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). Identified DPE-containing genes matching 6/6 positions in the motif that were identified in this analysis were cross-referenced with SAGA-regulated genes identified in microarray analysis of gene expression in nonstop, sgf11 and ada2b mutant third instar larvae relative to OregonR (wild-type) (Weake et al. 2008). SAGA-regulated genes are identified as having log2 expression ratios <-1 (p<0.05) in the any of the 3 mutant genotypes relative to the wild-type. The cDNA arrays analyzed contain 13664 unique genes.
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Supplemental references Florens, L., Carozza, M.J., Swanson, S.K., Fournier, M., Coleman, M.K., Workman, J.L., and Washburn, M.P. 2006. Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors. Methods (San Diego, Calif 40(4): 303-311. Florens, L. and Washburn, M.P. 2006. Proteomic analysis by multidimensional protein identification technology. Methods in molecular biology (Clifton, NJ 328: 159- 175. Guelman, S., Suganuma, T., Florens, L., Weake, V., Swanson, S.K., Washburn, M.P., Abmayr, S.M., and Workman, J.L. 2006. The essential gene wda encodes a WD40 repeat subunit of Drosophila SAGA required for histone H3 acetylation. Mol Cell Biol 26(19): 7178-7189. Kutach, A.K. and Kadonaga, J.T. 2000. The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters. Mol Cell Biol 20(13): 4754-4764. Mosley, A.L., Florens, L., Wen, Z., and Washburn, M.P. 2009. A label free quantitative proteomic analysis of the Saccharomyces cerevisiae nucleus. Journal of proteomics 72(1): 110-120. Weake, V.M., Lee, K.K., Guelman, S., Lin, C.H., Seidel, C., Abmayr, S.M., and Workman, J.L. 2008. SAGA-mediated H2B deubiquitination controls the development of neuronal connectivity in the Drosophila visual system. The EMBO journal 27(2): 394-405. Zeitlinger, J., Zinzen, R.P., Stark, A., Kellis, M., Zhang, H., Young, R.A., and Levine, M. 2007. Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo. Genes & development 21(4): 385-390.
4 Weake2009 Supplemental Figure and Table legends 1
Supplemental Figure and Table legends
Figure S1. (A) Multiple sequence alignment of the histone-fold domain of SAF6 and its closest homologs, TAF6 and TAF6L from various eukaryotes was performed using the MACAW program (Schuler et al. 1991). GenBank ID numbers are shown for each protein, followed by the amino acid positions of the aligned region within each protein. Yellow shading indicates conserved bulky hydrophobic residues (I, F, L, M, V, Y or W), red type indicates conserved negatively charged residues (D, E, N or Q), blue type indicates conserved positively charged residues (K or R), green type indicates conserved hydroxylic residues (S or T), and gray-shaded purple type indicates conserved small or kinky side chains (A, G, S or P). The -helical and loop regions assigned from the X-ray structure of TAF6 within the heterotetramer complex are indicated above the alignment (Xie et al. 1996). Asterisks in the heterodimer interactions line indicate residues in TAF6 that make contacts between the TAF6 and TAF9 heterodimer (Xie et al. 1996). Pound signs in the heterotetramer interaction line indicate residues that are involved in contacts between distinct TAF6/TAF9 heterodimers (Xie et al. 1996). Underlined asterisks and pound signs indicate residues conserved between SAF6 and D. melanogaster TAF6. Species abbreviations: Dm, Drosophila melanogaster; Ag, Anopheles gambiae; Os, Oryza sativa; At, Arabidopsis thaliana; Hs, Homo sapiens; Rn, Rattus norvegicus; Mm, Mus musculus; Cf, Canis familiaris; Bt, Bos taurus; Dr, Danio rerio; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Asg, Ashbya gossypii; Nc, Neurospora crassa; Mg, Magnaporthe grisea; Kl, Kluyveromyces lactis; Pt, Pan troglodytes.
Figure S2. (A) Schematic representation of the SAF6 locus, showing the position of the EY05869 P-transposon and the saf6303 deletion. The five exons are represented by boxes. Translated sequences are filled with gray, and 5' and 3' untranslated regions are open boxes. The adjacent CG3639 gene is shown in black and a schematic representation of the CG3639 genomic rescue construct is shown (pCasper4-CG3639+). (B) RT-PCR from OregonR (lanes 2 and 5), saf6 (saf6303; CG3639+) heterozygous (lanes 3 and 6) or homozygous mutant (lanes 4 and 7) first instar larvae with primers specific for SAF6, CG3639 or RpL32. Lanes 5 - 7 correspond to the minus reverse transcriptase (RT) control. (C) Developmental profile of SAF6 expression. RT-PCR was conducted using primers specific for SAF6 (upper panel) or RpL32 (middle and lower panel) on cDNA isolated from OregonR embryos 0 h (E0) and 12h (E12) after hatching, first instar larvae (L1), second instar larvae (L2), third instar larvae (L3), pupae (P), male (M) and female (F) adults. The negative PCR control (final lane) and no reverse transcriptase control (lower panel) are shown.
