Figure 1.
Rpd3L complex is required for establishment of heterochromatic boundaries. (A) Schematic diagrams of the chromosomal locations of three boundary-proximal genes, YPS6 and YIR042C, which are adjacent to telomere, and GIT1, which is proximal to HMR. The HMR-tRNAThr gene and STAR (sub-telomeric anti-silencing region) sequence are also labeled. (B) qRT–PCR results of mRNA levels of boundary-proximal genes, YPS6, YIR042C and GIT1, in wild type and various mutant strains. (C) qRT–PCR results of mRNA levels of YPS6, YIR042C and GIT1 in the individual component deletion mutants of Rpd3L and Rpd3S. Fold transcription is relative to wild type. The log2 ratio less than zero indicates repression of transcription, whereas greater than zero indicates enhancement of transcription. Error bars represent standard error of the mean for three independent RNA purifications. (D) Mating assay to test the effects of mutation of RPD3 on boundary activity of HMR-tRNA. The ∼1.0 kb region flanking the right side of HMR with the HMR tRNAThr boundary gene was cloned into the a2 gene, and these constructs (pRO363 or pRO466) were integrated into chromosome III in a MATα strain in the presence or absence of Rpd3p (see ‘Materials and Methods’ section). The resulting strains and a MATa strain were mated, serially diluted and spotted onto a YC plate or a Ura–/Lys– plate, followed by incubation at 30°C. The photograph was taken after 48 h.