wild-type cells, higher SUP4 RNA levels found in htz1D cells (Figure 3A), suggest its repressive role in SUP4 ex- pression. Decrease in occupancy upon activation of SUP4 is consistent with the reports showing that H2A.Z is lost during the active transcription of a gene (29,30). Shift in the position of +1 nucleosome modulates SUP4 transcription In vitro results from the Figure 1A suggest involvement of ATP-dependent chromatin remodelers in SUP4 transcrip- tional activation. Therefore, we monitored the SUP4 tRNA levels in cells mutated for a subunit of the major chromatin remodelling complexes of yeast. Deletion of Isw2 or Chd1 does not have any effect on SUP4 levels (Figure 3A), and both of them did not show enrichment near the gene (data not shown). In comparison, RSC lo- calizes to the region (Figure 3B) and an increase in RNA level is found (Figure 3A) in cells with an Rsc4 mutation Nucleic Acids Research, 2011, Vol. 39, No. 10 4027 known to cause a loss of interaction with pol III (31). Similar to repressed state (Figure 1B), higher RNA levels in both H2A.Z and RSC mutants could be due to higher transcription as well as defective processing. Decrease in occupancy of RSC (Figure 3B) along with increase of H2B (Figure 2A) under repression, only in the downstream region, suggests that the repressive effect of RSC on SUP4 expression may also be due to a remodelling of +1 nucleosome by RSC. We used IEL method, which can map the nucleosomes with fair accuracy (32), to see the remodelling, suggested by above data. Digestion pattern of chromatin in rsc4-D4 mutant does not show any apparent changes in the chro- matin structure (Figure 3C) except a reduced MNase sen- sitivity (cf. lanes 1–4) near the box B (asterisk), which could be either due to loss of nucleosome and its boundary or further encroachment by a nucleosome from the downstream region. Comparison of the proﬁles of similarly digested DNA in lanes 2 and 4 (Figure 3D) Figure 3. RSC has a repressive role in SUP4 expression. (A) RNA was extracted from different mutants under active transcription conditions and SUP4 levels estimated as given under the ‘Material and Methods’ section. U4 was used as the internal control to normalize the SUP4 transcript levels. Average and standard deviations calculated from three independent isolations are plotted. (B) Relative enrichment of myc-tagged Sth1 subunit of RSC complex on SUP4 in both active and repressed conditions. (C) IEL analysis of the chromatin structure in RSC mutant and wild-type yeast cells. Ovals represent the nucleosomes; arrow and rectangle mark the gene position. Bent arrow marks the TSS, asterisk marks the MNase hyper- sensitive site on the gene region. Digestions with two MNase levels are shown for each strain. (D) Comparison of the digestion proﬁles of the similarly digested DNA in lanes 2 and 4 from the panel C. Proﬁles were generated using the PhosphorImager and the Image Gauge (Fuji) software. Asterisk marks the MNase hypersensitive site on the gene region while short arrow marks the direction of nucleosome shift. Box marks the gene region, bent arrow the TSS and Term the gene terminator. Light and dark ovals represent the positioned nucleosomes in wild-type and mutant cells, respectively. (E) Relative enrichment of myc-tagged Nhp6 in rsc4-D4 mutant in active condition. (F) High-resolution MNase footprinting. Samples were prepared and primer extension reactions carried out as described under the legends for the Figure 2D. The primer hybridizes to the bottom strand, 92 bp downstream of the TSS. Nucleosomes in rsc4-D4 mutant are restricted to downstream of the terminator. Three levels of MNase digestions are shown for wild type (lanes 1–3) and mutant (lanes 4–6) cells. Dark grey oval represents the downstream nucleosome in mutant while light grey ovals represent fuzzy nucleosomes in wild-type condition. Numbers in the right hand side denote bp with respect to TSS while short vertical bar marks the position of MNase hypersensitivity in lanes 4–6.