4030 Nucleic Acids Research, 2011, Vol. 39, No. 10 Figure 5. FACT is a H2A.Z chaperone. (A) In vivo analysis of SUP4 expression in mutants of Spt16 and Pob3. Total RNA was isolated and intron-speciﬁc primer extension products of SUP4 were visualized by phosphorImaging of the gel as described under methods. RNA levels were normalized against U4 and average from three independent experiments with scatter are shown. (B) Relative enrichment of HA-tagged H2A.Z in the absence of functional Spt16 in active condition. Comparative levels of H2A.Z are low in both the nucleosomes in Spt16 mutant cell. (C) HA-H2A.Z levels around the SUP4 gene in wild type cells change with time in parrallel to transcriptional repression under nutrient starvation. (D) Co-immunoprecipitation of H2A.Z and Spt16 in vivo and in vitro. Mock was as described under Supplementary Data. Input lanes are loaded with one-third (in vivo) or one-fourth (in vitro) of the sample as compared to IP (immunoprecipitate) lanes. Spt16 was at increasing amounts in IP1, 2 and 3. Upper panel: In vivo, H2A.Z-HA is immunoprecipitated with anti-HA antibody and IP is probed with anti-Spt16 antibody. Lanes 3 and 4 are the duplicates of the immunoprecipitation (IP). Lower panel: (i) and (ii) show in vitro pull-downs, using puriﬁed Spt16/Pob3 or 6XHis-tagged H2A.Z; probed with anti-H2A.Z or anti-Spt16 antibodies, respectively. Control shows the immobilized bait protein alone on beads while mock lanes show incubation of the prey with bare beads. (E) Relative enrichment of HA-tagged H2A.Z in rsc4-D4 mutant in active condition. (F) Relative enrichment of H2A.Z-HA on tDNA loci present on three different chromosomes. Higher RNA and lower H2A.Z levels in Spt16 mutant and higher H2A.Z but lower Spt16 in repressed wild-type cells suggest a close relation between H2A.Z dynamics, FACT and transcription of SUP4. Unlike SUP4, H2A.Z on CMD1 and U4, two Spt16-independent genes transcribed by pol II (6, Supplementary Figure S4) shows lower levels under active as well as repressed states in mutant cells (Supplementary Figure S5C). Therefore, Spt16 is required for the deposition of H2A.Z in active state although the transcription of these genes does not require Spt16. As Spt16 is a known chaperone for other H2A variants, loss of the variant H2A.Z from the genes in the mutant cells reﬂects a direct outcome of defect in H2A.Z deposition by Spt16 in active state. FACT is a chaperone of H2A.Z for pol III-transcribed genes A chaperone role would require direct interaction between H2A.Z and Spt16. Using yeast cells carrying HA-tagged H2A.Z, we found that Spt16 co-immunoprecipitates with H2A.Z in vivo (Figure 5D, upper panel), while an N-terminal mutant of Spt16 could not pull down H2A.Z (Supplementary Figure S6A), re-enforcing the possibility of Spt16 working as H2A.Z chaperone in vivo. For con- ﬁrming direct interaction of the two in vitro, we puriﬁed TAP-tagged Spt16-Pob3 heterodimer from yeast cells and 6XHis-tagged recombinant yeast H2A.Z from E. coli cells (Supplementary Figure S6B). We used the tagged proteins immobilized on Calmodulin sepharose or Ni-NTA agarose beads, respectively, and added the other partner as pure protein (Figure 5D, lower panel). We found that both Spt16 and H2A.Z can efﬁciently pull-down each other, demonstrating a direct physical interaction between the two. As Nhp6/FACT are enriched speciﬁcally in the upstream region (Figure 4C and D), H2A.Z levels also show larger changes only in the 1 nucleosome (Figures 2F and 5D). All these results support a H2A.Z chaperone role for FACT on SUP4 locus. In agreement with Nhp6 requirement for Spt16/Pob3 binding to nucleo- some (45) and loss of Nhp6 in rsc4-D4 mutant; H2A.Z is speciﬁcally lost from the 1 nucleosome in this mutant (Figure 5E), suggesting Nhp6/FACT may be a link between RSC and H2A.Z. Nucleosomes ﬂanking NFRs containing tDNA sequences (9) are reported to have H2A.Z and recently human FACT was shown to associate with some pol III-transcribed genes (46). We monitored H2A.Z levels in the absence of functional Spt16 on some more tRNA genes (Figure 5F). Similar to SUP4, H2A.Z levels in the mutant cells show signiﬁcant loss on all these genes, suggesting FACT may have a general role in H2A.Z dynamics on pol III-transcribed genes.