Spt16 maintains the DS nucleosome position at tDNA loci. All occupancies were measured by the ChIP and real time PCR (% Input) method. The dots mark nonsignificant changes. (A) Average nucleosomal (histone H3) occupancy profiles (True et al. 2016) aligned by the TSS of tRNA genes in the wild-type and spt16-197 cells. Profiles were normalized by setting the NFR minima to zero. (B) Comparison of relative H3 occupancies in the DS region of selected tRNA genes at 37°C, normalized with the respective H3 levels at 30°C in wild-type cells. (C) The relative H3 occupancy in the DS region of selected tRNA genes in the wild-type and spt16-197 cells show gene-specific changes with inactivation of Spt16 in spt16-197 cells at 37°C. The P-values are (*) 0.002582, (**) 0.000107, (***) 0.000079, and (****) <0.000001. (D) Occupancies of Spt16 in the DS region of the tRNA genes in the wild-type and mutant cells. The gene-specific occupancies show a significantly reduced level in the spt16-197 cells at 37°C on all the tested genes. The P-values are (***) 0.0008796, (**) 0.005094, (*) 0.017963, (**) 0.005608, and (**) 0.005002 in order of their appearance on the graph panel. (E,F) Nucleosome occupancy profile (H3 tag counts per million reads) on the tRNA genes (E) tP(UGG)O3 and (F) tT(CGU)K from the ChIP-seq data (True et al. 2016). The shaded box marks the gene body. (E) The US/DS nucleosome peaks are marked. (F) Asterisk marks the DS nucleosome peak. (G) Schematic representation of the positions of real time PCR amplicons on SCR1 and RPR1 genes. Bent arrow depicts the position of TSS. Gray boxes mark the transcribed region and numbers in parentheses denote the size of the primary transcript. Amplicon D in the case of RPR1 and A4 in the case of SCR1 map the 3′-end of the genes whereas A5 overlaps the strongly positioned DS nuclesosome on the SCR1 gene. (H,I) Nucleosome occupancies at the non-tRNA gene bodies increase in the mutant cells. Relative H3 levels at different positions on the (H) SCR1 gene and (I) RPR1 gene are shown. The P-values are for the amplicons (**) A1 = 0.000061, (****) A2 = 0.000002, (*) A3 = 0.000012, (***) A4 = 0.000004 in panel H and B (*) = 0.000053, (**) C = 0.000015 in panel I.
