In plants, DNA methylation is one of the major epigenetic marks regulating activity of plant genes and transposable elements. Maintenance of established DNA methylation is well understood, but the precise mechanism of de novo methylation of naïve loci is still unclear. Recent publication (Sigman et al. 2021, doi.org/10.1038/s41477-021-01008-7) confirmed our long-standing suspicion that the initial recognition of the methylation target locus is mediated by RNA polymerase II (Pol II) and not by plant specific Pol V, which was mostly associated with this process in literature.

The Ph.D. project is focused on precise dissection of the role of Pol II and Pol V in de novo DNA methylation using Arabidopsis thaliana and tobacco BY-2 cell cultures as models. Detailed analysis of dynamics and efficiency of de novo transgene methylation in WT and hypomorphic and knock-out mutants of selected subunits of these two polymerases and their associated factors allow to determine the successiveness of individual steps of the process and to find the factors involved. We offer an up-to-date topic that includes modern experimental approaches such as generation of genetically modified lines, CRISPR/Cas9 mutagenesis and chromatin-immunoprecipitation analyses. We expect applications from highly motivated students capable of independent work.

Five relevant publications of the research group:

Čermák V, Tyč D, Přibylová A, Fischer L (2020) Unexpected variations in posttranscriptional silencing induced by differentially produced dsRNAs in tobacco cells. BBA – Gene Regulatory Mechanisms 1863(11):194647.

Přibylová A, Čermák V, Tyč D, Fischer L (2019) Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells. Epigenetics & Chromatin 12: 54

Čermák V; Fischer, L (2018) Pervasive read-through transcription of T-DNAs is frequent in tobacco BY-2 cells and can effectively induce silencing. BMC Plant biology 18: 252

Tyč D, Nocarová E, Sikorová L, Fischer L (2017) 5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level. Plant Cell Reports 36: 1311-1322

Nocarová E, Fischer L (2009) Cloning of transgenic tobacco BY-2 cells; an efficient method to analyze and reduce high natural heterogeneity of transgene expression. BMC Plant Biology 9:44.

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