Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana

Abstract

BackgroundDNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis.ResultsA dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor.ConclusionWe developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level of LUCL expression provides an easy readout in genetic and chemical genetic screens that will dissect the mechanisms of RdDM and methylation maintenance.

HSI2/VAL1 PHD-like domain promotes H3K27 trimethylation to repress the expression of seed maturation genes and complex transgenes in Arabidopsis seedlings

“…Previous reports showed that treatment with 5 μM/mL of the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) was effective in derepressing the transcriptional silencing of auxin-responsive ß-GUS reporter lines [ 35 ]. Treatment of Arabidopsis seedlings with 7 μM/mL 5-azadC also caused global changes in gene expression and derepression of silenced transgenes [ 36 , 37 ]. To investigate whether DNA methylation is involved in repressing GSTF8::LUC transgene expression, WT LUC , hsi2-2 LUC and hsi2-4 LUC seedlings were grown on media containing 5-azadC at various concentrations.…”

Tetrahydrofolate Modulates Floral Transition through Epigenetic Silencing

“…Tetrahydrofolate and its derivatives have long been recognized as one of the essential B vitamins in maintaining normal life because of their nutritional significance as well as crucial roles in one-carbon metabolism and epigenetic modification in living organisms (Hanson and Roje, 2001;Crider et al, 2012;Fitzpatrick et al, 2012;Lucock, 2000;Groth et al, 2016;Dinh et al, 2013). Thus, exploring folate homeostasis-associated biochemical reactions, cellular modifications, and underlying regulatory mechanisms are critical for people to improve the understanding of the important physiological functions of folates in plant development control and stressevoked plant adaptive responses.…”

A novel histone H3K4me1-specific binding protein is required for siRNA accumulation and DNA methylation at a subset of loci targeted by RNA-directed DNA methylation

“…RdDM is an important DNA methylation pathway in plants and has been extensively studied during the past two decades. Several forward and reverse genetics screens, including the ros1 suppressor screen in this study, were used to identify RdDM components 20,[31][32][33][34] . Through this ros1 suppressor screen, several novel RdDM mutants, such as rdm1, rdm4, and ktf1, were identi ed [21][22][23] .…”