Silencing recombination hotspots via RNA-directed DNA methylation

: Plant genomes are epigenetically modified and this information is critical for gene expression and chromosome function. For example, DNA cytosine methylation is a heritable silencing mark that maintains repression of repetitive sequences, including transposable elements (Henderson & Jacobsen, 2007). We have recently demonstrated that loss of DNA methylation causes epigenetic remodeling of CO frequency along Arabidopsis thaliana chromosomes (Yelina et al., 2012). We have also described the presence of CO hotspots, which are narrow regions with highly elevated recombination activity (Yelina et al., 2012). This project will further investigate control of meiotic CO by epigenetic information.

The student will directly test whether DNA methylation is sufficient to silence hotspot activity. They will generate lines where CO hotspots are targeted for DNA methylation, via expression of complementary inverted-repeats (Henderson & Jacobsen, 2007). The student will validate for the presence of DNA methylation at the hotspots using sodium bisulfite sequencing and methyl-sensitive restriction digestion & Southern blotting.
Once DNA methylation is confirmed they will measure CO activity using an allele-specific PCR technique, termed pollen-typing (Yelina et al., 2012).
This will test whether de novo DNA methylation of recombination hotspots is sufficient to silence CO activity. The student will further investigate control of recombination by combining fluorescent CO reporter lines into epigenetic (eg met1, rdr2, arp6) and recombination (eg fancm, msh4, mus81) mutants to investigate epistatic relationships.

The project will provide the student with training in genetics, epigenetics, molecular biology and microscopy. The laboratory also performs computational genomics and training will be available for interested students.

References:

Henderson, I.R. & Jacobsen, S.E. (2007) Epigenetic inheritance in plants.
Nature. 447:418-24. Yelina N.E., Choi, K., Chelysheva, L., Macaulay, M. de Snoo, B., Wijnker, E., Miller, N., Drouaud, J., Grelon, M., Copenhaver, G.P., Mezard, C, Kelly, K.A. and Henderson, I.R. (2012) Epigenetic remodelling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants. PLoS Genetics 8:e1002844.