Taken together, results presented in this study show while Nhp6 is required for ﬁdelity; loss of RSC activity, H2A.Z or the FACT components results in higher SUP4 RNA levels in active state, suggesting they may be working in the same pathway, probably to reduce the SUP4 expression. This may be a novel mode of gene regu- lation whereby all chromatin related activities target the gene to repress it rather than activate in active state. Importantly, regulation of H2A.Z levels in 1 nucleosome by FACT as H2A.Z chaperone and shift in position of +1 nucleosome by RSC in direct response to transcription repression show a differential role of both the gene ﬂanking nucleosomes in the gene expression. DISCUSSION According to the general belief, the tRNA genes are largely devoid of well-deﬁned, positioned nucleosomes obviating the need for epigenetic modes of regulation. In the genome-wide sequence-directed nucleosome position predictions (http://genie.weizmann.ac.il/software/nucleo_ prediction.html), the SUP4 gene sequence is predicted to exclude nucleosomes from the gene region. Actively transcribed SUP4 gene on a multicopy plasmid, was shown to resist incorporation into a positioned nucleo- some in vivo (47). Using salt-dilution chromatin assembly method, we found that the SUP4 gene excludes nucleosome deposition over itself in vitro while a single nucleosome is positioned in the upstream region (not shown). Therefore, sequence-directed nucleosomal absence near the SUP4 tDNA in vivo could potentially exclude role of all epigenetic mechanisms in regulating SUP4 transcription. Results presented in this study have shown a central role for H2A.Z, RSC and FACT in tRNA transcription and suggest a complex interplay of a number of epigenetic processes in regulating SUP4 gene. H2A.Z as stress-receptor in SUP4 expression Presence of H2A.Z may have important biological impli- cations (48). Yeast H2A.Z, present in many loci through- out the genome, is implicated in locus-speciﬁc activation and repression of genes (48–50). The initial increase of SUP4 transcription under repression is similar to induc- tion of many stress-response genes shortly after stress signal is received (51,52). During ﬁrst 2 h, when pol III levels drop, increase in Swr1 levels and loss of Spt16 may increase the H2A.Z levels in 1 nucleosome. The drop in H2A.Z levels after prolonged repression probably poises the gene for the activation process through a quick H2A.Z-independent trigger. H2A.Z presence in the nucleosome reduces its mobilization by a chromatin remodeller (27). Thus, increase in H2A.Z under repressed condition may help keep the 1 nucleosome in its place while loss of H2A.Z and increase of RSC in active state would delocalize the downstream nucleosomes making the +1 nucleosome fuzzy. Thus, H2A.Z confers differential behaviour on these nucleosomes in response to repression while its dynamics on SUP4 is not related to active transcription. These observations suggest H2A.Z serves as a starvation-respone factor for SUP4, similar to Nucleic Acids Research, 2011, Vol. 39, No. 10 4031 its recently suggested thermosensory role (53). Signiﬁcantly, on two of pol II-transcribed genes as well, which show a delayed repression, late increase of H2A.Z levels is seen (Supplementary Figures S4 and S5C), sug- gesting H2A.Z may be a general stress-response factor. Role of FACT components on SUP4 gene Our results suggest that FACT may be responsible for the reported presence of H2A.Z near tDNAs in NFR-ﬂanking nucleosomes (9), a role not attributed to the FACT till now. Different core histone dimers can be exchanged by more than one histone chaperone in the chromatin assembly/disassembly reactions in vivo on different genes, suggesting a broad range of carriers for them (44). Events like histone exchange or nucleosome loss on the transcribed or promoter regions of the genes may be either the cause or the consequence of transcription. Nevertheless, these events are neither spontaneous nor autonomous and require the assistance of a chromatin remodeler and chaperone. Moreover, transcription- associated loss/exchange of histones due to passage of the elongating RNA polymerase II takes place with the help of FACT only in the transcribed region of a gene (2–5). The N-terminal domain of Spt16 is known to interact with H3 and in its absence a rapid loss of H3 is seen on both the active and inactive genes (43). Thus, on SUP4, the observed effect of Spt16 on the dynamics of H2A.Z in the ﬂanking nucleosomes, which are regulatory and not structural in nature, need not be transcription process-associated, but very well be due to the Spt16 chaperone activity. Spt16 was shown to co-purify with H2A.Z in the absence of Nap1 and H2A.Z-speciﬁc chap- erone Chz1 and help in SWR1-mediated exchange of H2A.Z–H2B dimer in vitro (42). We have shown that FACT interacts with H2A.Z both in vitro and in vivo. Thus, FACT may be a chaperone for the dimer of H2B with H2A or its major variants on different genes which may or may not require FACT for their expression. Nhp6 stimulates the transcription of pol III-transcribed U6snRNA, while the deletion of both the copies of Nhp6 abolishes the TFIIIB footprint in the TATA box region (54). Although de novo synthesis of bulk of tRNA is not affected in Nhp6DD strain (55), we have found multiple effects of Nhp6 on SUP4. It physically interacts with RSC and blocks remodelling action of RSC in vitro (33). As it is required to load Spt16/Pob3 (45), mutation in RSC or loss of Nhp6, results in probably the same effect; loss of Spt16/ Pob3 recruitment, and hence, H2A.Z. Therefore, increase in SUP4 RNA levels in RSC, Spt16 and Pob3 mutants could be due to the loss of H2A.Z in these strains, a condition similar to htz1D cells. RSC plays multiple roles in SUP4 gene expression Loss of RSC is reported to shrink NFR (56) on pol II-transcribed genes and increase the nucleosome density on pol III-transcribed genes (57). We have shown that RSC is required to keep the nucleosome immediate down- stream of the SUP4 terminator, fuzzy in vivo (Figure 6A). It changes the local density of nucleosmes near the SUP4 gene (Figure 6B and C), maintains nucleosome positions