Fig 1: Leo1 aggravates the effects of siRNA loss Spotting assay to test TBZ sensitivity of the indicated strains. Tenfold serial dilutions of the indicated cultures were grown on rich medium (YEA) in the presence (17 μg/ml) or absence of TBZ.Loss of minichromosome Ch16. Two independent experiments were performed; error bars show the range.Representation of centromere 1 in S. pombe. Integration sites of ura4 + are indicated with a blue vertical arrow (upper panel). Spotting assay of strains with the relevant genotypes indicated (lower panel).Spotting of strains lacking HP1 (swi6∆) or SHREC (clr3) with ura4 + integrated at the imr1.Spotting of strains lacking FACT (pob3∆) with ura4 + integrated at the imr1.
Fig 2: FACT facilitates H3K9me2 to H3Kme3 transition by suppressing histone turnover(A, B, and D) H3K9me2 (A), H3K9me3 (B), and Swi6 (D) ChIP-qPCR at pericentromeres and TEL1L in WT and spt16–1. The TEL1L gene array is shown above the graphs. ChIP was normalized to the average of three euchromatic regions. n = 3 biological replicates. Data are presented as the mean ± SEM. The p values were obtained by linear mixed effect regression (*p < 0.05; **p < 0.01; ***p < 0.001). ns, not significant (p = 0.05).(C and E) RT-qPCR analysis. Expression of dg and tlh1/2 transcripts in the indicated strains relative to WT after normalization to act1+. The spt16–1, fft3?, spt16–1fft3?, and corresponding WT were shifted to 37°C for 1.5 h. n = 3 biological replicates. Data are presented as the mean ± SEM. One-way ANOVA was done on log2-transformed values. Different letters denote significant differences with a Tukey post hoc test at p < 0.05.(F) Histone turnover assay scheme.(G) ChIP-qPCR of the new histone (H3-T7) at TEL1L in WT, pob3?, and pob3?epe1?. Input-normalized ChIP signals from the uninduced samples (0 h) were subtracted from the input-normalized signals from the b-estradiol-induced samples (4 h). Error bars represent ± SEM from three independent experiments. One-way ANOVA was done on log2-transformed values. Different letters denote significant differences with a Tukey post hoc test at p < 0.05.See also Figure S4 and Table S3.
Fig 3: Genomewide distribution of heterochromatin in leo1? cells A browser view of ChIP–exo signals in the WT and leo1? strains using an antibody against H3K9me2 showing the constitutive heterochromatin domains of chromosome 2 (indicated in red in the lower panel). The ratios of RPKM (reads per kilobase per million mapped reads) over the indicated region are shown. Two independent experiments were performed.Browser views of H3K9me2 ChIP–exo signals in WT and leo1? cells at five facultative heterochromatin islands. The RPKM values are shown for each gene and strain.ChIP–qPCR of H3K9me2 at Tf2s.ChIP–qPCR of Swi6 in WT, leo1?, and leo1::Hermes strains at four loci.Spotting assay of WT and leo1? with ura4 + integrated in the Tf2-3 locus. The top row shows a WT strain with functional endogenous ura4 +; all other strains have the non-functional ura4-D18 allele.Spotting assay using strains with ura4 + integrated close to the SPAC23H3.14 locus.ChIP–PCR of H3K9me2 at the SPAC23H3.14 locus. Numbers indicate ratios for the signal SPAC23H13.14/leu1.Data information: (C, D) ChIP was performed in three independent experiments; error bars indicate SD.
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