Supporting Information · D # PCNA foci 40 80 100 120 0 20 60 C 0 10 20 30 40 50 60 # TUNEL...

Supporting InformationMerlo et al. 10.1073/pnas.1419083111SI TextGenotypes.Fig. 1. ctr: elav-GAL4/+. p53 null: p5311-1B-1. tau: elav-GAL4/+;UAS-tauR406W/+. tau + p53 null: elav-GAL4/+;UAS-tauR406W,p5311-1B-1/p5311-1B-1.Fig. 4. tau: elav-GAL4/+;UAS-tauR406W/+. Clc-LOF: elav-GAL4/+;ClcDG23206/+ or elav-GAL4/+; UAS-tauR406W/ClcDG23206. Chc-1:(elav-GAL4/Chc1 or elav-GAL4/Chc1; UAS-tauR406W/+. Chc-4: elav-GAL4/Chc4 or elav-GAL4/Chc4; UAS-tauR406W/+. Rab26-OE: elav-GAL4/+; UAS-Rab26/+ or elav-GAL4/+; UAS-Rab26/+; UAS-tauR406W/+. UAS-Rab26-DN: elav-GAL4/+; UAS-Rab26T204N/+ orelav-GAL4/+; UAS-tauR406W/UAS-Rab26T204N. Rab26 RNAi: elav-GAL4/+; UAS-Rab26 RNAi 43730/+ or elav-GAL4/+; UAS-Rab26 RNAi 43730/+; UAS-tauR406W/+. Sytβ-LOF: elav-GAL4/+;SytβDG10711/+ or elav-GAL4/+; UAS-tauR406W/SytβDG10711. SytβRNAi: elav-GAL4/+; UAS-Sytβ RNAi JF02593 or elav-GAL4/+;UAS-tauR406W/UAS-Sytβ RNAi JF02593. Amph-LOF: elav-GAL4/+;AmphEY9339/+ or elav-GAL4/+; AmphEY9339/+; UAS-tauR406W/+.Amph RNAi: elav-GAL4/+; UAS-Amph RNAi 7190/+ or elav-GAL4/+; UAS-tauR406W/UAS-Amph RNAi 7190.Fig. S1. ctr: elav-GAL4/+. tau: elav-GAL4/+;UAS-tauR406W/+. tau +p5311-1B-1: elav-GAL4/+;UAS-tauR406W, p5311-1B-1/p5311-1B-1.p535A-1-4: p535A-1-4. tau + p535A-1-4: elav-GAL4/+; UAS-tauR406W,

p535A-1-4/p535A-1-4.Fig. S7. ctr – SCA3: elav-GAL4/+. ctr + SCA3: elav-GAL4/+;UAS-ATXN3-Q78/+. EGFP + SCA3: elav-GAL4/+; UAS-EGFP/+;UAS-ATXN3-Q78/+ . Amph-LOF + SCA3: elav-GAL4/+ ;AmphEY9339/+; UAS-ATXN3-Q78/+. Chc-LOF + SCA3: elav-GAL4/Chc1; UAS-ATXN3-Q78/+. Clc-LOF + SCA3: elav-GAL4/+; UAS-ClcDG23206/UAS-ATXN3-Q78. Sytβ RNAi + SCA3: elav-GAL4/+;UAS-Sytβ RNAi JF02593/UAS-ATXN3-Q78. Rab26-OE + SCA3:elav-GAL4/+; UAS-Rab26/+; UAS-ATXN3-Q78/+. Rab26-DN +SCA3: elav-GAL4/+; UAS-ATXN3-Q78/UAS-Rab26T204N.Fig. S8. ctr: elav-GAL4/+. p53 null: p5311-1B-1. tau: elav-GAL4/+;UAS-tauR406W/+. tau + p53 null: elav-GAL4/+;UAS-tauR406W,p5311-1B-1/p5311-1B-1.GFP-Atg8a: elav-GAL4/+; UAS-GFP-Atg8a/+. Tau + Atg8a:

elav-GAL4/+; UAS-tauR406W/ UAS-GFP-Atg8a. GFP-ref(2)P:elav-GAL4/+; UAS-GFP-ref(2)P/+. Tau + ref(2)P: elav-GAL4/+;UAS-tauR406W/UAS-GFP-ref(2)P.