Figure S3. Chromatin immunoprecipitation was conducted using -acetylated H3 Lys-9 (A) or -Gcn5 (B) antibodies from 12-18 h OregonR or saf6 embryos. Enrichments are shown as immunoprecipitated (IP)/input material for the SAGA-regulated genes Crc, exba and Oda normalized to a non-SAGA regulated gene, Or82a. Mean results are shown for two separate biological experiments.
Figure S4. SAGA was immunoprecipitated from nuclear extract prepared from OregonR or saf6 mutant embryos using antibodies against Gcn5, Ada3 or non-specific rabbit IgG.
1 Weake2009 Supplemental Figure and Table legends 2
Co-immunoprecipitated proteins were analyzed by SDS-PAGE and western blotting using antibodies against Gcn5 and Ada2b.
Table S1. List of SAGA subunits in Drosophila melanogaster and their corresponding subunits in the Saccharomyces cerevisiae and Homo sapiens SAGA complexes. Drosophila subunits identified in this study are indicated in bold type. The list of human and yeast proteins in the complex is based on recently published information (Nagy and Tora 2007; Nagy et al. 2009).
Table S2. List of SAGA, TFIID and a subset of ATAC proteins detected by MudPIT in affinity purifications. Proteins are listed by their locus ID (GeneBank accession number) as well as by their CG number, gene name as described in FlyBase and protein name used in this study. FLAG-HA baits are listed horizontally in bold, with additional columns included for the mock purification (Control-1) and non-specific nuclear protein CG6459 purification (Control-2). The length in amino acids and molecular mass (kDa) for each protein are indicated in the last two columns. The number of peptides (spectral count; dS), sequence coverage (SC), and distributive normalized spectral abundance factor (dNSAF) observed for each protein in a given affinity purification of a particular bait are listed. Complex-specific spectral abundance factor (cNSAF) calculated for SAGA subunits and TAF6 are shown for the affinity purifications of the SAGA subunits SAF6, Ada2B, WDA, Ada1 and Sgf29. Spectral counts, coverage and dNSAF/cNSAF values are highlighted in bold for the bait protein in each affinity purification. Only SAGA, TFIID and 3 ATAC-specific proteins are listed in this table, and proteins belonging to each complex are indicated by vertical lines to the right of each table. The table is split vertically, with the first 4 baits in the upper portion of the table and the second 4 baits (including the two controls) in the lower portion of the table.
Table S3. Percentage of SAGA-regulated genes containing DPE-motifs relative to the whole genome. Criteria used for identification of DPE-motifs and SAGA-regulated genes are described in the supplemental methods.
Table S4. Primers used for qRT-PCR analysis of gene expression and chromatin immunoprecipitation qPCR analysis.
Supplemental references Nagy, Z., Riss, A., Romier, C., le Guezennec, X., Dongre, A.R., Orpinell, M., Han, J., Stunnenberg, H., and Tora, L. 2009. The human SPT20-containing SAGA complex plays a direct role in the regulation of endoplasmic reticulum stress- induced genes. Mol Cell Biol 29(6): 1649-1660. Nagy, Z. and Tora, L. 2007. Distinct GCN5/PCAF-containing complexes function as co- activators and are involved in transcription factor and global histone acetylation. Oncogene 26(37): 5341-5357. Schuler, G.D., Altschul, S.F., and Lipman, D.J. 1991. A workbench for multiple alignment construction and analysis. Proteins 9(3): 180-190.
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Xie, X., Kokubo, T., Cohen, S.L., Mirza, U.A., Hoffmann, A., Chait, B.T., Roeder, R.G., Nakatani, Y., and Burley, S.K. 1996. Structural similarity between TAFs and the heterotetrameric core of the histone octamer. Nature 380(6572): 316-322.
