www.sciencemag.org/cgi/content/full/1139517/DC1

Supporting Online Material for

CREB-Binding Protein Modulates Repeat Instability in a Drosophila

Model for PolyQ Disease Joonil Jung and Nancy Bonini

*To whom correspondence should be addressed. E-mail: nbonini@sas.upenn.edu

Published 1 March 2007 on Science Express DOI: 10.1126/science.1139517

This PDF file includes

Materials and Methods Figs. S1 to S7 References

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Drosophila stocks and genetic crosses. Intergenerational repeat instability was studied primarily using flies homozygous for the SCA3tr-Q78 transgene (CAG78/CAG78; unless otherwise noted). Fly lines mus201D1, mus201KG01051, dCBP3, dCBPP, dCBPEP1179 and UAS-GFP-nls were obtained from the Bloomington Stock Center. mus201D1 is characterized (S1), mus201KG01051 was confirmed in the lab. The nos-GAL4 line was a gift of Dr. Steve DiNardo (University of Pennsylvania). UAS-Httexon1Q93 line was a gift of Dr. Leslie Thompson (University of California, Irvine) & Dr. Larry Goldstein (University of California, San Diego). Note that determination of CAG repeat size indicated that the actual size of the CAG repeat was ~ 60 for this line. UAS-CGG90-EGFP was a gift of Dr. Peng Jin (Emory University). SCA3 flies are described (S2, S3). Flies were grown at 25ºC on standard medium with dry yeast added. The CAG51 allele was isolated from CAG78 line 2 by crossing CAG78/CAG78 flies with gmr-GAL4, and selecting for males with reduced external eye degeneration among the progeny. The size of CAG repeat was determined by GenescanTM (Applied Biosystems) and expression of the protein was confirmed by immunoblot.

Trichostatin A (TSA) treatment. For TSA (Calbiochem) treatment, instant dry BLUE food (Carolina Biological Supply Co.) was used, with TSA dissolved in DMSO. To test the effect of TSA on CAG repeat instability in single generation studies, flies (nos-GAL4 UAS-SCA3tr-Q78 (line 2)/nos-GAL4 SCA3tr-Q78 (line 2)) were raised on medium containing the respective amount of TSA or DMSO, and emerging adult flies were individually crossed to w1118 flies, also in TSA-containing medium. Both parents and progeny were collected and processed to determine repeat length. Methyl-methane sulfonate (MMS) treatment. About 6-8 dCBPP/w1118 females were crossed with 5-6 w1118 males and kept for 40h at 25ºC before being removed. The vials were incubated for additional 8h at 25ºC. MMS solutions (0, 0.1, 0.5, 2.5, 5.0 mM) were prepared in ddH2O and 250µl of MMS solution was put directly into vials with eight ~48h old larvae/vial. The vials were kept at RT under a fume hood for safety reasons. Emerging flies were scored for genotype (dCBPP/w1118 or w1118/w1118). A total 8 vials were examined for each concentration of drug, for 40-90 flies total per genotype per drug concentration. Survival rates were calculated by determining the relative abundance of dCBPP/w1118 flies over w1118/w1118 flies. CAG repeat size determination. Single fly genomic DNA (gDNA) was extracted in 10mM Tris-HCl pH8.2, 1mM EDTA, 25mM NaCl, and 200µg/ml Proteinase-K in 96-well plates. CAG repeats from individual fly gDNA were amplified using the GC-Rich PCR system (Roche) with 0.8 M GC-Rich resolution butter (for SCA3 and Htt transgenes) or 2.0 M GC-Rich resolution buffer (for CGG transgenes). PCR primers used for amplification were as follows. For SCA3: 5’ primer, 5’CAT GGA TGT GAA CTC TGT CC, and fluorophor-labeled 3’ primer, 5’TTC GGA AGA GAC GAG AAG CC. For Htt: fluorophor-labeled 5’ primer, 5’ATG AAG GCC TTC GAG TCC CTC AAG TCC TTC, and 3’ primer, 5’GGC GGC TGA GGA AGC TGA GGA. For CGG: 5’ primer, 5’ACG GAG GCG CCG CTG CCA GGG GGC GTG, and fluorophor-labeled 3’ primer, 5’AGC CCC GCA CTT CCA CCA CCA GCT CCT CCA. PCR cycles for SCA3 were as follows: Initial 2 cycles (3min X 94oC, 30s X 54oC, and 2min X 72oC) were followed by additional 26-27 cycles (30s X 94oC, 30s X 54oC, and 1’45s X 72oC) with final extension of 7min X 72oC. PCR cycles for Htt and CGG were as follows: 5min X 95oC followed by 14 touch-down cycles (1min X 94oC, 1min X 72oC (-0.50C touchdown each additional step), and 2min X 72oC), and 22 additional regular cycles (1min X 94oC, 1min X 65oC, and 2min X 72oC) with

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final additional extension of 10min X 72oC. PCR products were mixed with Liz-500 size standard (Applied Biosystems) and were submitted for size determination on an ABI3100 or ABI3730 sequencer. The size of the repeats was determined with GenescanTM 3.1.2 software (Applied Biosystems).

