4024 Nucleic Acids Research, 2011, Vol. 39, No. 10 response to nutritional stress but H2A.Z, present in the upstream, regulatory nucleosome is not required for its activation in vivo (17). Much of the in vitro transcription by pol III has been studied using yeast SUP4, one of the tRNATyr gene family members. Like a typical pol III-transcribed gene, SUP4 has two internal promoter elements 55 bp apart, boxes A and B, but no upstream regulatory elements. The tran- scription complex formation is initiated with binding of the transcription factor (TF) IIIC to the boxes A and B, followed by the recruitment of the initiation factor IIIB, 30 bp upstream of TSS at +1 (18). Short length of genes facilitates terminator-dependent recycling of pol III which involves direct transfer of pol III to the TSS after termin- ation in each round, giving several fold increase in tran- scription in vitro (19). Transcription by pol III is tightly regulated in response to any stress by a central regulator Maf1 (20,21). Nutrient deprivation like nitrogen and carbon starvation leads to immediate downregulation of transcription by pol III (22) and pol III is lost from its target genes under the conditions of repression (23). In this study, we have shown the role of H2A.Z, RSC and FACT in modulating the transcription of the yeast tRNATyr gene. Working as H2A.Z chaperone, Spt16 with the help of SWR1 complex deposits as well as removes H2A.Z in the gene ﬂanking nucleosomes. RSC promotes loading of FACT on the gene, maintains the transcribed region of SUP4 nucleosome-free and keeps a nucleosome close to the gene terminator region in vivo, which probably prevents the facilitated recycling of pol III, resulting in lower transcription from SUP4 in active state. These studies show a novel mechanism of transcriptional regu- lation of SUP4 by all these activities which together keep the SUP4 expression at a low level. MATERIALS AND METHODS Plasmid DNAs, primers, yeast strains and growth Plasmid pLNwt containing the 256 bp DNA from the yeast SUP4 gene locus was gift. Sequences of the primers and list of the yeast strains used in this study are given under the Supplementary Tables S1 and S2, re- spectively. Yeast cultures were grown in YEP (yeast extract and peptone) media containing 2% glucose. Cells were nutritionally deprived by shifting to 0.15 YEP without any carbon source (23) and allowed to grow for further 1.5 h, unless otherwise stated. Yeast cultures of temperature sensitive mutants were rapidly shifted to 37 C by adding pre-warmed media to the cultures grown at 30 C. In vitro chromatin assembly and transcription Chromatin was assembled on pLNwt using S-190 extract of Drosophila embryos and in vitro transcription using pure TFIIIB, TFIIIC and pol III was performed as previ- ously described (24). 6 His-tagged Nhp6A was puriﬁed from the overexpression clone (gift from Ian Willis). Spt16–Pob3 dimer was puriﬁed from strain carrying TAP-tagged Spt16 using standard tandem afﬁnity puriﬁ- cation protocol (25). Antibodies Antibodies against H3 (Ab46765), H2A.Z (Ab4626) and Myc (9E10) were purchased from Abcam. Anti-HA, anti-myc (05-904, 05-724MG; Millipore), IgG sepharose (GE Healthcare), FLAG M2 agarose (Sigma) were purchased while anti-Spt16 and anti-Pob3 (Tim Formosa) were gifts. Chromatin structure, chromatin immunoprecipitation and RNA in vivo Chromatin structure analysis by the IEL method, chroma- tin immunoprecipitation (ChIP) and real time PCR, RNA extraction and quantiﬁcation were performed as described (17), and repeated at least three times for each experiment. Fold enrichments in ChIP and real time PCR assays were calculated as occupancy normalized against TELVIR region and average from three independent ex- periments with error bars are shown. For high-resolution footprinting, digestion of chromatin with MNase in vivo and sample preparations were as described for the IEL method. Samples were used for primer extension and products resolved on 8 M urea–6% polyacrylamide gel as described earlier (24). Further details for each method and primers can be found in Supplementary Data. Secondary digestion of DNA for IEL analysis was with a HindIII site present 860 bp downstream of the SUP4 gene and probe was 150 bp DNA abutting HindIII site. Co-immunoprecipitations and pull-downs Co-immunoprecipitation (IP) experiments (26) using yeast whole cell extract and in vitro pull downs to follow physical interactions of tagged H2A.Z and Spt16 proteins were performed as described under Supplementary Data. RESULTS We studied the role of chromatin in regulating SUP4 tran- scription, in vitro as well as in vivo. Recruitment of various activities to SUP4 in vivo under two different nutritional states in yeast was ascertained by ChIP and real time PCR assays. Chromatin inﬂuences the transcription of SUP4 gene Naked DNA transcription of SUP4 is known to be TFIIIC-dependent (Figure 1A, lanes 1 and 2). Transcription from SUP4 using pure TFIIIC, pol III and recombinant TFIIIB gives two additional transcripts, corresponding to downstream initiations from the pos- itions +4 and +8 with respect to the TSS at +1. Similar to SNR6 (24), addition of TFIIIC to the chromatin assembled on the plasmid pLNwt yields 5-fold more tran- script (Figure 1A, lane 2 versus 4). Chromatin formation suppresses the non-speciﬁc transcription from the plasmid backbone (data not shown) which may contribute to the observed higher than naked DNA (lane 2) level of tran- scription from chromatin (lane 4). Unlike SNR6, TFIIIC addition does not result in a nucleosome positioning on