3 α1 L1 α2 L2 α3 Weake_FigS1
Heterodimer interactions TAF6/TAF9 ** ** ** * * *** ** ** * *** ** **** ** * Heterotetramer interactions TAF6/TAF9 # ## # # ## CG3883/SAF6 Dm_19920480 128 : FDPRSIKIFWEQHDQtdvELSQDVCARLAEDASYKVWELI-NNVKIYSRHSGGVVTYDLVNEVLKDADVPPMLGAMDSDWDRIDYDGSFy------FHSDKIFELSAEF : 233 Ag_58389686 6 : LSSRSVRTAWEQHDKqetEVAPDVYTKLAEDATYKLWELT-NSIKTYSRHSSGRVTFDLVNEVLKDSDVPPIVGANSQPWDKIEYDGAYf------FNSDEVLDLREEY : 111 SAF6
TAF6 Dm_24666846 21 : ISAESMKVIAESIGVG--SLSDDAAKELAEDVSIKLKRIVQDAAKFMNHAKRQKLSVRDIDMSLKVRNVEPQYGFVAKDFIPFRFASGGgrelh------FTEDKEIDLGEIT : 129 Os_115436920 4 : VPKETIEVIGQSVGIa--NLPADVSAALAPDVEYRLREIMQEAIKCMRHAKRTVLTADDVDSALSLRNVEPVYGFASGDPLRFKRAVGHkdlf------YIDDREVDFKEII : 111 TAFII59 At_15220358 4 : VPKETVEVIAQSIGIt--NLLPEAALMLAPDVEYRVREIMQEAIKCMRHSKRTTLTASDVDGALNLRNVEPIYGFASGGPFRFRKAIGHrdlf------YTDDREVDFKDVI : 111 TAF6 Hs_21536355 14 : LPSESMKVVAESMGIA--QIQEETCQLLTDEVSYRIKEIAQDALKFMHMGKRQKLTTSDIDYALKLKNVEPLYGFHAQEFIPFRFASGGgrely------FYEEKEVDLSDII : 122 TAF6 Rn_34871482 14 : LPSESMKVVAESMGIA--QIQEETCQLLTDEVSYRIKEIAQDALKFMHMGKRQKLTTSDIDYALKLKNVEPLYGFHAQEFIPFRFASGGgrely------FYEEKEVDLSDII : 122 TAF6 Mm_6678215 14 : LPSESMKVVAESMGIA--QIQEETCQLLTDEVSYRIKEIAQDALKFMHMGKRQKLTTSDIDYALKLKNVEPLYGFHAQEFIPFRFASGGgrely------FYEEKEVDLSDII : 122 TAF6 Cf_73957898 14 : LPSESMKVVAESMGIA--QIQEETCQLLTDEVSYRIKEIAQDALKFMHMGKRQKLTTSDIDYALKLKNVEPLYGFHAQEFIPFRFASGGgrely------FYEEKEVDLSDII : 122 TAF6 Bt_62752037 14 : LPSESMKVVAESMGIA--QIQEETCQLLTDEVSYRIKEIAQDALKFMHMGKRQKLTTSDIDYALKLKNVEPLYGFHAQEFIPFRFASGGgrely------FYEEKEVDLSDII : 122 TAF6 TAF6 Dr_52218960 14 : LPTDSMKAMAESVGIG--SLQEDSCLTLSEEVSYRIKEIAQDALKFMHHGKRCKLTTGDIDHALKLKNVEPLYGFQSEEFIPFRFASGGgrelh------FYEEKEVDLSDII : 122 TAF6 Ag_58384288 6 : LSLESIKVIAESIGVG--SLPDDAAKELADDVSYKLKQIVQDAVKFMHHSKRMKLSIADVDHSLKVRNIEPQYGFVARDFIPFRFASGGgrelh------FIEEKEVDLTEMV : 114 TAF6 Sc_6321326 12 : SPQDTVKDVAESLGLE--NINDDVLKALAMDVEYRILEIIEQAVKFKRHSKRDVLTTDDVSKALRVLNVEPLYGYYDGSEVNKAVSFSKvntsggqsvy------YLDEEEVDFDRLI : 125 TAF6 Sp_19075428 6 : WNIESIKDVAEMLGIG--NLADEPAAAIAMDLEYRIHQVVQEATKFMVHSKRTVLTSADISSALRTLNVEPLYGFNNSRPLEFHEAAVGagqnsly------YLDDEEVDFEKII : 116 TAF6 Asg_45198499 17 : SPQDTVRDVADSLGIN--NVNDDVLRSLAMDVEYRILEIIEQAVKFKRHSKRDVLTTDDIARALRVLNVEPLYGYEDNSTRDKEVSYSKvtgqggqtmy------YVNDEEVDFDKLI : 130 TAF6 Nc_32415970 10 : WNPENVKDVAESIGI---NLTEEPLRVLTQDVEYRIGQVVVEALRFMRAANRTTLTVQDVSQALRVLDVEPLYGYDSTRPLRYGEASIGpgqplf------YIEDEEVDFEKVI : 118 TAF6 Mg_39940726 33 : WNTENVKDVAESVGIP--NINDDAVRCLAQDVEYRIGQVLVEALRFMRAANRTTLTVQDTSQAFKVLDVEPLYGYDSTRPLRYGEASLGpgqplf------YIDDEEVDFEKLI : 142 TAF6 Kl_50311083 15 : SPHDTVKDVADSLGIS--NTNDDVLRSLAMDVEYRILEIVEQAVKFKRHSKRSTLTTSDVAKALRVLNVEPLYGFEEGSAKNEPVKFNKlegangqtly------YLDDEEIDFEKLI : 128
