DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3–9 homologs, the transcriptional anti-silencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.
Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide (nt) small RNAs (siRNAs) that bind ARGONAUTE4 (AGO4) and target genomic regions for silencing. It also requires non-coding RNAs transcribed by RNA POLYMERASE V (Pol V) that likely serve as scaffolds for binding of AGO4/siRNA complexes. Here we utilized a modified global nuclear run-on (GRO) protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5′ adenine found in many AGO4-bound 24-nt siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expressing wild-type AGO4 in ago4/6/9 was able to restore slicing of Pol V transcripts but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required the little understood elongation factor SPT5L. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.
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