4028 Nucleic Acids Research, 2011, Vol. 39, No. 10 shows a change further downstream, showing two pos- itioned nucleosomes with new boundaries (right-side cartoon, Figure 3C; and darker ovals, Figure 3D), in place of a fuzzy +1 nucleosome. Physical and functional interaction of a subunit of yeast FACT, Nhp6 with RSC helps its loading onto the nucleosome in vitro (33). Figure 3E shows that the Nhp6 shows comparatively higher enrichment in the upstream region of SUP4 from where it is lost in rsc4-D4 mutant, suggesting RSC may help recruit Nhp6 on SUP4. Chromatin shows MNase hypersensitivity in the presence of Nhp6 in vitro (Supplementary Figure S3A) as Nhp6 binding changes the conformation of DNA (34). Thus, loss of MNase hypersensitivity of the gene region in the rsc4-D4 mutant (Figure 3D) may be due to loss of Nhp6 as well as the shift of boundary of the +1 nucleosome. To resolve this further, we used the high-resolution MNase footprinting in vivo (Figure 3F). The presence of a strong cut around +160 bp position in the mutant as compared to the wild-type condition, suggests the presence of the common boundary of the two positioned nuclesomes at 50 bp downstream of the terminator. Out of the two, positioning of the gene-proximal nucleosome places its terminator at the dyad axis, though its footprint is not easy to ascertain since the chromatin-speciﬁc cut in the gene region is broad and diffuse. Therefore, the remodelling activity of RSC in the wild type cells must be keeping the gene region nucleosome-free and +1 nucleosome fuzzy, making it encroach the second termin- ator of SUP4, T2 at +108 bp (35) in active state. SUP4 gene in vivo is ﬂanked by a Ty element in upstream and an ORF of unknown gene in the down- stream region. The TSS of both the divergent genes are found two nucleosomes away from the SUP4. We did not ﬁnd either transcript of the downstream gene or effect of repression or RSC mutation on the upstream transcript (data not shown), conﬁrming the observed changes in the gene downstream region are related directly to SUP4 expression. Nhp6 regulates the transcription initiation from the SUP4 chromatin Nhp6 is reported to suppress the downstream initiations from naked SUP4 DNA in vitro (36). In a titration experi- ment (Figure 4A), addition of puriﬁed, recombinant Nhp6A to chromatin also abolishes downstream initi- ations and the right SUP4 transcript increases with increasing Nhp6 (lanes 1–3). However, in contrast to steady increase in naked DNA transcription with addition of upto 300 ng Nhp6 (data not shown), tran- scription from chromatin drops after the saturation levels of Nhp6 at 60 ng (lanes 4–10, Figure 4A). Expecting a similar effect of Nhp6 in vivo, we used an intron-speciﬁc primer which also detects transcripts from two isogenes of SUP4, all differing from each other by 1 or 2 bp in TSS selection (36). As marked in the Figure 4B, 1 represents Figure 4. FACT has inhibitory effects on SUP4 transcription. (A) Effect of Nhp6 on SUP4 transcription in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery in the absence (lane1) and presence (lanes 2–10) of increasing amounts (36, 60, 72, 84, 99, 108, 120, 132 and 144 ng) of pure Nhp6A. Transcript and RM are as given for the Figure 1A. (B) Nhp6 confers the ﬁdelity of transcription in vivo. Total RNA was isolated and used to generate extension products using the intron-speciﬁc primer of SUP4. The three products obtained from the two yeast strains using 5 or 10 mg total RNA are resolved on a gel as described under methods. Arrow marks the major SUP4 transcript initiating from the +1 bp position. Numbers on the right-hand side denote the positions of all the transcripts with respect to +1 site of SUP4, as explained in the text. (C) Relative enrichment of myc-tagged Nhp6A in both active and repressed states. (D) Relative enrichment of myc-tagged Spt16 in active and repressed states. (E) Relative enrichment of TAP-tagged Pob3 in both active and repressed states. (F) Effect of FACT on the chromatin transcription of SUP4 in vitro. Transcription of S-190 assembled chromatin over SUP4 was followed using pure yeast pol III transcription machinery. Pure FACT components in excess were added as indicated.