TAF6L Hs_5453844 12 : IPRESVRLMAESTGL---ELSDEVAALLAEDVCYRLREATQNSSQFMKHTKRRKLTVEDFNRALRWSSVEAVCGYGSQEALPMRPAREGely------FPEDREVNLVELA : 117 TAF6L Pt_114638058 114 : IPRESVRLMAESTGL---ELSDEVAALLAEDVCYRLREATQNSSQFMKHTKRRKLTVEDFNRALRWSSVEAVCGYGSQEALPMRPAREGely------FPEDREVNLVELA : 219 TAF6L Rn_34861560 19 : IPRESVRLMAESTGL---ELSDEVAALLAEDVCYRLREATQNSSQFMKHTKRRKLTVEDFNRALRWSSVEAVCGYGSQEVLPLRPAREGELY------FPEDREVNLVELA : 124 TAF6L Mm_22122597 12 : IPRESVRLMAESTGL---ELSDEVAALLAEDVCYRLREATQNSSQFMKHTKRRKLTVEDFNRALRWSSVEAVCGYGSQEALPLRPAREGDLY------FPEDREVSLVELA : 117 TAF6L TAF6L Cf_73983375 19 : IPRESVRLMAESTGL---ELSDEVAALLAEDVCYRLREATQNSSQFMKHTKRRKLTVEDFNRALRWSSVEAVCGYGSQEVLPLRPAREGely------FPEDREVNLVELA : 124 TAF6L Bt_76657737 12 : IPRESVRLMAESTGL---ELSDEVAALLAEDVCYRLREATQNSSQFMKHTKRRKLTVEDFNRALRWSSVEAVCGYGSQEVLPLRPAREGely------FPEDREVNLVELA : 117 TAF6L Dr_68362968 12 : VPRESVKLMAESAGV---ELSEELAGLLAEDVCYRLREATQNSSQFMRHAKRRKLSVEDFNRALRWSNTETVCGYGAQDALPFRPLKEGELF------YVEDREINLVELA : 117 TAF6L/Mia Dm_45446367 32 : SPRSLSAIFFHSTGL---HLDDGVAQWLSINVKEDITNLLNEAGKYMRRIRDRRLQLSHIQHAVRMHDDLCYDIFFRLVHCDDCKMPPSqkvlktvreavtaekkdellvs-YPESVQESVPEPV : 156 Weake_FigS2 A EY05869 -43 CG3639 SAF6
-1145 -252 -121 +1 +2519 saf6∆303 -288 +15
CG3639 pCasper4-CG3639+ -1517 +116 [CG3639+]
B C WT saf6/CyO 1,000bp saf6/saf6 WT saf6/CyO 500bp saf6/saf6 SAF6 SAF6
1,000bp RpL32
500bp CG3639 -RT
1,000bp E0 E12 L1 L2 L3 P M F -
500bp RpL32
1 2 3 4 5 6 7 Lane + + + - - - RT Weake_FigS3 A B
H3K9ac ChIP Gcn5 ChIP
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E 1.00 E 0.00 0.00 Crc exba Oda Crc exba Oda Primer set Primer set
OregonR saf6 OregonR saf6 Weake_FigS4 OregonR saf6/saf6 IP IP input Gcn5 input Gcn5 Ada3 Ada3 IgG IgG α-Gcn5
α-Ada2b Weake_TableS1 Drosophila melanogaster Saccharomyces cerevisiae Homo sapiens CG no. Gene name Protein CG4107 Pcaf Gcn5 Gcn5 Gcn5/PCAF CG9638 Ada2b (isoform B) Ada2B Ada2 ADA2B CG7098 diskette Ada3 Ada3 ADA3 CG30390 Sgf29 Sgf29 Sgf29 SGF29 CG6506 Spt7 Spt7 Spt7 SPT7L CG31866 Ada1-2 Ada1 Ada1 ADA1 CG3169 Spt3 Spt3 Spt3 SPT3 X X X Spt8 X CG17689 Spt20 Spt20 Ada5/Spt20 p38IP CG33554 Nipped-A (isoform D) Tra1 Tra1 TRRAP CG6474 enhancer of yellow 1 TAF9 Taf9 TAF9 CG3069 TBP-associated factor 10b TAF10b Taf10 TAF10 CG17358 TBP-associated factor 12 TAF12 Taf12 TAF12 CG4448 will decrease acetylation WDA X X X X X Taf5 TAF5L CG3883 saf6 SAF6 Taf6 TAF6L CG4166 non-stop Nonstop Ubp8 USP22 CG13379 Sgf11 Sgf11 Sgf11 ATXN7L3 CG15191 enhancer of yellow 2 E(y)2 Sus1 ENY2 CG9866 CG9866 CG9866 Sgf73 ATXN7L3 X X X X SAP130 Bait: tandem FLAG-HA purification from S2 cell nuclear extract Mol. GeneBank CG