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Supplementary Methods Weake2009 Supplemental Methods 1 Supplemental methods Preparation of soluble nuclear extracts, affinity purification and MudPIT analysis Nuclear extracts were prepared and affinity purifications conducted from 4 L of S2 cells grown to a density of 1 x 107cells/mL in Hyclone SFX media with low/no copper induction. Cells were collected by centrifugation at 5000 rpm 15 min 4ºC and washed in Wash Buffer (10 mM HEPEs [Na+], pH 7.5; 140 mM NaCl). Cells were resuspended in + 40mL of Buffer I (15 mM HEPEs [Na ] pH 7.5; 10 mM KCl, 5 mM MgCl2; 0.1 mM EDTA; 0.5 mM EGTA; 350 mM sucrose; supplemented with 20 g/mL leupeptin, 20 g/mL pepstatin and 100 M PMSF) and disrupted by 40 strokes in a Dounce homogenizer with the loose pestle. Nuclei were collected by centrifugation at 10,400 x g 15 min 4ºC and washed once with 40 mL of Buffer I. The soluble nuclear fraction was isolated by resuspending nuclei in 20 mL of Extraction Buffer (20 mM HEPEs [Na+], pH 7.5; 10% glycerol; 350 mM NaCl; 1 mM MgCl2; 0.1% TritonX-100; supplemented with 20 g/mL leupeptin, 20 g/mL pepstatin and 100 M PMSF) for 1 h 4ºC with rotation, followed by centrifugation to pellet the insoluble chromatin fraction at 18,000 x g 10 min 4ºC. The soluble nuclear extract was cleared by centrifugation at 40,000 rpm 1.5 h 4ºC. Nuclear extracts were diluted to a final salt concentration of 300 mM NaCl and incubated with FLAG-agarose. FLAG-bound proteins were washed 3 times in binding buffer and eluted using 0.5 mg/mL FLAG3 peptide. For tandem FLAG-HA purification, FLAG- eluted proteins were incubated with HA-agarose, washed 3 times in binding buffer and eluted using 0.2 mg/mL HA3 peptide. The relative spectral abundance and identity of proteins present in the tandem affinity-purified complexes was determined using MudPIT (Florens et al. 2006; Florens and Washburn 2006). Proteins that non-specifically bind FLAG-HA beads were identified in control purifications from untagged extract, and from cells expressing the tagged nuclear protein CG6459, and excluded from further analysis. The dNSAF calculation provides a measure of the relative spectral abundance of each polypeptide within a given purification and distinguishes between different protein isoforms based on the identification of unique peptide spectra (Mosley et al. 2009). The cNSAF calculation also compares the relative spectral abundance for a set of polypeptides, although it is used on a defined set of proteins rather than on all polypeptides detected in the MudPIT analysis (Florens et al. 2006; Florens and Washburn 2006). Merged data are shown for Ada1, WDA, SAF6 and TAF6 representing two technical replicates of the MudPIT analysis. Co-immunoprecipitation Nuclear extract was prepared from OregonR or saf6 12-18 h embryos as described above, with minor modifications. Embryos were dechorionated in 50% bleach for 2 min, then homogenized in 1 mL of Buffer I in a 1 mL Dounce homogenizer and nuclear extract prepared as described for S2 cells. Antibodies were pre-bound to sheep anti-rabbit IgG Dynabeads overnight at 4ºC in (20 mM HEPEs [Na+], pH 7.5; 10% glycerol; 150 mM NaCl; 1 mM MgCl2; 0.1% TritonX-100; supplemented with 20 g/mL leupeptin, 20 g/mL pepstatin and 100 M PMSF). 100 g of soluble nuclear extract was incubated with bead-bound antibody overnight at 4ºC in the same buffer. Beads were washed 4 times in binding buffer and bound proteins analyzed by SDS-PAGE and western blotting. 1 Weake2009 Supplemental Methods 2 Co-expression and purification of histone-fold domain proteins Co-expressed HFD-GST or FLAG fusion proteins were purified from E. coli. GST-HFD fusions were only moderately soluble when co-expressed in the absence of another HFD- fusion protein. However, solubility of FLAG-HFD fusions was similar in the presence of GST-HFD fusion proteins or GST alone. Briefly, expression was induced using 0.1 mM IPTG and soluble bacterial lysate prepared using standard methods. Lysate was incubated with glutathione sepharose for 2 h 4ºC in Binding Buffer I (20 mM Tris-HCl, pH 7.5; 250 mM NaCl; 1% TritonX-100) and GST-fusions eluted with glutathione. Eluted fractions were incubated with FLAG-agarose for 2 h 4ºC in Binding Buffer II (20 mM HEPEs + [Na ], pH 7.5; 350 mM NaCl; 1 mM MgCl2; 0.1% TritonX-100), and eluted using 0.5 mg/mL FLAG3 peptide. Purified complexes were analyzed by SDS-PAGE and stained with Imperial™ Coomassie (Pierce). Histone extraction from larvae Histones were acid extracted from second instar larvae using a modified soluble nuclear extract procedure. Both Buffer I and the Extraction Buffer were supplemented with 10 mM sodium butyrate. Briefly, larvae