Mutations in the Arabidopsis H3K4me2/3 Demethylase JMJ14 Suppress Posttranscriptional Gene Silencing by Decreasing Transgene Transcription  

Abstract

Posttranscriptional gene silencing (PTGS) mediated by sense transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of transgenes that do not trigger S-PTGS. Moreover, expression of transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.

The Nuclear Ribonucleoprotein SmD1 Interplays with Splicing, RNA Quality Control, and Posttranscriptional Gene Silencing in Arabidopsis

“…Because many of the PTGS-deficient mutants previously identified in our screen are impaired in components of the cellular machinery producing endogenous siRNAs (Elmayan et al, 1998;Fagard et al, 2000;Mourrain et al, 2000;Morel et al, 2002;Boutet et al, 2003;Jauvion et al, 2010;Le Masson et al, 2012), we examined the accumulation of representative endogenous small RNAs in smd1 mutants. The accumulation of microRNAs (miR173 and miR390), trans-acting siRNAs (TAS1, TAS2, and TAS3), and p4-siRNAs (siRNA02 and siRNA1003) was not affected in the smd1a or smd1b mutants ( Figure 5), suggesting that SmD1 does not generally participate in the production of small RNAs but likely affects transgene PTGS at a different step.…”

“…A forward genetic screen directly based on line 2a3 identified three additional loci (SGS13, SGS14, and SGS15) required for 2a3 but not L1 silencing (Jauvion et al, 2010). So far, SGS2/RDR6, SGS3, SGS4/AGO1, SGS5/HEN1, SGS6/MET1, SGS7/SDE5, SGS8/ JMJ14, SGS9/HPR1, and SGS13/SDE3 have been characterized (Elmayan et al, 1998;Fagard et al, 2000;Mourrain et al, 2000;Morel et al, 2002;Boutet et al, 2003;Jauvion et al, 2010;Le Masson et al, 2012). During PTGS triggered by sense transgenes (S-PTGS), primary siRNAs are produced from an aberrant RNA , methylated at their 39 end by the methyltransferase HEN1 (HUA ENCHANCER1) (Boutet et al, 2003;Li et al, 2005) before loading into AGO1 (ARGONAUTE1), which cleaves complementary target RNAs (Morel et al, 2002;Baumberger and Baulcombe, 2005).…”

“…These secondary siRNAs are also loaded onto AGO1, which cleaves complementary transgene mRNAs, resulting in an amplification loop that reinforces silencing. MET1 and JMJ14 encode a DNA methyltransferase and a histone demethylase, respectively, which likely play a role in remodeling chromatin to allow the transcription of transgene-derived aberrant RNAs that induce PTGS (Le Masson et al, 2012). Also, SDE5 and HPR1 encode RNA trafficking proteins, which likely play a role in bringing RNA molecules at the right place during PTGS (Hernandez-Pinzon et al, 2007;Jauvion et al, 2010;Yelina et al, 2010).…”

Arabidopsis miR156 Regulates Tolerance to Recurring Environmental Stress through SPL Transcription Factors

“…miRNAs typically regulate their target genes posttranscriptionally through transcript cleavage. Thus, we investigated whether the HS-dependent repression occurred at the transcriptional or posttranscriptional level by quantifying unspliced SPL transcripts as a proxy for transcriptional activity (Bäurle et al, 2007;Le Masson et al, 2012). Unspliced transcript levels of SPL2 and SPL11 were not repressed by HS in the wild type or ago1-25 mutants ( Figure 5A, middle and bottom panels; for primer positions, see Supplemental Figure 2C).…”

A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis

“…For example, several characterized JMJ genes regulate flowering time through repression or activation of different target genes and different modifications (Noh et al, 2004;Yang et al, 2012;Crevillén et al, 2014;Gan et al, 2014). The histone H3 K4 demethylase JMJ14 is required for RNA-mediated DNA methylation (Deleris et al, 2010;Searle et al, 2010;Le Masson et al, 2012;Greenberg et al, 2013). A H3K4 demethylase from rice (Oryza sativa) has been implicated in the repression of TE sequences (Cui et al, 2013).…”