no. Gene name Protein SAF6 Ada2B WDA Ada1 Length Mass. accession no. dS SC dNSAF cNSAF dS SC dNSAF cNSAF dS SC dNSAF cNSAF dS SC dNSAF cNSAF (kDa) Weake_TableS2 gi|24663348 CG4107 Pcaf Gcn5 141 26.1 0.00502 0.0336 346 53.5 0.02315 0.0916 123 40.5 0.00148 0.0099 78 33.3 0.00192 0.0195 813 92.2 gi|78706696 CG9638 Ada2b (isoform B) Ada2B 199 32.1 0.01039 0.0694 472 61.6 0.04627 0.1831 84 34.8 0.00148 0.0099 36 37.3 0.00130 0.0132 555 62.0 gi|17647341 CG7098 diskette ADA3 116 20.3 0.00604 0.0404 280 46.6 0.02740 0.1084 65 31.7 0.00114 0.0077 32 52.7 0.00115 0.0117 556 59.9 gi|24656885 CG30390 Sgf29 Sgf29 121 53.3 0.01213 0.0810 110 56.1 0.02071 0.0819 51 59.5 0.00173 0.0116 35 30.5 0.00243 0.0246 289 32.1 gi|85726390 CG6506 Spt7 Spt7 269 39.0 0.02171 0.1450 153 41.5 0.02319 0.0917 477 45.7 0.01300 0.0873 98 32.6 0.00547 0.0554 359 39.5 gi|24583773 CG31866 Ada1-2 ADA1 113 26.3 0.01063 0.0710 118 38.6 0.02084 0.0825 324 49.4 0.01029 0.0692 1161 54.6 0.07550 0.7656 308 35.4 gi|21356843 CG3169 Spt3 Spt3 116 12.0 0.00875 0.0585 61 28.9 0.00864 0.0342 269 42.2 0.00685 0.0461 32 27.6 0.00167 0.0169 384 43.5 SAGA gi|85726476 CG17689 Spt20 Spt20 337 19.8 0.00521 0.0348 173 18.4 0.00503 0.0199 70 18.7 0.00037 0.0025 44 7.7 0.00047 0.0048 1873 201.0 gi|161076330 CG33554 Nipped-A (isoform D) Tra1 676 25.1 0.00517 0.0345 89 14.7 0.00128 0.0051 190 20.6 0.00049 0.0033 113 14.1 0.00060 0.0061 3790 435.4 gi|17647743 CG4166 non-stop Nonstop 45 11.6 0.00177 0.0118 2 1.5 0.00015 0.0006 13 10.3 0.00017 0.0012 11 8.4 0.00030 0.0030 735 82.5 gi|24666394 CG13379 Sgf11 Sgf11 19 20.6 0.00247 0.0165 4 15.3 0.00098 0.0039 12 26.0 0.00053 0.0035 9 13.0 0.00081 0.0082 223 24.3 gi|17864496 CG15191 enhancer of yellow 2 E(y)2 57 34.7 0.01635 0.1092 8 34.7 0.00431 0.0171 12 34.7 0.00116 0.0078 X X X 0.0000 101 11.5 gi|24581122 CG9866 CG9866 47 16.6 0.00140 0.0094 40 14.5 0.00224 0.0089 32 15.0 0.00032 0.0022 18 10.9 0.00037 0.0038 971 104.2 gi|24649265 CG4448 will decrease acetylation WDA 331 36.7 0.01291 0.0862 207 55.7 0.01516 0.0600 5341 73.0 0.07031 0.4726 69 31.9 0.00186 0.0189 743 83.7 gi|19920480 CG3883 Saga factor like TAF6 SAF6 255 27.2 0.01030 0.0688 148 48.5 0.01123 0.0444 849 50.6 0.01158 0.0778 63 27.5 0.00176 0.0178 717 79.3 gi|45549141 CG6474 enhancer of yellow 1 TAF9 140 39.9 0.01459 0.0975 79 33.5 0.01546 0.0612 403 67.3 0.01418 0.0953 21 25.9 0.00151 0.0153 278 29.3 gi|17137648 CG3069 TBP-associated factor 10b TAF10b 16 22.6 0.00317 0.0212 53 51.4 0.01975 0.0782 141 66.4 0.00945 0.0635 6 41.1 0.00082 0.0083 146 15.8 gi|24646044 CG17358 TBP-associated factor 12 TAF12 11 23.0 0.00163 0.0109 25 23.5 0.00694 0.0275 77 38.8 0.00384 0.0258 6 25.5 0.00061 