Initially, 96 X 10 single fly gDNAs were analyzed twice with no discrepancies between repeat sizes in duplicate analyses. Therefore, when heterozygous F1 progeny were analyzed, a plate of pooled gDNA (2 to 1) was generated to amplify two CAG repeats in a single reaction. Many of the samples with altered repeat lengths were re-examined using the original single fly gDNA: > 99% of results were confirmed. For 9-generation studies, 100-200 homozygous progeny (200-400 CAG repeat sequences) were examined. For 1-generation studies (Figures 1, 2 & 3), each experiment consisted of 6-9 individual crosses (600-900 progeny per experiment). From each cross, 96 progeny were examined. For each genotype, experiments were repeated 2 to 4 times as indicated. Pooled data are presented for Supplemental Figures S3B (~30 crosses) and S6 (7~8 crosses). Crosses to test the effect of dCBPP on repeat instability with SCA3tr-Q78 line 3 (Fig. S3A) and Httexon1Q93 (Fig. S7) produced mostly fewer than 50 progeny per cross, and the data was pooled from ~30 crosses.

Realtime PCR analysis. In order to determine whether various genetic modifications and pharmaceutical treatments of flies affected SCA3 transgene expression levels, the steady-state level of the transgene mRNA was determined using realtime PCR analysis. Total RNA from testes or ovaries was prepared using the QiaQuick RNAeasy kit with DNaseI treatment (Qiagen). Size of ovaries was matched between samples for RNA extraction. cDNA synthesis and PCR amplification were done using the SYBR Green PCR kit (Qiagen) and Opticon realtime PCR machine (MJ research). Rp49 was used as the loading control. Primers for SCA3 were: 5’ primer, 5’CTA TCA GGA CAG AGT TCA CAT and 3’ primer, 5’CAG ATA AAG TGT GAA GGT AGC. Primers for Rp49 are from Foley et al. (S4). Immunohistochemistry and histone acetylation analysis. Flies bearing nos-GAL4 UAS-SCA3tr-Q78 line 2 were crossed with flies bearing UAS-GFP-nls. Tissue from progeny flies was dissected in cold PBS and fixed in 4% paraformaldehyde 30min RT. DAPI was used to counterstain nuclei. Tissue was mounted in Vectashield. To determine the level of acetylated histones, acid-extracted histone preparations were used. 60-70 female flies were ground in a Pyrex 2ml dounce homogenizer in 500µl buffer A (0.1M Tris pH7.4, 150mM NaCl, 1% NP-40, 1mM EDTA, 1X proteinase inhibitor cocktail (Sigma, P8340)). Nuclei were pelleted at 2,500g and resuspended in 500µl buffer B (10mM HEPES, pH7.9, 1.5mM MgCl2, 100mM KCl, 0.5mM DTT, 1X proteinase inhibitor cocktail). Histones were extracted by adding 0.25N HCl, and precipitated with Trichoacetic acid (10% final concentration). Antibodies for Western blotting were: anti-acetylated H3 (1:10,000, Catalog #06-599,Upstate), anti-acetylated H4 (1:500, Catalog #06-598, Upstate) and anti-H3 antibodies (1:12,500, Catalog #07-690, Upstate). To test the efficacy of the antibodies, histones extracted from HeLa cells with or without sodium butyrate treatment were used in control immunoblots. Statistics. Student’s t-test, Chitest, Fisher’s exact test or ANOVA were used as appropriate for the experimental design and comparison being examined. Mean ± SD or SEM was used as appropriate and is indicated in the figure legends. ExcelTM (Microsoft Inc.), JMP InTM (SAS Institute Inc.) or PRISMTM programs were used for statistical analysis.

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Figure S1

Figure S1. Genetic crossing schemes and profile of CAG repeat amplicons.

(A) Two different crossing schemes were used. The 9-generation scheme allowed analysis of instability over multi-generations, while the single generation scheme was used to study modifiers and parent-of-origin effects. Flies homozygous for transgenes of the same CAG repeat size were typically used (CAG78/CAG78). (B) Elution profiles of PCR-amplified SCA3 CAG repeat regions, analyzed by GenescanTM software (Applied Biosystems, Inc.). Three major peaks, each ~3 bases apart, were seen for each homozygous (CAG78/CAG78) or heterozygous (CAG78/+) sample; this pattern was highly reproducible. The 2nd to the last major peak was the strongest in intensity and considered representative of the CAG78 repeat. The actual size of the repeats was determined against a size standard (orange). The presence of a new repeat size allele was represented by the appearance of a new group of peaks, either to the right (expansions) or to the left (shrinkages) of the CAG78 peaks.

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Figure S2

Figure S2. Expression of the SCA3 transgene in the germline.

