RFTS mutants conditionally disrupt heterochromatin integrity.

<p><b>A.</b> Assay for silencing at <i>cen1:ade6+</i>. Diagram shows the position of the <i>cen1:ade6+</i> marker gene within centromere 1, relative to the outer repeat (otr) <i>dg</i> and <i>dh</i> elements, innermost repeats (<i>imr</i>), and central core (<i>cnt</i>). Wild-type cells with the marker gene repressed form red colonies on low adenine whereas cells with disrupted heterochromatin such as <i>clr4Δ</i> or <i>raf2Δ</i> cause marker gene expression and form white colonies. <i>Raf2-I98A</i> and <i>raf2-S100F</i> disrupt marker gene silencing specifically at 36°C but not at 25°C. <b>B.</b> ChIP analysis of H3K9me2 levels associated with <i>cen(dg)</i> relative to <i>act1+</i> in <i>clr4Δ</i> and raf2 mutant cells normalised to wild-type at 25°C or 36°C. Error bars: SEM. <b>C.</b> qRT-PCR analysis of <i>cen(dg)</i> transcript levels relative to a control transcript <i>act1+</i>, normalised to wild-type at 25°C or 36°C. Error bars: SEM. <b>D.</b> Swi6 localisation in wild-type or mutant cells at 25°C or 36°C. Representative images of fixed cells with Swi6 (green), CENP-A^Cnp1(red) and DNA (DAPI-blue). Numbers shown denote % cells with Swi6 localised at centromeres, as determined by colocalisation with Cnp1, from a total number of 80 cells per sample.