0.0062 196 21.5 gi|28571540 CG17603 TBP-associated factor 1 TAF1 X X X 4 2.0 0.00011 15 7.2 0.00007 2 0.7 0.00002 2065 232.2 gi|24662032 CG6711 TBP-associated factor 2 TAF2 X X X 5 4.2 0.00022 43 17.4 0.00034 2 2.8 0.00003 1221 139.5 gi|24638625 CG2009 bip2 TAF3 X X X 4 2.99 0.00015 3 2.49 0.00002 X X X 1406 154.5 gi|24665038 CG5444 TBP-associated factor 4 TAF4 X X X 1 3.4 0.00006 1 3.4 0.00001 X X X 921 99.3 gi|17136870 CG7704 TBP-associated factor 5 TAF5 X X X 5 4.6 0.00039 7 9.5 0.00010 X X X 704 79.3 gi|24666846 CG32211 TBP-associated factor 6 TAF6 1 2.2 0.00005 0.0003 X X X X 26 35.5 0.00042 0.0028 2 6.9 0.00007 0.0007 606 65.7 gi|21358419 CG2670 TBP-associated factor 7 TAF7 X X X X X X 10 14.8 0.00020 X X X 479 55.1 TFIID gi|17647803 CG7128 TBP-associated factor 8 TAF8 X X X X X X 6 7.3 0.00018 X X X 328 36.6 gi|17137724 CG2859 TBP-associated factor 10 TAF10 X X X 3 8.4 0.00098 X X X X X X 167 18.5 gi|24583119 CG4079 TBP-associated factor 11 TAF11 X X X X X X 1 6.6 0.00005 X X X 196 22.1 gi|19921578 CG10756 TBP-associated factor 13 TAF13 X X X X X X X X X 1 8.1 0.00015 136 15.7 gi|17647997 CG9874 TATA binding protein TBP X X X 6 5.4 0.00092 25 15.3 0.00069 X X X 353 38.5 gi|17136834 CG7562 TBP-related factor TRF X X X X X X 6 21.4 0.00026 3 13.8 0.00027 224 25.5 gi|45554500 CG18009 TBP-related factor 2 TRF2 X X X X X X 3 5.1 0.00005 X X X 632 68.5
gi|20129303 CG9200 ATAC component 1 Atac1 X X X X X X X X X X X X 356 41.0 gi|20129591 CG10414 ATAC component 2 Atac2 X X X X X X X X X X X X 774 89.2 ATAC gi|21356259 CG10238 CG10238 X X X X X X X X X X X X 367 41.9
Bait: tandem FLAG-HA purification from S2 cell nuclear extract Mol. GeneBank CG no. Gene name Protein Sgf29 TAF6 Control-1 Control-2 Length Mass. accession no. dS SC dNSAF cNSAF dS SC dNSAF dS SC dNSAF dS SC dNSAF (kDa) gi|24663348 CG4107 Pcaf Gcn5 153 42.8 0.00473 0.0237 1 1.9 0.00001 X X X X X X 813 92.2 gi|78706696 CG9638 Ada2b (isoform B) Ada2B 127 55.9 0.00573 0.0287 3 3.2 0.00005 X X X X X X 555 62.0 gi|17647341 CG7098 diskette ADA3 256 68.2 0.01158 0.0580 7 11.5 0.00011 X X X X X X 556 59.9 gi|24656885 CG30390 Sgf29 Sgf29 1679 74.7 0.14608 0.7319 X X X X X X X X X 289 32.1 gi|85726390 CG6506 Spt7 Spt7 82 33.4 0.00574 0.0288 5 14.8 0.00012 X X X X X X 359 39.5 gi|24583773 CG31866 Ada1-2 ADA1 42 37.7 0.00343 0.0172 3 13.6 0.00008 X X X X X X 308 35.4 gi|21356843 CG3169 Spt3 Spt3 35 36.7 0.00229 0.0115 5 7.3 0.00011 X X X X X X 384 43.5 SAGA gi|85726476 CG17689 Spt20 Spt20 102 21.0 0.00137 0.0069 X X X X X X X X X 1873 201.0 gi|161076330 CG33554 Nipped-A (isoform D) Tra1 332 34.7 0.00220 0.0110 2 0.3 0.00000 3 0.4 0.00004 X X X 3790 435.4 gi|17647743 CG4166 non-stop