(A-F) Expression pattern in germline stem cells in the testes (A-C) and ovary (D-F), using the nos-GAL4 driver line with a GFP-nls reporter. Highly mitotic germline stem cell populations (asterisks) were strongly labeled with GFP. Genotypes: nos-GAL4/UAS-GFP-nls. (G) Fold induction of SCA3 transgene (line 2) mRNA by nos-GAL4 driver, measured by realtime RT-PCR (Student’s t-test: *P<0.05, **P<0.01; mean +/- SD; N= 3). Genotypes: UAS-SCA3tr-Q78/ UAS-SCA3tr-Q78 vs. nos-GAL4 UAS-SCA3tr-Q78/ nos-GAL4 UAS-SCA3tr-Q78.

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Figure S3

Figure S3. Additional evidence that CBP regulates repeat instability.

(A) Reduction in dCBP leads to an increase in repeat instability in SCA3tr-Q78 line 3 (1.94% (control), 3.70% (dCBPP/+). Chitest for rate of repeat expansions, *P<0.05. Genotypes: +/+;; nos-GAL4 UAS-SCA3tr-Q78 line 3 /TM6,Tb vs. dCBPP/+;; nos-GAL4 UAS-SCA3tr-Q78 line 3 /TM6,Tb. (B) Upregulation of dCBP with dCBPEP1179 overexpression line in male germ cells reduced repeat instability (1.92% (control), 0.98% (dCBPEP1179); Chitest for total repeat instability, **P<0.01). Genotypes: +;; nos-GAL4 UAS-SCA3tr-Q78/ nos-GAL4 UAS-SCA3tr-Q78 vs. dCBPEP1179;; nos-GAL4 UAS-SCA3tr-Q78/ nos-GAL4 UAS-SCA3tr-Q78.

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Figure S4

Figure S4. Reduction of dCBP gene dosage modified the survival rate of larvae on MMS.

Methyl-methane sulfonate (MMS) is a strong alkylating reagent that causes DNA damage and chromosomal aberrations; survival on MMS therefore reflects the activity of DNA repair pathways (S5, S6). Larvae with reduced levels of dCBP (dCBPP/+) showed a significant change in the survivability compared to placebo-treated animals (ANOVA, P< 0.001; N=8.). In particular, the rate of survival was significantly increased when dCBP/+ animals were raised on a low level of MMS treatment (ANOVA Dunnett test, *P< 0.05), while an opposite trend was observed when the mutant animals were raised on a 5.0 mM MMS, compared to placebo-treated animals. At intermediate levels of MMS, we observed no difference in survival. This complex pattern of changes in survival with decreased level of dCBP indicates that dCBP likely modulates multiple DNA repair pathways. For example, chromosomal aberrations occur only at a higher concentrations of MMS, thus potentially stimulating different DNA repair pathways than are stimulated by low concentrations of MMS (S7, S8).

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Figure S5

Figure S5. SCA3 transgene expression levels were not affected by reduction of dCBP or treatment of flies with TSA.

The level of SCA3 transgene mRNA was not significantly altered in the ovaries of (A) wildtype and dCBP mutants (Gentoypes: dCBP3/+;; nos-GAL4 UAS-SCA3tr-Q78/ nos-GAL4 UAS-SCA3tr-Q46 vs. +/+;; nos-GAL4 UAS-SCA3tr-Q78/nos-GAL4 UAS-SCA3tr-Q46; N= 2; dCBPP/FM7c;;nos-GAL4 UAS-SCA3tr-Q78/TM6, Tb vs. +/FM7c;;nos-GAL4 UAS-SCA3tr-Q78/TM6, Tb, N= 3), and (B) TSA-treated flies (Genotypes: nos-GAL4 UAS-SCA3tr-Q78/ nos-GAL4 UAS-SCA3tr-Q78; N= 3) measured by realtime RT-PCR. (Mean +/- SD).

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Figure S6

Figure S6. Additional Drosophila models of trinucleotide repeat diseases show transcription-dependent repeat instability.

Repeat instability increases with germline transcription in (left) HD flies bearing the transgene Httexon1Q93 (Chitest for total repeat instability, P<0.05*), and (right) in flies modeling fragile X, bearing a 5’UTR premutation CGG expanded repeat (CGG90-EGFP; Chitest for the rate of repeat expansions, **P<0.01). Genotypes for HD model: UAS-Httexon1Q93/ UAS-Httexon1Q93 vs. UAS-Httexon1Q93/ UAS-Httexon1Q93; nos-GAL4/ nos-GAL4. Genotypes for fragile X model: UAS-CGG90-EGFP/UAS-CGG90-EGFP vs. UAS-CGG90-EGFP/UAS-CGG90-EGFP; nos-GAL4/nos-GAL4.

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Figure S7

Figure S7. Reduction of dCBP levels enhances repeat instability in an HD model.

Reduction of dCBP levels caused increased repeat instability in flies bearing Httexon1Q93 (Chitest for total repeat instability, **P< 0.01). Genotypes: UAS-Httexon1Q93/+; nos-GAL4/+ vs. dCBPP/+; UAS-Httexon1Q93/+; nos-Gal4/+, with second and third chromosome balancers floating.

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