Dia2's F-box and LRR domains are indispensable for transcriptional silencing.
(A) A schematic drawing of the domain structure of Dia2. Dia2 contains a tetratricopeptide repeat (TPR) region, nuclear localization signal (NLS), F-box and leucine rich repeat (LRR) domain. (B) The Dia2 F-box and LRR domains are important for silencing at the HMR locus. The dia2Δ mutant cells containing the HMR::GFP reporter were transformed with empty vector or a plasmid expressing WT or different dia2 mutants, and the percentage of cells expressing GFP was determined as in Figure 1B. Note that the percentage of cells expressing GFP in dia2Δ cells was higher when using selective media compared to cells growing in YPD. (C) The Dia2 F-box and LRR domains are important for telomere silencing. The dia2Δ mutants containing the VIIL-URA3 reporter were transformed with empty vector or a plasmid expressing the indicated dia2 mutant and spotted onto selective media (-HIS) and media containing FOA (-HIS, FOA) to assay silencing. (D–E) The Dia2 LRR domain is important for HMR silencing (D) and telomere silencing (E). Assays were performed as described in B and C.