Primers.ChIP primers for Drosophila p53 gene.

dp53 ChIP For 5′-CGCTTGTACTTGCATCATTCG-3′;dp53 ChIP Rev 5′-GCGCCTTGGCTGGATAAAC-3′;

3′ UTR ChIP For 5′-GTGGCAGCCGGTCGAA-3′;3′ UTR ChIP Rev 5′-CAGCCAAAGCGGATGCA-3′.

ChIP primers for Drosophila rpr gene.

rpr ChIP For 5′-CGGAAAACTGATATGGCGATAAG-3′;rpr ChIP Rev 5′-CGGTCCCTCAGTCTCCAAGTC-3′.

ChIP primers for validation of candidates in Drosophila.

Amph ChIP For 5′-ATGTTCAGCCACACAAGTACACGT-3′;Amph ChIP Rev 5′-ATGTACATTAAAAACAAGGTTCCGC-3′;Clc ChIP For 5′-TCTGCTGCCAGTGAAAAACG-3′;Clc ChIP Rev 5′-CAAGTTCACCGTGGCATCAT-3′;Chc #1 ChIP For 5′-AGAAAAGAGGCAGCATTATCATCA-3′;Chc #1 ChIP Rev 5′-CGCAACTCAACATGGCTGACT-3′;Chc #2 ChIP For 5′-GCTGGATTGTCCGAATTTGAC-3′;Chc #2 ChIP Rev 5′-GCAGGAATGCGGCAGACT-3′;Rab26 #1 ChIP For 5′-GGTGCAGAGTTACCGTGCTACA-3′;Rab26 #1 ChIP Rev 5′-GGTGTGCGACACGGCTACTT-3′;Rab26 #2 ChIP For 5′-CACGACGATGCTATCATGGC-3′;Rab26 #2 ChIP Rev 5′-ACGATAGCCAGGATATCCGGT-3′;Sytβ ChIP For 5′-CCTCGCTGGGCATGTGAT-3′;Sytβ ChIP Rev 5′-CCTTTGTGCCAAATTTAATTGTCA-3′.

ChIP primers for human BIN1.

hBIN1 prom For 5′-AGGGAATGGCCTGTGTGAAC-3′;hBIN1 prom Rev 5′-GAGAAATCGAGTCCCAGGAATG-3′;hBIN1 intron1 For 5′-GCACCCGGTCAGTTGTCTATC-3′;hBIN1 intron1 Rev 5′-GCAGCTGGCGTGATCCA-3′.

Solutions.RIPA buffer was composed of 10 mM Tris·Cl (pH 8.0),1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodiumdeoxycholate, 0.1% SDS, and 140 mM NaCl. NEEM buffer wasmade up of 10 mM Hepes pH 7.6, 1.5 mM MgCl2, 0.1 mMEDTA, 10 mM KCl, and 300 mM sucrose. NEB buffer was madeup of 0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mMEDTA, and 10 mM Tris·HCl (pH 8).

Merlo et al. www.pnas.org/cgi/content/short/1419083111 1 of 7

www.pnas.org/cgi/content/short/1419083111

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Fig. S1. p53 is neuroprotective in a Drosophila tauopathy model. (A) Loss of p53 (p5311-1B-1) in the brain increases tau-induced cell death, as indicated byTUNEL staining (Left) and cell-cycle activation and as monitored with PCNA expression (Right), arrows. (Scale bar: 20 μm.) (B) Colabeling of PCNA, TUNEL-positive neurons in tau-transgenic flies, arrows. (Scale bars: 5 μm.) (C) Apoptosis, indicated by the number of TUNEL-positive neurons, increases in the brains oftau-transgenic, p535A-1-4 animals. (D) Cell-cycle reactivation, indicated by the number of PCNA-positive foci, increases in the brains of tau-transgenic, p535A-1-4

animals. Flies are 10-d-old (n ≥ 6 per genotype). In the graph, values represent mean ± SEM. ctr, control: elav-GAL4/+.

02468

1012141618

rpr p53

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Fig. S2. p53 antibody specifically immunoprecipitates p53 protein. ChIP assay showing that p53 binds regulatory regions of the p53 and rpr genes. Foldenrichment is expressed relative to IgG control. Values have been normalized to the total DNA and represent mean ± SEM. The graph is representative of oneof three independent triplicate assays. Chromatin for IP was taken from untreated (ctr) or irradiated (Irr) w1118 embryos.