were homogenized in 2 mL of Buffer I and filtered through a single layer of miracloth prior to centrifugation 10,400 x g 15 min 4ºC. Soluble nuclear proteins were extracted by incubation in 1 mL of Extraction Buffer for 1 h at 4ºC with rotation, followed by centrifugation to pellet the insoluble chromatin fraction at 18,000 x g 10 min 4ºC. Acid-soluble material was extracted from the insoluble chromatin fraction by resuspension of the pellet in 0.4 M HCl for 20 min 25ºC with rotation. Acid- insoluble material was pelleted by centrifugation at 425 x g 10 min 4ºC. The supernatant containing the histone proteins was neutralized by the addition of an equimolar volume of NaOH and used directly for SDS-PAGE and western blotting. Genetics The y1 w67c23; P{w+mC y+mDint2=EPgy2}CG3883EY05869 (Bloomington stock number 16681) and w1118; Df(2L)Exel6002, P{w+mC=XP-U}Exel6002/CyO (Bloomington stock number 7489) flies were kindly provided by the Bloomington Drosophila Stock Center at Indiana University. The EY05869 P-element was mobilized as described previously to generate deletions in SAF6 (Guelman et al. 2006). A genomic construct consisting of the adjacent CG3639 gene together with its regulatory regions was generated in pCaSpeR4. A homozygous viable third chromosome insertion of the P{w+mC=[CG3639]} rescue construct was crossed to the saf6303 deletion to generate w; saf6303/CyO, P{w+mC=GAL4-twi.G}2.2, P{UAS-2xEGFP}AH2.2; P{w+mC=[CG3639]}. Homozygous mutant embryos were identified as described previously (Guelman et al. 2006) and populations sorted either using the CoPas Plus (Union Biometrica) or by hand (chromatin immunoprecipitation experiments). The lethality phase of the saf6 mutation was determined by crossing w; saf6303/CyO, P{w+mC=GAL4-twi.G}2.2, P{UAS- 2xEGFP}AH2.2; P{w+mC=[CG3639]} to w1118; Df(2L)Exel6002, P{w+mC=XP- U}Exel6002/ CyO, P{w+mC=GAL4-twi.G}2.2, P{UAS-2xEGFP}AH2.2 and comparing the growth of GFP and non-GFP larvae. 2 Weake2009 Supplemental Methods 3 qRT-PCR RNA was isolated from three independent collections of 100 stage 14-16 embryos of the following genotypes: wda, saf6 and OregonR. RNA was treated with Turbo-DNAse (Ambion) and cDNA generated using Superscript III reverse transcriptase (Invitrogen). qPCR reactions on 10 ng-equivalent of cDNA were conducted using a 7900HT Fast Real-time PCR system (Applied Biosystems) with Perfecta SYBR Fast Mix (Quanta BioSciences). Samples were analyzed relative to OregonR cDNA standards for all primer sets. Candidate SAGA-regulated genes were identified by microarray analysis of gene expression (data not shown). The primer sequences used are listed in Table S4. Chromatin immunoprecipitation Chromatin immunoprecipitation was conducted as described in (Zeitlinger et al. 2007) with minor modifications. 12-18 h OregonR or hand-sorted saf6 embryos were fixed and homogenized in Buffer A1 (15 mM HEPEs [Na+], pH 7.5; 15 mM NaCl; 60 mM KCl; 4 mM MgCl2; 0.5% TritonX-100; 0.5 mM DTT) containing 1.8% formaldehyde for 15 min on ice. Cross-linking was quenched by the addition of 125 mM glycine and the chromatin pellet washed 3 times with Buffer A1, and once with Buffer A2 (15 mM HEPEs [Na+], pH 7.5; 140 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 1% TritonX-100; 0.1% sodium deoxycholate; 0.1% SDS; 0.5% N-lauroylsarcosine; supplemented with 20 g/mL leupeptin, 20 g/mL pepstatin and 100 M PMSF). 0.5 g equivalent embryo mass was sonicated in 1 mL Buffer A2 for 5 x 12 s at 30% power. Chromatin was centrifuged for 10 min 14,000 rpm 4ºC, and 0.2 g equivalent embryo mass of soluble chromatin (400 l) incubated with -Gcn5 or -acetylated Lys-9 H3 (Upstate; rabbit; #06-942) antibody overnight at 4ºC. 50 l of sheep anti-rabbit IgG Dynabeads was added to each chromatin/antibody solution and incubated for 2 h 4ºC, then beads were washed 5 x in RIPA Buffer (50 mM HEPEs [Na+], pH 7.5; 1 mM EDTA; 0.7% sodium deoxycholate; 1% NP-40; 0.5 M LiCl). DNA/protein complexes were eluted in TE Buffer containing 1% SDS and 250 mM NaCl 2 x 30 min at 65ºC. Cross-links were reversed at 65ºC 6 h, DNA was treated with RNAse and proteinase K, and purified by phenol-chloroform extraction and ethanol precipitation. Input and immunoprecipitated (IP) DNA samples were analyzed by qPCR relative to sonicated OregonR input DNA standards for all primer sets. The primer sequences used are listed in Table S4. DPE analysis The 5' UTR of all annotated Drosophila genes were searched for sense occurrences of the DPE motif described in (Kutach and Kadonaga 2000) with IUPAC ambiguity code sequence DSWYVY at position +28 relative to the transcription start site (+1).
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