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Supplemental Materials
and Methods

Immunofluorescence. YBC1093 was grown in 100 ml of YPD to an OD600 of 1.3. Cells were fixed by adding 1 M potassium phosphate [pH 6.5] to a final concentration of 0.1 M and formaldehyde (37% stock) to 4%, followed by incubation for 30 min at 30oC. An equivalent of 10 ml at OD 1.0 of cells were subjected to the following studies. The fixed cells were harvested, washed with 3 ml SP buffer (0.1 M potassium phosphate pH 7.5, 1 M Sorbitol), and resuspended in 1 ml of SP buffer containing 0.2% b-ME and 20 mg/ml Zymolyase 100T (Seikagaku America). The cell suspensions were incubated for 30 min at 35oC with gentle shaking for digesting cell walls. The spheroplasts were then recovered by centrifugation, washed twice with SP buffer and resuspended in 0.5 ml of the same buffer. The following studies were performed at room temperature. 40 ml of fixed spheroplasts were applied to each well of a poly-L-Lysine coated multiwell slide (Polyscience) and incubated for 10 min. The bound cells were washed five times with 0.5% BSA in PBS by aspiration and blocked for 30 min in the same solution. 20 ml of aProtein A antibody (1:100 dilution in 0.5% BSA-PBS) was applied to each well and incubated for 1 h in a humid chamber. After ten times of washes, cells were incubated with 20 ml of FITC-conjugated secondary antibody (Zymed Laboratories Inc.; 1:200 dilution) for 1.5 h in the dark. Cells were washed and mounted with mounting media prepared as described by Pringle et al., and analyzed with a Zeiss Axiophot2 fluorescence microscope. Images were collected with a Hamamatsu digital camera and analyzed using the OpenLab software.

NuA4 Purification and immunoprecipitations. 36 L of YBC925 were grown to OD 4.0 and spun down. Whole cell extract was made by bead beating followed by a PEI precipitation. The extract was then spun in a TI45 rotor at 42,000 rpm for 1 h. Extract containing 9.8 g of protein was diluted to 100 mM NaCl and was flowed over an SP column in buffer A (20 mM Hepes pH7.6, 12% glycerol, 0.02% NP-40, 1.0 mM EDTA, 0.5 mM DTT, 1x protease inhibitors) with 100 mM NaCl. Retained proteins were eluted with a linear gradient of buffer A with 0.1-0.5 M NaCl. Peak fractions containing Yaf9 (ascertained by anti-HA western blots) were pooled, dialyzed into buffer B with 50 mM NaCl, and bound to a DEAE Sephacel column. Bound proteins were eluted with a linear gradient of buffer B (20 mM Tris pH 7.9, 12% glycerol, 0.02% NP-40, 2 mM b-ME, 1´ protease inhibitors) with 0.1 – 1.0 M NaCl, peak fractions containing both Yaf9 and Esa1 (by western blot) were combined and diluted in buffer B to a final concentration of 100 mM NaCl and 20 mM Imidazol. This protein was bound to a Ni2+ NTA agarose column, and eluted with a step gradient of 0.02 - 0.2 M Imidazol. Fractions containing both Yaf9 and Esa1 were combined and loaded onto a Resource Mono-Q column in buffer B with 100 mM NaCl and eluted using a linear gradient from 0.1 – 0.5 M NaCl. Fractions containing both Yaf9 and Esa1 were combined and bound in batch to anti-HA sepharose beads or HA peptide blocked beads. The beads were washed 3´ in buffer C (25 mM Tris pH7.5, 15% glycerol, 0.5% Np-40, 1 mM EDTA, 400 mM NaCl, and 1´ protease inhibitors). Protein was eluted from the beads twice using buffer D with 5 M urea. The beads were then boiled in a 5% SDS solution and the supernatant retained. Eluates were TCA precipitated and run on a 12% acrylamide gel and silver-stained. Bands were excised and subjected to Maldi TOF mass spec. sequencing.

