Author Archives: Feng Wang

Control meiotic crossover by DNA methylation in Arabidopsis

We discussed a paper by Yelina et al. (2015) titled “DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis” (PMID: 26494791; doi: 10.1101/gad.270876.115) in our journal club today. It’s a pretty interesting paper with intriguing topic and smart experimental designs.

The authors previously identified two meiotic crossover hot spots, 3a and 3b on subtelomeric regions of Chromasome 3 in Arabidopsis (Yelina et al., 2012). These crossover hot spots have low CG methylation compared to average genome methylation level or regions in between 3a and 3b. They then tested if increasing DNA methylation in 3a and 3b could suppress meiotic crossover rate by expressing inverted-repeat (IR) transgene, which would trigger RdDM in targeted regions. Interestingly, meiotic crossover rate significantly decreased in several IR expressed lines (Figure 1 and 2, Table 1). Other RdDM markers, such as increased H3K9me2 and denser nucleosome occupancy were also detected in IR targeted regions (Figure 3). There results indicate that crossover rate is negatively correlated with DNA methylation in euchromatic regions.

It is thus obvious to ask that if crossover rate would significantly elevate if genome-wide demethylation occurred. The authors used met1/+ plants to test this hypothesis. However, overall crossover rates were similar in Col/Ler vs met1 Col/Ler. Regional remodeling of crossover around subtelomeric and pericentromeric regions was observed (Figure 4C). They showed that the remodeling of crossover in met1 was dependent on crossover interference pathway (Figure 4G). The analysis of crossover in met1 mutant suggests that genome-wide demethylation has different effect on crossover in euchromatic and centromeric regions. Very interestingly, the met1 mutation causes increased crossover in euchromatic regions, but vise versa in pericentromeric regions (Figure 5D). They further showed that double strand DNA breakage (DSB) was similar in met1 and WT in Arabidopsis (Figure 6), which ruled out the possibility that crossover remodeling in met1 was due to altered DSB.

A few brilliant technique/experiments were used in this research. I think it’s very smart to study meiotic crossover by studying pollen DNA. More information about pollen typing could be found in Drouaud and Mézard (2011). The crossover detecting system by using GFP/RFP that inserts into different positions on same chromosome is also very neat. More information about the GFP/RFP lines can be found in Yelina et al. (2012) paper.


Drouaud, J., & Mézard, C. (2011). Characterization of meiotic crossovers in pollen from Arabidopsis thaliana. DNA Recombination: Methods and Protocols, 223-249.

Yelina, N. E., Choi, K., Chelysheva, L., Macaulay, M., De Snoo, B., Wijnker, E., … & Mezard, C. (2012). Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants. PLoS Genet, 8(8), e1002844.

Yelina, N. E., Lambing, C., Hardcastle, T. J., Zhao, X., Santos, B., & Henderson, I. R. (2015). DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis. Genes Dev, 29, 2183-2202.


AGO4 and AGO6 are more specific in mediating RdDM than we expect

Paper: Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation by Duan et al.

EMBO J. doi: 10.15252/embj.201489453 PMID:25527293

The function of AGO6 has been considered redundant with AGO4 previously. This paper, however, shows that the redundancy of AGO4 and AGO6 in mediating RdDM is much smaller than we would expect. AGO4 and AGO6 dependent methylation is profiled by genome-wide bisulfite sequencing. Interestingly, DNA methylation in only a small subset of loci is redundantly regulated by AGO4 and AGO6. In more than half of the hypomethylation loci, DNA methylation is similarly reduced in either ago4-6 or ago6-2, and no significant reduction is observed in double mutant. This result indicates that AGO4 and AGO6 have related yet specific function in RdDM.

The authors also want to show the distinct function of AGO4 and AGO6 by studying their subcellular localization. The conclusion of the paper is that AGO4 and AGO6 show different co-localization patterns with DNA dependent RNA polymerases. However, I am not quite convinced by these immuno-staining figures. The localization of AGO4 and AGO6 are scattered in the nucleus and the co-localization signal with Pol IV or Pol V is not obvious. Even though the co-localization data is not convincing to me, I do agree with the authors that studying the localization of AGO4 and AGO6, especially the co-localization pattern with Pol IV or Pol V, is very important.

The other thing I am interested in is that the authors studied the accumulation of Pol V transcripts as well as Pol V occupancy in ago4 and ago6 mutant. A very interesting result is that Pol V occupancy at most tested IGN loci obviously decreases in ago6 mutant. The accumulation of most tested Pol V transcripts decreases in ago6 mutant but increases in ago4 mutant. These results indicate that AGO6 is required for Pol V recruitment. It is very intriguing that AGO4 and AGO6 show such distinct effect on Pol V occupancy. In my own study, I am trying to pull down AGO4 associated Pol V transcripts. It might be interesting to see if Pol V transcripts could also be pull down by AGO6. We have to notice that only a small number of Pol V transcripts are studied here. It remains unclear that whether this small subset can represent the real pattern.

Last thing to mention, another paper from the Slotkin lab (McCue et al. 2015) shows that AGO6 can load 21-22nt siRNAs and establish RdDM, which is also distinct from AGO4. In conclusion, these two proteins may have more specific functions than we expect.