The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis [SMARTSeq2]

ARABIDOPSIS THRITHORAX-RELATED PROTEINS 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements, chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, upregulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6 null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels. Fresh nuclei isolated from two-week-old cotyledons grown on 1%MS under long day conditions were isolated as follows. Roughly 50 cotyledons were finely chopped with razor into 50 ul Partec nuclei extraction buffer (Sysmex America, 05-5002), and stained with 400 ul Partec nuclei staining buffer. Samples were filtered once through a 35um nylon mesh (Falcon, 352235) and subjected to fluorescent activated nuclei sorting. Nuclei were sorted from 2C, 4C, 8C and 16C peaks based on DAPI fluorescence. 50 nuclei from each peak were sorted into individual wells of a 96 well plate. Three to four replicates per ploidy/genotype were collected. Negative controls, with no nuclei sorted into a well, were included in every plate and carried through library preparation and sequencing.