Supporting Information s27

Supporting information

Functional characterization of ATAD2 as a new cancer/testis factor and a predictor of poor prognosis in breast and lung cancers

Cécile Caron*, Cecile Lestrat*, Sandrine Marsal*, Emmanuelle Escoffier*, Sandrine Curtet*, Virginie Virolle#, Pascal Barbry#, Alexandra Debernardi*, Christian Brambilla*, Elisabeth Brambilla*, Sophie Rousseaux* and Saadi Khochbin*

* - INSERM, U823; Université Joseph Fourier - Grenoble 1; Institut Albert Bonniot, Grenoble, F- 38700 France. # - CNRS and Université de Nice Sophia Antipolis, Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, F-06560 France Supplementary materials and methods Cell culture, transfection, treatments and immunoflurescence Cos7, Saos2, A549 and H1299 cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (Cos7), McCoy5A (Saos) and RPMI1640 medium (H1299, A549) supplemented with antibiotics and 10% fetal calf serum. Plasmid transfections were performed with lipofectamine reagent (InVitrogen). The amounts of expression vectors were normalized with the corresponding empty vectors.For siRNA treatment, cells were plated in six-well plates (105 cells/well), cultured for 24 h at 37°C and transfected twice (with a gap of 24 h) with 10 µl of 0.02 mM siRNA using oligofectamine reagent (InVitrogen) according to the manufacturer’s instructions. Atad2 siRNA (purchased from Eurogentec) were as follow: ACUaacacugcugaagcug-dTdT(si1) ; GGUUGUAGCUCCUCCAAAU-dTdT (si2); GCUAAGGAUUUCGAGGUAG-dTdT(si3). Apoptosis was induced by treating cells with 20 µM etoposide up to 48h, or with 250 nM Actinomycin D for 14 h. Apoptotic cells were visualized by FACS after the detection of active caspase 3 using a BD Pharmingen kit, according to the vendor’s instructions. Immunofluorescence was performed as described in (Pivot-Pajot et al., 2003). Cloning of murine testis-specific Atad2 cDNA and plasmid constructs Murine full length Atad2-S cDNA was amplified by PCR from a Marathon-ready testis cDNA library (Clontech) using primers respectively generating BamH1 and XhoI sites in 5’ and 3’ of the coding sequence. It was inserted into pcDNA3.1 (InVitrogen) vectors previously modified by tag sequences insertion, in order to encode proteins tagged with Ha in N-terminus, or with Flag in C-terminus. Truncated forms of Atad2-S were then generated by PCR from pcDNA-Ha-Atad2-S using appropriate primers. Mutations in AAA and Brd domains were generated by PCR-based directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene), according to the manufacturer’s instructions. Reverse transcription, PCR and qPCR Total RNA from cell lines was extracted with Trizol reagent (InVitrogen). Total RNA from mouse and human tissues were purchased from BD Bioscience. 1 µg of total RNA was reverse- transcribed with the Super Script III First-Strand Synthesis System RT-PCR –RT (InVitrogen), using hexamer primers, according to the manufacturer’s instructions.30 PCR cycles were performed on 1/10 of the mouse tissues reverse transcription products using the following Atad2 primers: Forward: 5’-ARGAGCCTACTGAAAATGCGAAGCC-3’ for mAtad2-S and 5’- CAGTCTCCACTGGAAAGTAAACCTCG-3’ for mAtad2-L, and Reverse: 5’- CATGAAGAACGCCACTCTCTTATCCC-3’. PCR products (450 bp for mAtad2-S, and 570 bp for mAtad2-L) were analyzed on a 1.5% agarose gel. Real time quantitative PCR was performed on 1/50 of the human tissues or cell lines reverse transcription products, using the Brilliant SYBR Green qPCR MasterMix on an Mx3005p cycler (Stratagene). The data were normalized using U6 (human tissues) and GAPDH (human tissues and siRNA treated cell lines) cDNA abundance. Co-immunoprecipitation (IP) and chromatin IP and Peptide pull-down Briefly, Cos 7 cells were transfected with 5µg of Atad2 constructs and lysed 24 h after transfection by incubation for 15 min on ice in 100 µl LSDB500 (500 mM KCl, 20% glycerol, 3 mM MgCl2 and 50 mM Hepes pH 7.9, 0.1% NP-40, 1 mM DTT, and protease inhibitors cocktail (complete mini EDTA-free; Roche)). After centrifugation, the lysate was diluted in the same buffer without KCl in order to obtain a final 200 mM KCl concentration.Extracts were incubated with 1 µg of anti-Ha or anti-flag antibody for 1h on ice. Protein G-Sepharose beads (Amersham) were then added and incubated at 4°C on a rotoshake for 1h. After three washes with LSDB200 (same as the lysis buffer with 200 mM KCl), complexes were recovered by adding Laemmli sample buffer and analysed by Western blots.For chromatin IP, extracts were similarly prepared, except that 50 ng/ml TSA was added in the lysis buffer when required, and that chromatin fibres were solubilized by sonication before centrifugation. Peptides (60 µl of a 30 µM solution) corresponding to H4 N-terminal tail (SGRGKGGKGLGKGGKRH-GSGSK-biotin) non acetylated, tetra-acetylated on K5, K8, K12 on K16, or mono-acetylated on K5, K8, K12 or K16 were bound by incubation (30’, 4°C) on 20 µl streptavidine sepharose beads, followed by 3 washes in Phosphate Buffer Saline (PBS). After equilibration in LSDB200, beads were incubated (2h, 4°C) with the LSDB200 soluble extracts of transfected Cos cells or with the soluble nucleic testis extracts, washed 3 times in LSDB200, and re-suspended in Laemmli buffer (5 min, 90°C). Pulled-down proteins were analyzed by western blot.

