunit but has a unique carboxy-terminal domain (CTD) of unknown function. We show that the NRPD1 CTD is critical for transcriptional silencing of target loci and for producing 24 nt siRNAs at high levels. However, the CTD is surprisingly dispensable for near wild-type levels of Pol IV-dependent genomic cytosine methylation. These results suggest that low levels of 24 nt siRNAs, produced at only 20-30% of wild-type levels, are sufficient for full RNA-directed DNA methylation, yet insufficient for silencing, suggesting additional roles for siRNAs beyond DNA methylation. Moreover, at a subset of target loci, neither siRNA levels nor cytosine methylation are impaired upon deletion of the CTD, yet silencing is lost. Collectively, the non-linear relationships between siRNA levels, cytosine methylation and silencing suggest the existence of additional mechanisms of silencing dependent on Pol IV transcription and mediated by the CTD, such as promoter occlusion to inhibit the activities of other polymerases.

molecular biology↗

bioRxiv2017

RNA polymerases IV and V influence the 3’ boundaries of Polymerase II transcription units in Arabidopsis

Nuclear multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved in plants as specialized forms of Pol II. Their functions are best understood in the context of RNA-directed DNA methylation (RdDM), a process in which Pol IV-dependent 24 nt siRNAs direct the de novo cytosine methylation of regions transcribed by Pol V. Pol V has additional functions, independent of Pol IV and 24 nt siRNA biogenesis, in maintaining the repression of transposons and genomic repeats whose silencing depends on maintenance cytosine methylation. Here we report that Pol IV and Pol V play unexpected roles in defining the 3 boundaries of Pol II transcription units. Nuclear run-on assays reveal that in the absence of Pol IV or Pol V, Pol II occupancy downstream of poly A sites increases for approximately 12% of protein-coding genes. This effect is most pronounced for convergently transcribed gene pairs. Although Pols IV and V are detected near transcript ends of the affected Pol II - transcribed genes, their role in limiting Pol II read-through is independent of siRNA biogenesis or cytosine methylation. We speculate that Pols IV and V (and/or their associated factors) play roles in Pol II transcription termination by influencing polymerase bypass or release at collision sites for convergent genes.

plant biology↗

bioRxiv2017

Hybrid incompatibility caused by an epiallele

Hybrid incompatibility resulting from deleterious gene combinations is thought to be an important step towards reproductive isolation and speciation. Here we demonstrate involvement of a silent epiallele in hybrid incompatibility. In Arabidopsis thaliana strain Col-0, one of the two copies of a duplicated histidine biosynthesis gene, HISN6B is not expressed, for reasons that have been unclear, making its paralog, HISN6A essential. By contrast, in strain Cvi-0, HISN6B is essential because HISN6A is mutated. As a result of these differences, Cvi-0 x Col-0 hybrid progeny that are homozygous for both Col-0 HISN6B and Cvi-0 HISN6A do not survive. We show that HISN6B is not a defective pseudogene in the Col-0 strain, but a stably silenced epiallele. Mutating HISTONE DEACETYLASE 6 (HDA6) or the cytosine methyltransferase genes, MET1 or CMT3 erases HISN6Bs silent locus identity in Col-0, reanimating the gene such that hisn6a lethality and hybrid incompatibility are circumvented. These results show that HISN6-dependent hybrid lethality is a revertible epigenetic phenomenon and provide additional evidence that epigenetic variation has the potential to limit gene flow between diverging populations of a species.\n\nSignificance statementDeleterious mutations in different copies of a duplicated gene pair have the potential to cause hybrid incompatibility between diverging subpopulations, contributing to reproductive isolation and speciation. This study demonstrates a case of epigenetic gene silencing, rather than pseudogene creation by mutation, contributing to a lethal gene combination upon hybridization of two strains of Arabidopsis thaliana. The findings provide direct evidence that naturally occurring epigenetic variation can contribute to incompatible hybrid genotypes, reducing gene flow between strains of the same species.

genetics↗