4026 Nucleic Acids Research, 2011, Vol. 39, No. 10 Figure 2. Effect of Repression and H2A.Z deposition on the SUP4 gene locus in vivo. Chromatin organization at the SUP4 locus is probed by ChIP/ Real Time PCR and footprinting methods. (A) Relative occupancy of FLAG-tagged H2B. (B) Relative occupancy of Myc-tagged H3. (C) Nucleosomes ﬂank the histone-free SUP4 gene: Cartoon showing positions of the nucleosomes 1 and +1, relative to the cis elements of the gene, as marked in the panel 1C. (D) High-resolution MNase footprinting in vivo. MNase digested chromatin and naked DNA samples were used for extension with a primer which hybridizes to the bottom strand, 13 bp downstream of the TSS. Lane M shows a 50 bp ladder used as molecular size marker while GATC represent the sequencing reaction over genomic DNA. Two levels of MNase digestions are shown for SUP4 as naked DNA (lanes 1 and 2) and chromatin (lanes 3 and 4). Positions of the box B and terminator are marked while grey ovals represent nucleosomes. Position of MNase hypersensitivity immediate upstream of the terminator is marked with a short vertical bar in the left-hand side. (E) Low-resolution chromatin structure analysis by the IEL method. Grey ovals denote the nucleosomal size protections and arrows mark the MNase cut sites in the chromatin. Gene region is marked with a rectangle. All numbers represent bp with respect to TSS at +1. Numbers on the left-hand side mark the MNase cuts seen on the naked DNA in lanes 1 and 2. Numbers on the right-hand side mark the MNase cuts seen on chromatin and 1, +1 mark the gene ﬂanking nucleosomes. MNase cleavage pattern of wild type (lanes 3–6) and Htz1 cells (lanes 7–10) without (lanes 3, 4 and 7, 8) or with nutritional stress (lanes 5, 6 and 9, 10) for 2 h is shown. (F) H2A.Z deposition in the nucleosomes around the SUP4 gene in vivo. Relative occupancies of FLAG-tagged H2A and H2A.Z on SUP4 against TELVIR region are shown. H2A.Z levels increase with repression. suggests a differential histone dynamics in both the nucleosomes. Higher association of H2A.Z with SUP4 gene under repressed state Nucleosome mobility and occupancy on the promoter regions are often associated with the state of activity of a gene (11). As above data show absence of nucleosomes on the SUP4 in its active state, we followed the nucleo- some dynamics on the SUP4 gene locus under repression conditions using IEL method. No change in the arrange- ment of nucleosomes upstream or downstream of the gene after repression (Figure 2D, lanes 5 and 6) is seen. But, the MNase sensitivity of the gene at +98 bp is reduced, sug- gesting a change in +1 nucleosome region, probably resulting in further terminator exposure and higher tran- scription in the repressed state (Figure 1B). Nucleosomes ﬂanking the tRNA genes (9) are reported to have H2A.Z. We looked for the presence of H2A and its only variant in the budding yeast, H2A.Z, in both the nucleosomes. As opposed to negligible H2A levels, H2A.Z shows signiﬁcant enrichment in both, especially the 1 nucleosome (Figure 2F, Supplementary Figure S2). This proﬁle of H2A.Z is similar to the general pattern of H2A.Z-containing nucleosomes ﬂanking an NFR around the TSS, reported for active pol II-transcribed genes (10). However, IEL analysis shows that similar to SNR6 (17); the chromatin structure remains unperturbed in the absence of H2A.Z, even under repression (Figure 2D, lanes 7–10), supporting a previous report showing no role of H2A.Z in general nucleosome positioning or main- tenance of nucleosomal organization (27). H2A.Z associ- ates with both active and inactive genes (28). On SUP4, H3 levels do not change (Figure 2B) but H2A.Z (Figure 2F) levels increase under repression. As compared to