Transcriptome analysis

The DNA microarray analysis was performed on three distinct cell lines (H1299, A549, and Saos) treated with either a scrambled or an anti-Atad2 siRNA. RNA labelling was performed as described in (Hofman et al., 2007). The quality of the total RNA was controlled on an Agilent Bioanalyzer 2100 as previously described (Moreilhon et al., 2005). Pangenomic microarrays were printed using human RNG/MRC oligonucleotide collection (corresponding to 25,299 distinct probes) as previously described (Le Brigand and Barbry, 2007) (http://www.microarray.fr/). All calculations were performed with the Bioconductor packages (Gentleman et al., 2004), limma (Wettenhall and Smyth, 2004) and topGO (Alexa et al., 2006). Differentially expressed genes were selected using a Benjamini-Hochberg correction of the p- value for multiple tests, based on a p-value below 0.01.

Experimental data and associated microarray designs have been stored into Mediante, a web- based microarray data manager (Le Brigand and Barbry, 2007), and deposited in the NBCI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under series GSE12279.

Analysis of ATAD2 expression in normal somatic tissues and cancers The expression of the gene Atad2 was analysed by using available data online and obtained from a large set of 764 cancer samples of different origins (Exp O, International Genomic Consortium, https://expo.intgen.org/geo, GEO data sets: GSE series 2109), as well as a randomized subset of 112 control somatic tissue samples (from GEO data sets: GSE series 3526). The CEL files were imported using the Genespring software (RMA normalization was performed on the series of 112 control samples). The raw expression data of the four probes corresponding to Atad2 were extracted, and all expression values were normalized on the mean values obtained in the control somatic tissue samples. Using a similar strategy, the expression of Atad2 was also correlated with survival data in a series of 271 samples of lung tumour corresponding to patients included in clinical studies associated with genome-wide transcriptomic analysis (unpublished data obtained using HG_U133_Plus_2 Affymetrix microarrays, Ligue Contre le Cancer “CIT project”).

References

Alexa A, Rahnenfuhrer J, Lengauer T (2006). Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics 22: 1600-7.

Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S et al (2004). Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80. Hofman VJ, Moreilhon C, Brest PD, Lassalle S, Le Brigand K, Sicard D et al (2007). Gene expression profiling in human gastric mucosa infected with Helicobacter pylori. Mod Pathol 20: 974-89.

Le Brigand K, Barbry P (2007). Mediante: a web-based microarray data manager. Bioinformatics 23: 1304-6.

Moreilhon C, Gras D, Hologne C, Bajolet O, Cottrez F, Magnone V et al (2005). Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells. Physiol Genomics 20: 244-55.

Pivot-Pajot C, Caron C, Govin J, Vion A, Rousseaux S, Khochbin S (2003). Acetylation- dependent chromatin reorganization by BRDT, a testis-specific bromodomain-containing protein. Mol Cell Biol 23: 5354-65.

Wettenhall JM, Smyth GK (2004). limmaGUI: a graphical user interface for linear modeling of microarray data. Bioinformatics 20: 3705-6.

