4032 Nucleic Acids Research, 2011, Vol. 39, No. 10 Figure 6. Relationship of transcription from SUP4 with H2A.Z level and nucleosome positions. Ovals represent nucleosomes, boxes and T1, T2 denote two terminators, bent arrow marks the TSS. Only down- stream nucleosomes are shown with H2A.Z levels and transcriptional state of the gene shown on the right-hand side. (A) In wild-type cells, active state, nucleosmes are fuzzy and block T2. (B) In wild-type cells, following 2 h repression, +1 nucleosome is positioned at a short distance from the terminators. (C) In wild-type cells, after repression longer than 2 h, two nucleosomes may be positioned similar to that seen in rsc4-D4 cells. Both terminators are near the dyad axis of the gene-proximal nucleosome. to suppress the transcription in active state, helps loading Nhp6/FACT for transcriptional ﬁdelity and differential marking with H2A.Z according to the expression state. While leaving the +1, RSC persists on the 1 nucleosome under repression, keeping the gene poised for activation under repressed state. Loss of Sth1, the catalytic subunit of RSC, from the downstream region of SUP4 within ﬁrst hour during re- pression, may result in a nucleosome positioning similar to that seen in the rsc4-D4 mutant (which shows loss of remodelling activity on SNR6, 17). On some of pol III-transcribed genes, loss of Sth1 activity was earlier found to result in gradual increase of histone density but as RSC was not lost under repression, histone density on these genes did not show any change (57). However, despite an increase in histone density near the gene ter- minator at +100 bp (Figure 2A) we could not see a similar nucleosomal protection in the gene proximal region after 2 h of repression. As Sth1 is lost from the downstream of SUP4, a gradual increase in histone density in the gene region may take place with further repression. Accordingly, a nucleosome was seen to encroach the gene after 4 hr repression (not shown), suggesting an or- ganization similar to rsc4-D4 cells may be generated after prolonged satrvation. Regulation of SUP4 from downstream Absence of the transcription terminator of pol III-transcribed genes results in loss of the facilitated recycling of pol III and transcript yield (19). As the cartoon in the Figure 6 depicts, due to its fuzzy nature, the +1 nucleosome encroaches the gene terminator T2 at +108 bp (Figure 6A). This may allow transcription termin- ation only at T1, ensuring utilization of only one gene terminator in vivo; but interfere with terminator-directed recycling of pol III (19), resulting in reduced transcription rate in active state. Under initial hours of repression, +1 nucleosome positioning away from the gene would make both the terminators equally exposed (Figure 6B) and the resultant increase in the transcription rate would give higher RNA levels from SUP4 as observed under repres- sion for some time (Figure 1B). Interestingly, presence of the gene terminators at the dyad axis of a positioned nucleosme in the rsc4-D4 mutant, similar to that seen on the SNR6 gene in active state (17), may allow the terminator-dependent recycling of pol III, giving higher transcription rate. In comparison, prolonged stress may completely abolish the transcription despite the same pos- itioning in the wild type cells. This may be related to dif- ferent signals from H2A.Z dynamics in both conditions (Figures 5C and F, and 6C). Thus, a ﬁne tuning of the +1 nucleosome position by RSC along with H2A.Z as the stress-sensor, probably regulates the gene output under different states from pol III-transcribed genes. These observations suggest SUP4 may be an example where diverse chromatin related activities come together and co-ordinately keep the gene expression low in active state generating a milieu in which gene remains in a poised state for release from the re- pressed state. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Tim Formosa, Phillipp Korber, Hiten Madhani, Sebastian Chavez, Frank Pugh, David Stillman, Kevin Struhl, Toshio Tsukiyama, Michel Werner, Ian Willis and Jerry Workman for kind gifts of some of the yeast strains used in this study. FUNDING Council of Scientiﬁc and Industrial Research (CSIR); Department of Science and Technology, Govt. of India, research grant (SR/SO/BB-71/2003 to P.B.); CSIR senior research fellowship (to S.M. and P.S.D.). Funding for open access charge: Waived by Oxford University Press. Conﬂict of interest statement. None declared. REFERENCES 1. Biswas,D., Yu,Y., Prall,M., Formosa,T. and Stillman,D.J. 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