ONCOGENE [ONC-2012-00522-EBMR] resubmission Version 2012, August 15th

The Cul4A-DDB1 E3 ubiquitin ligase complex represses p73 transcriptional activity

Martina Malatesta, Angelo Peschiaroli, Elisa Maria Memmi, Jianxuan Zhang, Alexey Antonov, Pengbo Zhou, Douglas R. Green, Nickolai Alexander Barlev, Alexander Vasilievich Garabadgiu, Gerry Melino and Francesca Bernassola

Supplementary information

Supplementary information Materials and Methods

Immunostaining

Cells grown on glass coverslips were fixed in 4% paraformaldehyde in PBS for 20 min at RT, and then permeabilized for 5 min with 0.1% Triton-X 100. After fixation, cells were washed four times with PBS and blocked with 5% goat serum for 1 hour at RT. Cells were incubated with primary antibody for 4 hr at RT. Cells were then washed five times with PBS, incubated with a 1:500 dilution of Alexa Fluor 594- or 488-conjugated secondary antibodies for 40 min at room temperature, and then washed 4 times with PBS. Nuclei were labeled with DAPI in PBS for 3 min at RT. After a final wash in PBS, cells were mounted onto slides with mounting medium and analysed with a Nikon microscope.

Subcellular fractionation

Cells were resuspended in buffer A [20 mM Hepes (pH 7.9), 10 mM KCl, 2.5 mM MgCl, 1 mM EDTA, 1 mM DTT, 0.2% NP40, and protease inhibitors], incubated on ice for 5 min, and centrifuged at 14,000 rpm, 4°C for 1 min. The supernatant is the soluble cytoplasmic fraction. To obtain the insoluble nuclear fraction, the pellet was resuspended in buffer B (buffer A, 20% glycerol, 20 mM NaCl) and incubated on ice for 30 min. After centrifugation at 14,000 rpm, 4°C for 20 min, the supernatant was collected.

Quantitative Real Time PCR assays

qPCR reactions were performed in the 7500 Fast System apparatus (Applied Biosystems, Foster, CA, USA) by using SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer's protocol. cDNA products were amplified with the indicated primer sets: Puma: Fw 5’-GGACGACCTCAACGCACAGT-3’ and Rv 5’-GGAGTCCCATGATGAGATTGTACA-3’; p21: Fw 5’-TGAGCGATGGAACTTCGAC-3’ and Rv 5’-ACAAGACAGTGACAGGTCC-3’; Gadd45: Fw 5’-TCAGCGCACGATCACTGTC-3’ and Rv 5’-CCAGCAGGCACAACACCAC3-3’. GADPH was used as an internal control.

Semiquantitative RT-PCR analysis

PCR amplification was carried out using the following paired-primers and conditions: human TAp73 Fw 5’-CCACGTTTGAGCACCTCTGG-3’ and Rv 5’-CTGCTCAGCAGATTGAACTGG-3’ (56°C, 30 Cycles); human PUMA Fw 5’-CTGTGAATCCTGTGCTCTGC and Rv 5’-AATGAATGCCAGTGGTCACA-3’ (56°C, 30 cycles); human b-actin Fw 5′-CTGGCACCACACCTTCTACAATG-3’ and Rv 5′-AATGTCACGCACGATTTCCCGC (55°C, 22 cycles); mouse p21 Fw 5’-TGTTCCGCACAGGAGCAAAGT-3’ and Rv 5’-CGTCTCCGTGACGAAGTCAA-3’ (55°C, 24 cycles); mouse Gadd45 Fw 5’-CAGAGCAGAAGACCGAAAGGA-3’ and Rv 5’-GCAGGCACAGTACCACGTTATC-3’ (55°C, 26 cycles); mouse c-Jun Fw 5’-GCAGAGGGAAGCGCATGA-3’ and Rv 5’-TGAGCATGTTGGCCGTGGAT-3’ (56°C, 24 cycles). The levels of transcripts were normalized against the levels of b-actin mRNA that were used as internal control.

BrDu staining and cell cycle analysis

Bromodeoxyuridine (BrDu) was added to the cells and allowed to incorporate for 30 min followed by cell harvesting and fixation with 70% EtOH for 30 min on ice. After denaturation, cells were labelled with mouse anti-BrdU antibody (1:5) (Pharmingen) for 1 hour. Cells were then incubated with a FICT-conjugated anti-mouse antibody (1:50) (Pharmingen) for 1 hour. After washing, cells were resuspended in 1 ml of propidium iodide (PI) solution (2.5 µg/ml) containing RNase (250 mg/ml) and incubated overnight at 4oC, before FACS analysis.

Bioinformatical analyses

Datasets (see supplementary table S1) were downloaded from GEO repository (http://www.ncbi.nlm.nih.gov/geo/). The datasets were united in one metadataset with 980 samples in total. The rank normalization was applied before analyses. Based on normalized expression values of Cul4A, samples were split into 2 groups, those with high expression of Cul4A and with low expression of Cul4A. Absolute value of Pearson correlation coefficient between expression profile of p73 and expression profiles of its targets was computed in both groups.

To demonstrate that differences in correlations in both groups are significant, we used Monte Carlo simulation, which is commonly employed in biological studies when the null distribution could not be derived analytically. The samples from the metadataset were split randomly into groups in the same proportion as in original split based on Cul4A expression value. The correlation between p73 and its targets was computed and the difference (absolute value) was computed for each pair (p73->target gene). The procedure was repeated 1000 times, thus generating a background distribution of difference in correlation between randomly sampled groups. The difference in correlation between p73 and its targets for original split of samples based on Cul4A expression value was compared to generated distribution. In most cases, in no one case of random split the difference in correlation coefficients was greater or equal to the one related to original split based on Cul4A expression value.

Double ChIP assays

Cells were double cross-linked with1.5 mM EGS for 20 min followed by 1% formaldehyde for 15 min. Cross-linking was neutralized with 0.125 M glycine, and cells were scraped in PBS. Chromatin was sonicated followed by incubation overnight with the appropriate antisera. To precipitate p73 anti-HA monoclonal-conjugated sepharose beads (Sigma) pre-blocked with 2.5% BSA and 0.1 mg/ml sheared salmon sperm DNA were used. A DDB1-specific polyclonal rabbit serum (Santa Cruz) was used to precipitate the corresponding immune complex. Cell extracts were incubated with antibodies overnight following addition of protein G-Sepharose beads blocked with 0.1 mg/ml sheared salmon sperm DNA and 2.5% BSA for 4 hrs at 4°C (in case of DDB1). Bead-bound immune complexes were washed three times, and then eluted. Immunoprecipitated DNA was purified and quantitative PCR was performed with 1 μl of DNA to assess p73 and DDB1 binding levels.