Primer sequences used for RT-qPCR on human cell lines :

(F=forward, R=reverse). hAtad2: F-AGGCTCCATTGGAAAAACCT, R-CCTGCGGAAGATAATCGGTA; HSF1: F-AAGTGGTCCACATCGAGCAG, R-TCCTGGCGGATCTTTATGTCT; ODC1: F-CTGAGGTGGTTAAAAGCTCTTCC, R-TCTGCACCAACTGTATTTCAGTC; RRM2: F-CCACGGAGCCGAAAACTAAAG, R-CTCTGCCTTCTTATACATCTGCC; PPP1R7: F-AAACTCACGATGTTGGACATTGC, R-GCTCCAGCTCTCAAGGAGATT; DNAJB6: F-AAAGAGAATTGTCGAGAACGGTC, R-GTCGGCCACACCATTTATTGT; MRPL15: F-AACCGGAGAGAAGACCAAGAG, R-TTCGGATGTAAAATGGAGTCTGG; LYPLA1: F-ACTGCCCTTACCACACAGC, R-ATTAGCACCACCGATAGGACC; MAF1: F-ACTGTGGAGACCGGAGATG, R-CTGGGGCTCAGTCCTGAAG; SNRPA1: F-TGCTACGTTAGACCAGTTTGATG, R-CCCTCACCTATACGGCATATTCT; HOXB6: F-CTACCGCGAGAAAGAGTCGG, R-GTAGACCGGAGTGGAGCAC; GAPDH: F-CGAGATCCCTCCAAAATCAA, R-ATCCACAGTCTTCTGGGTGG; U6: F-CTCGCTTCGGCAGCACA, R-AACGCTTCACGAATTTGCGT. Legend to supplementary Figures:

Figure 1.

Identification of a new bromodomain-containing AAA ATPase conserved during evolution. (a) Schematic representation of the short (Atad2-S) and long (Atad2-L) Atad2 transcript variants and proteins. AAA: AAATPase domain (white); BrD: Bromodomain (black); D/E: Acidic stretch. (b) Phylogenic tree of Atad2 homologs in other species. The accession numbers of the proteins are indicated.

Figure 2.

Detailed analysis if the expression of ATAD2-related gene, ATAD2B and ATAD2 in various cancer population and sub-populations.

(a) ATAD2B is not overexpressed in cancer. The expression of the ATAD2B gene (see supp. Fig. 1) was monitored in control normal tissues and the indicated cancers, exactly as described in the legend of Fig. 2a. (b) MK167 expression is significantly higher in the group of lung cancer patients expressing ATAD2 at high levels compared to either control samples or low ATAD2 expressing lung cancer patients (unpaired t-test p<0.001). The expression of Ki67-encoding gene, MK167, was monitored in the two ATAD2-expressing lung cancer populations described in the legend of Figure 2b and is presented here as box plots, comparing the expression of MK167 in patients with low (below percentile 30; middle box plot) or high (above percentile 30; right box plot) expression of ATAD2 (c) The expression of ATAD2 was evaluated in lung patients populations corresponding to the indicated subtypes compared to the control normal tissues and presented exactly as described in the legend of Figure 2a. Ctrl; control adult somatic tissues; All: all of the 271 lung tumors; ADK: adenocarcinoma; SQC: squamous cell carcinoma; BAS: basaloid tumours; LCNE: Large NeuroEndocrine tumours (d) High and low ATAD2-expressing populations were determined and correlated to the survival data for lung cancers patients belonging to the indicated sub-populations exactly as described in Figure 2b. (e) Prognosis of breast cancer is correlated to the expression of ATAD2. GEO data sets respectively corresponding to 204 tumor samples from breast cancer patients, all at a metastatic stage (GSE12276) and to 77 tumor samples from ER+ breast cancer patients (GSE9195) were used to correlate ATAD2 expression and breast cancer survival (please see corresponding GEO references for more information on the studies). In both studies the survival probabilities of patients with high ATAD2 expressing breast tumors with that of patients with low ATAD2 expressing tumors. The thresholds between high and low expression are indicated (percentile 30 or 50).

Figure 3.

Control of transcriptomic data using two additional and unrelated siRNAs.

Real Time quantitative PCR (RT-qPCR) analysis of ATAD2-dependent gene expression. Ten of the genes overexpressed after ATAD2 knock-down according to the microarray data were randomly chosen. Total RNA was extracted from H1299 cells treated by 3 different ATAD2- targetting siRNA (si1, si2, si3) or a scramble siRNA as a control, and reverse-transcribed using hexamer primers. The expression level of the selected genes was analyzed by RT-qPCR using specific primers, and normalized to GAPDH expression. The results are the mean of four values corresponding to two independent experiments of siRNA treatment, each containing qPCR duplicates and expressed as fold variations compared to the scramble siRNA control.

Figure 4.

ATAD2 down-regulation does not affect general histone H4 acetylation.

H1299 cells were transfected with control or two unrelated anti-ATAD2 siRNAs and the acetylation of histone 4 monitored as indicated. The same blot was probed with anti-ATAD2 and anti-H2B antibodies to show the efficacy of the siRNAs and the loading.

Supplementary table 1:

List of genes up and down regulated in each of the three lines used upon ATAD2 knock-down after siRNA treatment.

Supplementary table 2:

List of genes corresponding to different functional categories according to Ingenuity Pathway Analysis of the genes presented in supplementary table 1