| Across all eukaryotes DNA packaging affects gene expression. Mutations affecting DNA packaging can have drastic effects on cellular function. In Arabidopsis thaliana, knock-out of the methyltransferases ATXR5 and ATXR6 that mono-methylate Histone H3 at Lysine 27 (H3K27me1) in constitutive heterochromatin leads to de-repression of transposons, re-replication of heterochromatic regions, DNA damage, and large-scale chromatin decondensation. Though H3K27me1 is specific to constitutive heterochromatin and H3K27me2/3 to facultative heterochromatin, we found that induction of H3K27me2 or H3K27me3 in constitutive heterochromatin is sufficient to rescue all atxr5 atxr6 associated phenotypes. This suggests that these chromatin marks are interchangeable. Parallel work has demonstrated that over-replication and DNA damage in atxr5 atxr6 lead to formation of over-replication-associated centers (RACs) that have decondensed chromatin and enrichment of DNA repair proteins. We showed that replication stress induced by hydroxyurea was not sufficient to induce RACs in wild-type plants. To further investigate RACs, we developed a high-throughput antibody-based screen for use in genetic screening of nuclear phenotypes. This screen utilized an antibody raised against phosphorylated Tetrahymena linker histone H1, which we found is associated with RACs and DNA damage. Overall, this research underscores the many ways that cellular function is supported by proper functioning chromatin. |
