Generation of Atad5+/m Mice
Mice used in this study were derived from the ES cell line (ID: RRF055, 129/Sv background). Chimeric male mouse generated from the ES cell injected C57BL/6 blastocytes were transferred into pseudopregrant CD1 female recipients. All mice works were conducted under in compliance with federal regulations and guidelines. All mice work in this study was approved by the NHGRI Animal Care and Use Committee (NIH animal study proposal: G-05-2, KM as a PI).
We followed the method described previously 
. Briefly, six-week old male mice were exposed to 0.7 Gy of γ-ray irradiation from a 137
Cs source. Normochromatic erythrocytes (NCEs) and reticulocytes (RETs) in erythrocytes were distinguished by their negative and positive expressions of CD71 in cell surface, respectively. The RETs carrying micronuclei were detected by the nucleic acid binding agent propidium iodide. At least 10,000 RETs and 500,000 NCEs were analyzed in each blood sample.
Anonymized primary endometrial tumor tissues (n
102) and matched histologically normal tissues were obtained from the Cooperative Human Tissue Network, or from the Biosample Repository at Fox Chase Cancer Center, Philadelphia PA. Six cases of matched tumor and normal DNAs were purchased from Oncomatrix. All primary tumor tissues were pretreatment specimens, snap-frozen within 30 minutes of surgical removal. A hematoxylin and eosin (H&E) stained section was cut from each tumor specimen and reviewed by a pathologist to verify histology and to delineate regions of tissue with a tumor cell content of ≥70%. All specimens and accompanying clinicopathological information were anonymized, and procured with appropriate IRB approval. The Office of Human Subjects Research declared exempt for human specimen usage because the specimens were anonymized when investigators received them: The exemption numbers are #3529, #3456, and #3534.
To confirm that tumor-normal pairs were consistent with derivation from the same individual, all human DNA samples were genotyped using the Coriell Identity Mapping kit (Coriell). Genotyping fragments were size separated on an ABI-3730xl DNA analyzer (Applied Biosystems). Alleles were scored using GeneMapper software.
Primer Design, PCR Amplification, and Nucleotide Sequencing
Primer pairs to amplify ATAD5
were designed using published methods 
). PCR products were subjected to bidirectional Sanger sequencing using the BigDye Terminator Version 3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were run on ABI 3730xl
DNA Analyzers (Applied Biosystems). Sequence data was analyzed as described in the Supplemental Experimental Procedures.
Laser Capture Microdissection and RT-PCR
Pure populations of tumor cells were isolated from heterogeneous tissue sections by laser capture microdissection (LCM) using an Arcturus PixCell IIe system. RT-PCR products, generated from microdissected tumor cells were sequenced to discriminate between monoallelic and biallelic expression of mutant ATAD5 alleles.
Analysis of Atad5+/m ES Cells, MEFs, and Mice
The wild-type and mutant Atad5 alleles were distinguished by PCR using primers: 1298NF (5′- GAG ACT GTC TCA CCA TGT ACA GGG-3′), 1372NR (5′- ATA AAT TGT AAA AAA TCG ATC TCT-3′) and pGKR (5′- GTG GCC TGT CCC TCT CAC CTT CTA-3′). PCR products of 542 bp and 900 bp distinguished the wild-type and mutant alleles, respectively (). PCR conditions were an initial denaturation step at 94°C for 5 minutes, followed by 35 cycles of a denaturation step at 94°C for 30 seconds, an annealing step at 55°C for 30 seconds, and an elongation step at 72°C for 60 seconds and by a final elongation step at 72°C for 7 minutes. There were no additional insertions of the retroviral vector in the genome of RRF055 ES cells, as confirmed by Southern hybridization with a NEO probe. The ES cells were injected to into C57BL/6 blastocytes that were transferred into pseudopregnant CD1 female recipients. A resulting chimeric male mouse was mated with C57BL/6 females. Germline transmission was confirmed in progeny of agouti mice. The germline transmitted heterozygous mice were crossed with wild-type 129/Sv mice to increase the heterozygous population. In addition, two heterozygous mice were mated to generate Atad5m/m mice. Mice were bred and maintained under a protocol (#G-05-02) approved by the Institutional Animal Care and Use committee in the NHGRI, US NIH Animal Care Facility. Young Atad5+/m mice did not show any distinct phenotypic features and were fertile. However, when the Atad5+/m mice were intercrossed, we did not observe any postnatal progeny that were homozygous for the Atad5 allele (Atad5m/m).
Mouse embryonic fibroblast (MEF) cells were isolated from both wild-type and Atad5+/m embryos at day 13.5 p.c. and cultured in Dulbecco's modified Eagle's medium (DMEM) (high glucose, w/o L-glutamine, w/o sodium pyruvate, GibcoBRL) supplemented with 2 mM glutamine, 1% penicillin/streptomycin, 15% fetal calf serum, 2 mM non essential amino acids, 0.1 mM 2-β mercaptoethanol in 4% oxygen concentration. RNAs isolated from wild-type and Atad5+/m MEFs were analyzed by RT-PCR with primer sets specifically amplifying the region of mRNA containing exons 4 and 5, exons 18 and 19, or exons 19 and 20 respectively from 500 ng of total RNA isolated with TRIzol reagent (Invitrogen). The expression of β-actin was used as an internal expression control. For quantitative RT-PCR, reactions were performed using the ABI7300 sequence Detection System (Applied Biosystems) with an annealing temperature of 60°C and the following primer pairs: Atad5-Ex4F (5′-GAC TGA AGA AAC AGT GGT ACC-3′) and Atad5-Ex5R (5′-CAA AGA CAG GAA TGG CTG CTC-3′); Atad5-Ex14F (5′-GCC TCT TCA CAG CGA AGT GG-3′) and Atad5-Ex15R (5′-GTG CTC GCT TCT GCC CAC T-3′); Atad5-Ex18F (5′-GTC TAG TGT TTG ATG GCT GCT TTG-3′) and Atad5-Ex19R (5′-CAC TTG TAG ATA GCT GGC AAC-3′); Atad5-Ex19F (5′-GTT GCC AGC TAT CTA CAA GTG-3′) and Atad5-Ex20R (5′-GAG CCA TCT TCT GAA CAA ACC-3′); and LacZR (5′-CTT CGC TAT TAC GCCAGC TGG-3′).