Nonstop 26 22.3 0.00089 0.0045 X X X X X X X X X 735 82.5 gi|24666394 CG13379 Sgf11 Sgf11 8 40.8 0.00090 0.0045 X X X X X X X X X 223 24.3 gi|17864496 CG15191 enhancer of yellow 2 E(y)2 11 17.8 0.00274 0.0137 2 16.8 0.00017 X X X X X X 101 11.5 gi|24581122 CG9866 CG9866 56 26.1 0.00145 0.0073 X X X X X X X X X 971 104.2 gi|24649265 CG4448 will decrease acetylation WDA 96 54.2 0.00325 0.0163 6 7.7 0.00007 X X X X X X 743 83.7 gi|19920480 CG3883 Saga factor like TAF6 SAF6 57 31.1 0.00200 0.0100 1 2.9 0.00001 X X X X X X 717 79.3 gi|45549141 CG6474 enhancer of yellow 1 TAF9 23 25.2 0.00208 0.0104 784 65.1 0.02443 X X X X X X 278 29.3 gi|17137648 CG3069 TBP-associated factor 10b TAF10b 4 27.4 0.00069 0.0035 345 32.2 0.02047 X X X X X X 146 15.8 gi|24646044 CG17358 TBP-associated factor 12 TAF12 18 34.7 0.00231 0.0116 75 14.8 0.00331 X X X X X X 196 21.5 gi|28571540 CG17603 TBP-associated factor 1 TAF1 3 2.7 0.00004 1543 43.4 0.00647 X X X X X X 2065 232.2 gi|24662032 CG6711 TBP-associated factor 2 TAF2 6 3.4 0.00012 1433 52.6 0.01017 X X X X X X 1221 139.5 gi|24638625 CG2009 bip2 TAF3 8 3.84 0.00014 876 41.04 0.00540 X X X X X X 1406 154.5 gi|24665038 CG5444 TBP-associated factor 4 TAF4 X X X 825 30.2 0.00776 X X X X X X 921 99.3 gi|17136870 CG7704 TBP-associated factor 5 TAF5 4 9.9 0.00014 741 43.2 0.00912 X X X X X X 704 79.3 gi|24666846 CG32211 TBP-associated factor 6 TAF6 3 9.9 0.00012 0.0006 4697 71.5 0.06715 X X X X X X 606 65.7 gi|21358419 CG2670 TBP-associated factor 7 TAF7 X X X 643 53.0 0.01163 X X X X X X 479 55.1 TFIID gi|17647803 CG7128 TBP-associated factor 8 TAF8 X X X 175 32.3 0.00462 X X X X X X 328 36.6 gi|17137724 CG2859 TBP-associated factor 10 TAF10 1 8.4 0.00015 72 44.9 0.00373 X X X X X X 167 18.5 gi|24583119 CG4079 TBP-associated factor 11 TAF11 1 13.3 0.00013 189 49.0 0.00835 X X X X X X 196 22.1 gi|19921578 CG10756 TBP-associated factor 13 TAF13 X X X 35 25.0 0.00223 X X X X X X 136 15.7 Control-1: nonspecific FLAG-HA tagged nuclear protein (CG6459) gi|17647997 CG9874 TATA binding protein TBP 2 2.3 0.00014 67 11.3 0.00164 X X X X X X 353 38.5 Control-2: S2 cell nuclear extract (untagged) gi|17136834 CG7562 TBP-related factor TRF 1 7.6 0.00011 11 26.3 0.00043 X X X X X X 224 25.5 dS: Distributed spectral count gi|45554500 CG18009 TBP-related factor 2 TRF2 X X X 18 22.2 0.00025 X X X X X X 632 68.5 SC: Sequence coverage (%) dNSAF: Normalized Distributed Spectral Abundance Factor gi|20129303 CG9200 ATAC component 1 Atac1 47 38.2 0.00332 1 3.09 0.00002 X X X X X X 356 41.0 cNSAF: Complex-specific Normalized Spectral Abundance Factor gi|20129591 CG10414 ATAC component 2 Atac2 65 34.1 0.00211 X X X X X X X X X 774 89.2 ATAC cNSAF calculated for shown SAGA subunits and TAF6 gi|21356259 CG10238 CG10238 101 63.5 0.00693 X X X X X X X X X 367 41.9 Bait proteins are indicated in bold type Weake_TableS3