Preparation of extracts and immunoblot analysis. WCE were prepared as described previously {Cairns, 1999 #43}. Samples were separated on SDS 10-20% acrylamide gradient or 7.5% acrylamide gels, transferred to PVDF membranes and immunoblotted following standard procedures. All primary antibodies were used at 1:1000 dilution and secondary (goat anti-mouse or anti-rabbit antibody HRP conjugates (Bio-Rad)) at 1:10,000 dilution.

Assembly studies with the Yaf9 C-terminus. Strains YBC1093 and YBC1740 express Yaf9-TAP and yaf91-186-TAP, respectively, from the endogenous YAF9 promoter. Cultures were grown to an OD600 of 0.8 to 1. Cells were harvested, washed with ddH2O, and resuspended in 1 ml of buffer A (50 mM Tris-HCl pH 7.5, 250 mM KoAc pH7.5, 10% glycerol, 2 mM EDTA, and PIs). Extracts were prepared as described previously and equivalent amount of WCE from each strain were subjected to IgG binding and TEV cleavage. Briefly, WCE (1 mg) was combined with 25 ml of pre-washed IgG beads and incubated for 4 h at 4oC. IgG beads were then washed four times with IgG washing buffer (10 mM Tris-HCl pH 8.0, 200 mM KOAc pH7.5, 10% glycerol, 0.5 mM EDTA, 0.1% NP40, and PIs) and three times with TEV cleavage buffer (10 mM Tris-HCl pH 8, 200 mM KOAc pH7.5, 10% glycerol, 0.5 mM EDTA, 0.1% NP40), separated with 5 min of incubations at 4oC. 4 ml of TEV protease (Invitrogen; 10 units/ml) in 120 ml TEV cleavage buffer was added to the beads followed by incubation for 1 h at 4oC and 1 h at 15oC. The TEV eluate was recovered by centrifugation. Samples were separated on an SDS 12% acrylamide gel followed by immunoblot analysis.

G-ChIP analysis. The purified ChIP samples and the input materials were amplified and labeled with Cy5-dCTP and Cy3-dCTP (Amersham Pharmacia catalog no. PA55321), respectively, and competitively hybridized on glass slides containing segments spanning the entire yeast genome as described in Roberts et al., 2003. Further, normalized ratios were calculated as described by Roberts et al., 2003. Segments identified as occupied by Sir3 are provided as supplemental information.

RT-QPCR analysis.

YBC 1665 (WT) and 1582 (yaf9D) strains were grown in YPD at 30oC to an OD600 of 0.8. Cells were collected and frozen in liquid nitrogen. Total RNA was isolated by acid phenol and chloroform extraction. 45 µg total RNA was incubated with 2 units RNase-free RQ1 DNase (Promega) for 1 hour at 37 degrees Celcius, and the RNA was cleaned up using the Qiagen RNeasy MinElute cleanup kit. Fist-strand synthesis Reverse Transcription was performed using 5 µg total RNA added to Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences) with 0.5 µg oligo (dT)20 primer and incubated at 37 degrees Celcius for 1.5 hours. The RT reaction product from YBC 1665 was diluted 10, 100, and 1000 fold, and product from YBC 1582 was diluted ten fold. Dilutions were used as templates in quantitative PCR reactions using a Bio-Rad iCycler. 5 loci near the telomere of Chromosome 14 and Actin were interrogated for down-regulation of gene expression in the yaf9D compared to WT. Q-PCR reactions were performed with 2 µl template, 1X iQ SYBR Green Supermix (Bio-Rad) and 100nM of each gene specific primer.







Actin was compared between WT and yaf9D strains for each primerset. Dilutions of genomic DNA standards were run for each primerset, and the cycle threshold number for WT and yaf9D RT products were plotted against the concentrations of the standards. Ratio of yaf9D/WT were Log (2) transformed.