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Fig. S3. (Continued)



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Fig. S3. (Continued)



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Fig. S3. Representative high-resolution ChIP-chip analysis of p53 binding to the promoter regions of the Amph, Clc, Chc, Rab26, and Sytß genes. The log2 ratiofluorescence values for control flies (Top) and tau flies (Middle) chromatin hybridization is shown for a region of 30 kb. Gene positions in the genome (Bottom)are represented with rectangles (exons), connected by lines (introns). ctr, control: elav-GAL4/+.

Genomic sequence gene

cacaCAAAcactcgacatgctcacaagagcgcgc Amph

tgccccatcctttgtCATGccCTTGatcagtcaagtcaaChc

ccgagtcgatctggtCATGtccgtctgtccgtat

tagtccacaaggtcaCATGtcct

Rab26ctcccCTTGcctcgaccagtcat

ctacgatgaagtggaCATGcacccagccccgtcc

atggggctgtaatcaCATGcccaSytb

gcaaCAAGttgcgaaaaagcttt

Fig. S4. Genomic sequences of p53-responsive elements identified in synaptic gene promoters. Bold and uppercase characters indicate potential p53 consensus.

a-H3K4me3 taua-H3K4me3 ctrIgG

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Fig. S5. ChIP assay for H3K4me3 histone modification showing that the selected synaptic genes are actively transcribed. Fold enrichment is expressed relativeto negative control (IgG). Values have been normalized to the total DNA and represent mean ± SEM. The graph is representative of one of the three in-dependent triplicate assays. Flies are 10-d-old.



***

ClcDG23206

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Fig. S6. Altering the function of synaptic genes in tau-transgenic animals modifies locomotion, measured as walking speed. Flies are 2-d-old (n = 18 flies pergenotype). In the graph, values represent mean ± SEM. Asterisks indicate significant differences compared with tau-transgenic flies by ANOVA with Student–Newman–Keuls post hoc test for multiple comparisons. ***P < 0.001. ctr, control: elav-GAL4/+.

+ + + + + + +EGFP LOF

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Fig. S7. Synaptic function modifiers do not alter neuronal toxicity of mutant ataxin 3. The number of Kenyon neurons per 250 μm2 was counted. Flies are10-d-old (n = 6 flies per genotype). In the graph, values represent mean ± SEM. ctr, control: elav-GAL4/+; SCA3, mutant ataxin 3: UAS-ATXN3-Q78.



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Fig. S8. Synaptic abnormalities in tau-transgenic animals. (A) There is a significant reduction in the expression of synaptic markers in the brains of tau-transgenic, p53-null flies. (B) Quantification of the protein expression reduction relative to control. In the graph, values represent mean ± SEM of at least threeWestern blots per antibody. Asterisks indicate significant differences compared with control flies by ANOVA with Bonferroni post hoc test for multiplecomparisons. *P < 0.05; ***P < 0.001. ctr, control: elav-GAL4/+. (C) Ubiquitin accumulates at synapses when tau is expressed in the absence of p53. (D) Au-tophagy markers accumulate at synapses in tau-transgenic flies. Flies are 10-d-old. (Scale bars: 10 μm.) Syn, Synapsin; Cha, Choline acetyltransferase; Syx,syntaxin; dlg1, discs large 1; Elav, embryonic lethal abnormal vision; Csp, Cysteine string protein; Atg8a, Autophagy-specific gene 8a, detected with a transgenicreporter (UAS-GFP-Atg8a) and anti-GFP immunofluorescence; ref(2)P, refractory to sigma P, also known as p62 and SQSTM1, detected with a transgenic re-porter (UAS-GFP-ref(2)P) and anti-GFP immunofluorescence.

Dataset S1. List of genes bound by p53 in tau-transgenic flies throughout the whole Drosophila genome

Dataset S1

Dataset S2. Gene Ontology analysis of the genes bound by p53 in tau-transgenic flies. To reduce the redundancy in gene setannotation, we used functional annotation clustering, which groups similar annotations together

Dataset S2

Dataset S3. Detailed list of the synaptic-transmission–related terms

Dataset S3


http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1419083111/-/DCSupplemental/pnas.1419083111.sd01.xlsx




Supporting Information · D # PCNA foci 40 80 100 120 0 20 60 C 0 10 20 30 40 50 60 # TUNEL...

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Transcript of Supporting Information · D # PCNA foci 40 80 100 120 0 20 60 C 0 10 20 30 40 50 60 # TUNEL...