A. Diagram of the Reb1:dT₇ signal used in this study. Circles indicate nucleosomes. Yellow circle indicates H2A.Z variant nucleosome.
B. Models for relationships between NFR formation and H2A.Z deposition
C. Conditional degron alleles of ABF1, REB1 and STH1 display protein depeletion under degron-inducing conditions. Strains were shifted to YPA media containing 2% galactose for the indicated times and analyzed by immunoblotting with anti-HA or anti-Abf1 antibodies. Ponceau staining of blots demonstrated equal protein loading (Fig. S1)
D. Growth of degron strains under noninducing and inducing conditions. Shown are serial dilutions of strains plated on the indicated media. Plates were photographed after 2 days of incubation.

A. Difference map analysis of effects of Reb1 depletion on nucleosome positions. Map represents nucleosome positioning data from a control strain lacking the Reb1-degron subtracted from a strain with the Reb1-degron. See Experimental Procedures for further details. Nucleosome positioning data 1kb upstream and downstream of the ATG of 150 genes are shown and orientated such that the direction of transcription is to the right. Asterisks indicate center of the +1 nucleosome downstream of the NFR in the control strain. The x-axis represents the distance (in bp) from the center. Data are organized into two clusters using the k-means method.
B. Line traces of average nucleosome positions of the two clusters shown in A The indicated strains were grown under degron-inducing conditions.
C. Scatter plots of the lowest probe signal in NFRs. Points indicate the lowest probe signal in the NFR for a locus in control versus degron strains grown under the indicated conditions.
D. Correlation between Reb1 binding and changes in nucleosomal enrichment at NFRs at promoters. The significance values (log10 p-value) of Reb1 binding at promoters (Harbison, et al. 2004) are compared against the highest fold changes of nucleosome positioning signals at the associated NFR.
E. Correlation between Reb1 consensus sites in promoters and the highest fold change of nucleosome enrichment at the associated NFR under the indicated promoters.
F. Enrichment of Reb1 sites in clusters. Shown are the p-values (hypergeometric testing) of the significance of the indicated Reb1 motifs in the indicated clusters.

A-C. These panels are analogous to those of Figure 3 except that a strain with the Sth1-degron was compared to the same control strain used for analysis of nucleosome positions upon Reb1 depletion.
D. Gene expression analysis. Shown is the correlation between mRNA and NFR changes in cells depleted of Sth1. Ploted are the values for genes on chromosome III. Shown on right is the p-values (hypergeometric testing) of the significance of the enrichments in the indicated gene groups using a 1.5-fold cutoff for decreases in mRNA levels. Expression data were not available for all genes in the clusters; hence, Cluster I N=79, Cluster II N=64.
E. NPS signature averages. Lines traces of NPS predictions (Ioshikes et al., 2006) for genes in the indicated gene clusters are shown in green. These predictions were smoothed using a 51bp moving average window. Experimental nucleosome position averages (control is blue, Sth1-degron is red) are shown as in panel B.
F. Effect of transcription on nucleosome positioning. Shown are the average nucleosome positions for the indicated gene clusters in rpb1-1 strains grown under permissive conditions or for 1 hr under nonpermissive conditions.

A. Clustering analysis of difference maps. Shown are difference maps for the indicated strains including the Abf1-Reb1 “double degron” strain. K-means (K=15) clustering was applied. Line traces of cluster averages are shown in Fig. S14.
B. Scatter plots of double degron. Analysis was performed as in Fig. 3C.
C. Gene expression analysis. Shown is the correlation between mRNA and NFR changes in cells depleted of both Reb1 and Abf1. The p-value (hypergeometric testing) of the significance of the enrichments in the group of genes (N=20) consisting of those affected in NFR size by Abf1-or Reb1-depletion (Cluster I in Fig. 3 and Cluster I in Fig. 4; corresponding points are colored red in this figure) using a 1.5-fold cutoff for decreases in mRNA levels was 1.2×10^-6. The p value for the remaining, unaffected genes (N=123) was 0.99.

Panels A and B are analogous to Fig. 3B and 3C except that data were derived from cells lacking H2A.Z (htz1Δ) or the Swr1 ATPase (swr1Δ).

A. Schematic of H2A.Z intein constructs. An engineered M. tuberculosis RecA intein controlled by the human estrogen receptor ligand binding domain was placed at a chosen site in HTZ1 gene (encoding H2A.Z). The HTZ1 promoter was replaced with the galactose-inducible pGAL1 promoter. Protein splicing would occur in the presence of 4-hydroxytamoxifen (4-HT) and leave a cysteine residue (“scar”) at the splicing junction.
B. Splicing of the H2A.Z intein construct occurs with an allele that replaces Ala46 with the intein construct. The 4-HT-regulated intein was inserted in place of four different residues in H2A.Z, and these constructs each were placed on a 2μ plasmid under the control of a pGAL1 promoter and transformed into a htz1Δ strain. Strains were grown to mid-log phase in the presence of 2% galactose and, when indicated, 10 μM 4-HT. Shown is a Western using polyclonal antibody specific to the C-terminus of H2A.Z. A strain with a chromosomal-based, wild-type copy of HTZ1 and a htz1Δ strain are included as controls.
C-F: Analysis of H2A.Z deposition requirements. A strain carrying the Sth1 degron was shifted to degron-inducing conditions which also induces synthesis of unspliced H2A.Z intein construct. After 5 hrs, 4-HT was added to 10μM to induce splicing. Cells were collected after 3 hrs of further incubation, and H2A.Z enrichment at select loci was determined relative to histone H3 enrichment and normalized to a locus in the middle of the large BUD3 ORF. H2A.Z/H3 enrichment profiles under Sth1-depleted conditions were compared against control profiles. The promoters of DCC1/BUD3 are analyzed in C; the PRM1 ORF containing the Reb1:dT₇ insertion is analyzed in D; the promoter of YCR016W is analyzed in E, and the promoter of YCRO23C is analyzed in F. Top panels of C-F indicate nucleosomal DNA enrichment in various conditions (blue: degron-OFF conditions, red: 5 hrs of degron-ON, green: 8 hrs of degron-ON, with the last 3 hrs in the presence of 4-HT). Bottom panels of C-F represent normalized H2A.Z/H3 enrichment values at the indicated amplicons.

A. Analysis of the effect of Reb1:dT₇ sequence insertion on nucleosome positioning. Shown are line traces of a moving average of mononucleosome/genomic probe signals across the PRM1 gene with and without the indicated sequence insertion. Triangles indicate the insertion site.
B. Analysis of effects of Reb1 and Sth1 depletion on NFR formation mediated by Reb1:dT₇. Indicated strains containing the Reb1:dT₇ insertion in the PRM1 gene were analyzed as described in A.
C. Analysis of effects of htz1Δ and swr1Δ mutations on NFR formation mediated by Reb1:dT₇. Indicated strains containing the Reb1:dT₇ insertion in the PRM1 gene were analyzed as described in A.

A-F. These panels are analogous to those of Figure 3 except that a strain with the Abf1-degron was compared to the same control strain used for analysis of nucleosome positions upon Reb1 depletion.