barely visible SUP2 transcript; SUP3 and SUP4 appear together at +1; while +2 and +3 represent downstream initiated transcripts from SUP3 and SUP2, respectively. Deletion of Nhp6 is found to reduce transcription from the right positions in all three genes ( 1 and +1 bands in lanes 3, 4 versus lanes 1, 2). In Nhp6DD cells, the down- stream initiated transcripts from +2 and +3 (transcripts from SUP3 and SUP2 respectively), show an increase and a transcript initiated from +8 position, probably from SUP4, is also found, as seen in vitro (cf. Figures 1A and 4B). Nhp6 can recruit Spt16 and Pob3 to nucleosome to form the stable FACT complex (6). Accordingly, presence of Nhp6 specially in the upstream region of SUP4 is matched with levels of other two FACT subunits Spt16 and Pob3 in active state (Figure 4C–E) and its increase in all three regions under repression (Figure 4C) is in agreement with its inhibitory role in SUP4 transcription in vitro, at higher levels. However, unlike Nhp6, Spt16 and Pob3 show loss from the re- pressed gene (Figure 4D and E) and mutations in Spt16 and Pob3 did not affect the ﬁdelity of pol III (not shown), suggesting the functions of Nhp6 and Spt16/Pob3 are in- dependent of each other. In agreement to this, when puriﬁed Spt16 and Pob3 are added for in vitro transcrip- tion, downstream-initiated transcripts from chromatin are not abolished (lane 3 versus 1, Figure 4F). This result is in contrast to reported association of Spt16 with the increase in transcription ﬁdelity for many of the pol II-transcribed genes (5,37). Being in excess, Nhp6, wherever added, shows increase in ﬁdelity (lanes 2 and 4) but decrease in total transcript, whether added alone (lane 2) or with Spt16/Pob3 (lane 4). These results show that all the three components of yFACT occupy the SUP4 gene simultaneouly in vivo but the ability to ameliorate the downstream initiated transcripts resides with Nhp6 alone. Repressive role of FACT in tDNA transcription Apart from being a part of the FACT complex, Spt16 has a speciﬁc role in chromatin organization on certain pol II-transcribed genes (38). The IEL analysis of Nhp6 as well as Spt16 mutants did not show any change in gross chromatin structure around SUP4 (Supplementary Figure S3B), suggesting their association with SUP4 locus may have effect at some other level. Spt16 is known to differentially affect transcription of the pol II-transcribed genes (38). The SUP4 RNA levels in cells defective in either Spt16 or Pob3 (Figure 5A) show an increase while pol II-transcribed genes CMD1 and U4 do not show change in their transcript levels in Spt16 mutant cells (Supplementary Figure S4). In contrast to roles of FACT subunits in pol II transcription activation (5,6,39,40), the higher SUP4 levels in Spt16 and Pob3 mutants suggest a repressive role for them in SUP4 expression. Spt16 is required for H2A.Z deposition on SUP4 locus Spt16 has been shown to work as chaperone for H2A (4) and H2A.X (41). It co-puriﬁes with H2A.Z in the absence of Nap1 and H2A.Z-speciﬁc chaperone Chz1 and helps in Nucleic Acids Research, 2011, Vol. 39, No. 10 4029 SWR1-mediated exchange of H2A.Z–H2B dimer in vitro (42). We found a loss of H2A.Z levels on SUP4 in the absence of Chz1 (data not shown) and Swr1 (Supplementary Figure S2) suggesting Swr1 and Chz1 are required for H2A.Z deposition at SUP4 locus in vivo. A loss of Spt16/Pob3 (Figure 4D and E) but increase of H2A.Z (Figure 2F) under repression suggest Spt16 may be required for H2A.Z removal from nucleo- somes. Similarly, a loss of H2A.Z in the absence of func- tional Spt16 (Figure 5B) indicates an involvement of Spt16 in the deposition of H2A.Z. Spt16 is known to interact with histone H3. In the absence of Spt16, a rapid loss of H3 is seen on both active and inactive genes (43). This suggests that the observed H2A.Z dynamics on SUP4 could be indirectly due to H3/nucleosome dynamics and may not have any direct correlation with Spt16. However, total H3 as well as H2A.Z levels in mutant and wild type cells did not show any signiﬁcant difference (Supplementary Figure S5A), suggesting lack of Spt16 activity may be directly respon- sible for the lower H2A.Z levels in nucleosmes ﬂanking the SUP4 (Figure 5B). Mutant cells showed a differential be- haviour of H3 in 1 and +1 nucleosomes (Supplementary Figure S5B), again suggesting both the nucleosomes follow different dynamics under repression, and may be targets of different chromatin modiﬁers. Preferential oc- cupancy of FACT in the upstream region and NFR on the transcribed region of SUP4 advocate for requirement of FACT activity only in the 1 nucleosome, which shows enrichment of H2A.Z. While general pol III transcription is repressed within minutes of nutrient deprivation, the SUP4 transcript (Figure 1B) and H2A.Z levels (Figure 5C) show steady increase till 2 h. The increase probably reﬂects the initial cellular response to the stress wherein H2A.Z plays a positive role as discussed later. The H2A.Z shows signiﬁ- cantly higher level in the 1 nucleosome after 2 h repres- sion. Levels drop after 2 h, in parallel to transcription levels but remain at higher than active state in the later stages of repression, suggesting its loss may be necessary for activation. Unlike H3, H2A.Z levels in both nucleo- somes are comparatively lower in Spt16 mutant cells (Figure 5C) but time course of H2A.Z dynamics looks similar in both types of cells. While a slight increase in H3 could be the reason behind the small increase of H2A.Z in the 1 nucleosome in mutant cells (Supplementary Figure S5B) under repression, it could also be due to activity of the H2A.Z speciﬁc chaperone Chz1 (42). These differences in dynamics of the two suggest that H2A.Z does not follow H3 dynamics on SUP4. While Chz1 may be an alternate chaperone, Spt16 is required on SUP4 for H2A.Z deposition during active state and removal in repressed state. Thus, working as H2A.Z chaperone along with Chz1, Spt16 may deposit as well as remove H2A.Z from the SUP4 ﬂanking nucleo- somes. This is not surprising, as most of the histone chap- erones show broad speciﬁcity and often work in redundant manner in vivo (44). Therefore, H2A.Z levels at any point of time may represent the outcome of the removal/deposition by Spt16 and Chz1.