Nucleic Acids Research, 2011, Vol. 39, No. 10 4025 Figure 1. Transcription of SUP4 gene and chromatin. (A) Transcription from chromatin is higher than naked DNA in vitro. TFIIIC-dependent transcription of naked DNA (cf. lanes 1 and 2) and chromatin (cf. lanes 3 and 4) produces two additional transcripts due to downstream initiations from the positions +4 and +8 with respect to TSS at +1. RM represents the position of recovery marker in the gel. Transcript from +1 site is marked. (B) SUP4 RNA synthesis under nutritional stress. The total SUP4 transcript levels at different time points of repression are normalized against the U4 transcript used as an internal control. Average and scatter of RNA levels estimated from three independent experiments are plotted. (C) Schematic diagram showing the amplicon sizes and primer pairs used in real time PCR and chromatin immunoprecipitation studies. Positions of the gene sequence elements with respect to the three amplicons representing the upstream, gene and downstream regions of SUP4 gene are shown. Bent arrow represents TSS. For all ChIP data, averages from three independent experiments with error bars, normalized against TELVIR region, are calculated. (D) Relative occupancy of FLAG-tagged C160 subunit of pol III on SUP4 in both active and repressed conditions. Nutrient deprivation with 0.15 YEP medium lacking glucose was used to repress the pol III transcription for 80 min. the transcribed region of the gene (Supplementary Figure S1) suggesting absence of nucleosome on the gene may be due to the ATP-dependent chromatin remodelling activities present in the S-190 extract, which probably mobilize the nucleosome away from the gene region, making it transcriptionally active in the presence of TFIIIC. These in vitro observations suggest that chroma- tin may inﬂuence SUP4 expression in vivo as well. We measured SUP4 RNA levels under active and re- pressive conditions of pol III transcription (Figure 1B). Surprisingly, SUP4 pre-tRNA levels increase for the ﬁrst 2 h while levels of 50-end processed transcript do not change after 1 h of repression. Levels do not change much with prolonged starvation due to high stability of tRNAs. This suggests pre-tRNA levels increase after 1h of repression because processing stops but the increase during the ﬁrst hour may be due to increased tran- scription. Starvation induced repression is accompanied by loss of pol III from the target genes (23). ChIP of Flag-RPC160 subunit using amplicons shown in the Figure 1C showed that after 80 min of nutritional stress, pol III levels on the gene drop by only 50% on the transcribed region (Figure 1D). This suggests that transcription from the gene may be continuing during initial repression and a change in the transcript level may be related to even chromatin structure around the gene. A nucleosome-free gene region is ﬂanked by two nucleosomes Using micrococcal nuclease (MNase) digestion instead of sonication, we probed the SUP4 gene region (Figure 1C) for the presence of core histones by the ChIP and real time PCR method. As compared to nucleosome-dense TELVIR region, the gene region is found histone-depleted where levels appear close to background level (Figure 2A and B). Comparatively lower enrichment of H2B and H3 in downstream region as compared to upstream, suggests that the nucleosomes may be present on both sides of the SUP4 gene in vivo ( 1 and +1, Figure 2C), but the +1 nucleosome is not well positioned. We conﬁrmed the histone-depleted nature of the gene region in vivo by high-resolution MNase footprinting. As compared to naked DNA, chromatin shows MNase hypersensitivity at the position from +93 to +96 in the gene region (Figure 2D), while the region downstream of the termin- ator is protected (lanes 3, 4 versus 1, 2), suggesting presence of nucleosomes. The MNase digestion proﬁle of rest of the gene sequence is same for both chromatin and naked DNA, suggesting the observed hypersensitivity probably represents the 50-boundary of the +1 nucleo- some, downstream of the gene. Nucleosome positions near the gene were further con- ﬁrmed by analysing the chromatin structure of SUP4 tDNA locus in vivo using the indirect-end-labelling (IEL) approach (Figure 2E). As compared to MNase digestion pattern of naked DNA, the chromatin structure in vivo shows presence of an array of positioned nucleosomes upstream of the gene sequence (cf. lanes 1, 2 and 3, 4). The transcribed region of the gene is not cut well by MNase, but the broad band around +98 bp, downstream of the box B at+80 bp, shows a MNase hypersensitivity of the gene, not seen on naked DNA. Thus, considering the results from the Figure 2A and B, and the low-resolution nature of the IEL mappings, the region from 47 to +98 (±20) bp encompassing the SU4 transcribed region is devoid of a nucleosome. Protection of naked DNA sites at 151 and 63 bp positions shows the presence of the 1 nucleosome positioned between 242 and 47 bp giving a median at 140 bp as opposed to 130 bp mapped by the ChIP-seq method (http://h2az.atlas.bx .psu.edu) for the same nucleosome. The protection of naked DNA sites, including the +192 bp position but absence of any strong boundaries between +100 and +350 bp positions (cf. lanes 3, 4 and 1, 2) suggests that as compared to the 1 nucleosome, the boundaries of +1 nucleosome are comparatively diffused. The span of this nucleosome matches well with the position of a fuzzy nu- cleosome between +90 and +300 bp (http://h2az.atlas.bx .psu.edu). Fuzzy nature of the +1 nucleosome may also be the reason that H2B and H3 levels in the downstream region are lower as compared to the upstream region in active state of the gene (Figure 2A and B). Increase in H2B levels only in +1 nucleosome under repression (Figure 2A)