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

Abstract

Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs. Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c. Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c. Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs. Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity.

Arabidopsis JMJ29 is involved in trichome development by regulating the core trichome initiation gene GLABRA3

“…Recent studies demonstrated that JMJ25/IBM1 and JMJ27 have histone H3K9 demethylase activity (Saze et al, 2008;Dutta et al, 2017). Although JMJ24 has no demethylase activity in vitro, it can repress H3K9me2 and CHG methylation indirectly at transposons in vivo (Kabelitz et al, 2016).…”

Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling

“…The PHD domains of FGT1 (aa 667–757) and ING1 ( Lee et al, 2009 ) were subcloned into pGEX-4T-1 (Amersham Biosciences, Pittsburgh, PA), expressed in E. coli BL21 (DE3) as GST fusion proteins and purified using Glutathione affinity resins (Thermo Fisher Scientific, Waltham, MA). The histone peptide binding assay was performed as described ( Kabelitz et al, 2016 ). In brief, 1 µg of biotinylated histone peptides (Merck-Millipore, Darmstadt, Germany) were incubated with 10 µg of GST-fusion protein in binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF, protease inhibitors) overnight at 4°C with rotation.…”

“…After incubation with Streptavidin Dynabeads (Thermo Fisher Scientific) and extensive washing with TBST, bound proteins were analyzed by SDS-PAGE and immunoblotting with anti-GST antibodies (Merck-Millipore). Results for GST-ING1 were taken from Kabelitz et al (2016) , as the experiments were performed in parallel.…”

F‐box protein CFK1 interacts with and degrades de novo DNA methyltransferase in Arabidopsis

“…DNA methylation and repressive histone marks, H3K9me, are highly correlated with gene silencing (Du et al ., 2015). Recent studies revealed that the RING motif of JMJ24, instead of the JmjC domain that is well characterised with histone demethylase activity (Klose et al ., 2006; Deng et al ., 2016), is required for regulating CHG methylation and H3K9me2, to reinforce transcriptional silencing (Klose et al ., 2006; Deng et al ., 2016; Kabelitz et al ., 2016). Rbx1, the core subunit in SCF complex, contains the RING domain (Gray et al ., 2002; Zheng et al ., 2002).…”