, S.cryophilus and S. japonicus) only encode RFTS proteins devoid of a methyltransferase domain. The genomes of other fungi for instance Coccoides immitis encode each RFTS only and RFTS plus methyltransferase domain proteins (Figure S1A). The majority of those fungal proteins happen to be identified even though homology and remain uncharacterised. Indeed, in spite of the fact that Raf2 is necessary for heterochromatin integrity in S. pombe the function of the RFTS and its contribution to heterochromatin integrity and centromere function has not been investigated.Marker gene silencing is disrupted by mutations within the RFTS domain of RafTo determine the role of the Raf2 RFTS domain, we very first deleted the complete domain (residue M1 to N203) from C-terminally FLAG-tagged Raf2 expressed from its personal locus. However, the resulting Raf2-RFTSD-FLAG protein was unstable plus the anticipated 52.6 kD band was not detectable by western (Figure S1B). For that reason, to study the function of RFTS, we introduced distinct point mutations into residues conserved among each fungal (I98A) or mammalian proteins (E104A). As well as mutating these residues, we previously isolated a conditional temperature sensitive (ts) allele, raf2-1, in a screen forThe RFTS Domain of Raf2 Is Necessary for Heterochromatin IntegrityFigure 2. RFTS mutants conditionally disrupt heterochromatin integrity. A. Assay for silencing at cen1:ade6+. Diagram shows the position of the cen1:ade6+ marker gene within centromere 1, relative for the outer repeat (otr) dg and dh elements, innermost repeats (imr), and central core (cnt). Wild-type cells with all the marker gene repressed kind red colonies on low adenine whereas cells with disrupted heterochromatin for example clr4D or raf2D result in marker gene expression and form white colonies. Raf2-I98A and raf2-S100F disrupt marker gene silencing particularly at 36uC but not at 25uC. B. ChIP evaluation of H3K9me2 levels connected with cen(dg) relative to act1+ in clr4D and raf2 mutant cells normalised to wild-type at 25uC or 36uC.Formula of 912331-75-0 Error bars: SEM. C. qRT-PCR evaluation of cen(dg) transcript levels relative to a manage transcript act1+, normalised to wild-type at 25uC or 36uC. Error bars: SEM. D. Swi6 localisation in wild-type or mutant cells at 25uC or 36uC. Representative pictures of fixed cells with Swi6 (green), CENPACnp1(red) and DNA (DAPI-blue). Numbers shown denote cells with Swi6 localised at centromeres, as determined by colocalisation with Cnp1, from a total quantity of 80 cells per sample.1246761-84-1 web doi:10.PMID:23910527 1371/journal.pone.0104161.gPLOS One | plosone.orgThe RFTS Domain of Raf2 Is Essential for Heterochromatin IntegrityFigure three. Raf2 conditional mutants show defective chromosome segregation in the restrictive temperature. Analysis of lagging chromosomes in anaphase by fluorescence microscopy. Cells with defective heterochromatin show lagging chromosomes in anaphase. Shown here are representative photos of fixed cells stained with DAPI (red) and tubulin (green). The numbers shown denote anaphase cells with lagging chromosomes. 100 mitotic cells were counted for each and every sample. doi:10.1371/journal.pone.0104161.gmutants which disrupt heterochromatin at 36uC, but not at 25uC [23]. The raf2-1 mutant expresses a protein with a missense mutation (S100F) within a conserved fungal residue inside the RFTS domain, identified hereafter as raf2-S100F. These 3 mutated residues, I98, S100 and E104, reside inside a versatile looped region between the key a-helix of the RFTS domain and also the b-sheet regi.