Total genes in arraysa SAGA-regulated genesb Total gene number 13664 926 DPE-containing genesc 648 35 % DPE-containing genes 4.7% 3.8%
a Unique genes represented in cDNA microarrays. b Defined as down-regulated log2 <-1 (p<0.05) in not, sgf11 or ada2b mutant third instar larvae relative to OregonR. c Match 6/6 positions of the consensus sequence DSWYVY at position +28 relative to the transcription start site. Weake_TableS4
qRT-PCR Primer Name FB accesion number Gene Sequence (5' - 3') CG3883.1 TATTCTCCCAGGGCAATGAG FBgn0031281 SAGA factor like TAF6 (saf6) CG3883.2 AGCTGGCATCTCAAAGTGGT CG4448.1 TGATGATGAGCTTCCAGCAC FBgn0039067 will decrease acetylation (wda) CG4448.2 ATCACTCGGCGAGCATTAGT CG3639.1 CCACGTAAGGCATGAAGGTT FBgn0031282 CG3639 CG3639.2 ATGGGGGAGTTTGAGGATTC CG5680.1 TTAGAGTGCAGTCGGCCTTT FBgn0000229 basket (bsk) CG5680.2 CTCTGCCAGGTCATCCAAAT CG8532.1 CCCCAGAGAAATATCCAGCA FBgn0028582 liquid facets (lqf) CG8532.2 GCCAGAGTGAGCAGGATTTC CG16747.1 GACTCCAAGTCGGAGCAATC FBgn0014184 Ornithine decarboxylase antizyme (Oda) CG16747.2 CCACCACAGAACAGATCACG CG8884.1 CCCTTGATGAAAGCTTCCTG FBgn0013334 Synapse-associated protein 47kD (Sap47) CG8884.2 TAACACAGCAGGCCAAACAC CG5390.1 CTGGTTGACAGCTCCAGTGA FBgn0032213 CG5390 CG5390.2 CGCCGAATGCAAGAACTATT CG2922.1 GCATGCTCTCCATGCTCTC FBgn0250753 extra bases (exba) CG2922.2 GGGCAACAAGCTCGACTATC CG9429.1 TGAGGACGAATTTTCCGAAC FBgn0005585 Calreticulin (Crc) CG9429.2 TGCCGAGGTTTATCTGAAGG CG1151.1 AGGCACTTTGTTCGTCTCGT FBgn0027527 Osiris 6 (Osi6) CG1151.2 TCGCCTACCAGGGATACAAG CG11797.1 ACTTGGAGGCGGTATCACAC FBgn0034468 Odorant-binding protein 56a (Obp56a) CG11797.2 AGGAAAAGGCCAAGGTCAAT CG1934.1 GGCTGCTCTTTGATTTCAGG FBgn0001254 Ecdysone-inducible gene E2 (ImpE2) CG1934.2 GCCATTGTCGAGGATAAGGA CG4702.1 CCAGCAAGGTGTTGATGATG FBgn0037992 CG4702 CG4702.2 CAACCAAGTCCGATGATGTG CG5820.1 GTAGGATAGTTCGCCGTGGA FBgn0013272 Gp150 CG5820.2 GACACGTTCGTGAACAAGGA CG9689.1 CCCAATTGGCTCTGTAGTCA FBgn0030159 CG9689 CG9689.2 CGAACTCGTTTGCTGATTCAT
ChIP-qPCR Primer Name FB accesion number Gene Sequence (5' - 3') CG9429_ChIP.1 CTTCTCCAACGAGGACAAGC FBgn0005585 Calreticulin (Crc) CG9429_ChIP.2 AGCGGATGTCTTTGCTGATT CG2922_ChIP.1 ATGACTAATCTGGCCTTAGGTACG FBgn0250753 extra bases (exba) CG2922_ChIP.2 GAAAGACAAATAGGGCGAGTAAAA CG16747_ChIP.1 CACAAATTGCTGGGAGGTTT CG16747_ChIP.2 FBgn0014184 Ornithine decarboxylase antizyme (Oda) CCGCTTTGGACACCATTACT neg_Or82a.1 AACGTTGTCTTTGAATTATGAGCA FBgn0041621 Odorant receptor 82a (Or82a) neg_Or82a.2 TTCTACATTTTTGGCAAACAGAAA