Total cell extract was prepared by sonication of cells in lysis buffer (100 mM Tris-Cl (pH 7.5), 50 mM NaCl, 8% glycerol, and protease inhibitor cocktail (Roche) with either 0.5% Triton X-100 or 0.5% NP40 as a detergent). Supernatant after centrifugation was used as total cell extract. Proteins in total cell extract were separated by electrophoresis using 4–12% gradient polyacrylamide gels (BioRad) and transferred to PVDF membranes (BioRad). Proteins on PVDF membranes were detected by Western blotting with antibodies. Atad5 protein was detected by anti ELG1 antibody raised in rabbits against human N-terminal 1–197 amino acid fragments of ELG1. Fused Elg1 protein was detected by anti-Beta galactosidase antibody (Abcam).
The TritonX-100-insoluble fraction (chromatin bound fraction) was isolated from the Triton X-100-soluble fraction using the traditional methods with slight modifications. Harvested cells were resuspended in buffer A (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM Pipes (pH 6.8), 1 mM EGTA, 0.2% Triton X-100, 100 mM NaVO4, 50 mM NaF, and protease inhibitors (RocheApplied Science)) and incubated for 5 minutes on ice with gentle inverting. The supernatants were recovered as the “soluble fraction” after centrifugation. Followed by washing with buffer A, the pellet was resuspended either in buffer X (100 mM Tris-HCl (pH 8.5), 250 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 100 mM NaVO4, 50 mM NaF, 2 mg/ml bovine serum albumin, and protease inhibitors) or in buffer B (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 100 mM 100 mM NaVO4, 50 mM NaF, and protease inhibitors) for immunoblotting. Followed by a 10 min incubation on ice, the samples were sonicated and then incubated for another 10 min on ice before centrifugation to isolate the “chromatin-bound fraction.” Immunoblots were stained with PCNA (Santa Cruz) and Rad9 (Cell Signaling) antibodies.
Generation of Atad5m/m Mouse Embryos
All embryos were generated by natural mating of Atad5+/m mice. The morning of the day on which a vaginal plug was detected was designated as day 0.5 p.c. and 3.5 p.c. embryos were collected by flushing uteri with M2 medium (Sigma). To genotype embryos collected at other embryonic days, individual yolk sacs were lysed in 20 µl of 1xPCR lysis buffer with 0.2 mg/ml of proteinase K at 55°C for overnight.
Chromosomal Abnormalities and Cell Survival Assay
MEFs were treated with the indicated doses of methyl methane sulfonate (MMS) for 2 hours, washed with PBS three times, and incubated in media for one day. Colcemid (0.1 µg/ml) was added to the medium over night before cells were collected. For each cell culture, 50 metaphases were analyzed for chromosomal abnormalities. MEFs were plated in 96 well plates (1,000 cells/well) in triplicate. The following day, cells were treated with MMS at different doses, ranging from 0.00012 to 0.01% for one hour. Cells were washed with PBS and incubated for ten more days. Cell survival was determined using the CyQuant cell proliferation assay kit (Invitrogen).
Immunofluorescence and Confocal Microscopy
MEF cells were cultured in 2 well Lab Tek chamber slides (Nunc) chamber slide. The next day cells were fixed with 3.7% para-formaldehyde, treated with 0.2% tritonX-100 and stained with phospho-H2AX (α-γ-H2AX; Millipore) and Rad51 (Santa Cruz) antibodies. Fluorescence conjugated anti IgG antibodies (Invitrogen-Molecular Probes) were used as a secondary antibody. Confocal images were collected with a Ziess LSM 510 NLO meta system mounted on a Ziess Axiovert 200 M microscope with an oil immersion Plan-Apochromat X 63/1.4 differential interference contrast objective lens.
Sister Chromatid Exchange for MEF Cells
MEF cells cultured in 10 cm culture dish for one day were incubated with BrdU for 36 hours. Cells were then treated overnight with Colcemid (0.1 µg/ml). For each cell culture, 50 metaphases were analyzed for sister chromatid exchange abnormalities.
Cell Cycle Analysis for MEF Cells
MEF cells cultured in 10 cm culture dishes for a day were harvested and washed three times with PBS. Following fixation with 70% ethanol, cells were washed with PBS, stained with PI solution, and kept at 37°C for 30 minutes before sorting. Data were analyzed by ModFIT software (http://www.vsh.com/products/mflt/index.asp
Generation of the Heterozygous ATAD5-Deficient Chicken DT40 Cells
The full-length chicken cDNA (DDBJ accession number AB511312) encodes a protein of 1,816 amino acids with 45.6% identity to human ATAD5
. Partial chicken (ch) ATAD5
cDNA and genomic sequences were identified by searching databases and cloned by PCR. To knock out ATAD5
in chicken DT40 cells, a chATAD5
targeting vector was designed to replace a region that is homologous to the RFC large subunit, RFC1 (PRKO4195) as assigned by conserved domain database with a drug-resistant gene cassette (Figure S1A
). The ATAD5
-targeting vector was created by replacing a 2.7 kb genomic fragment containing 3 exons that correspond to chATAD5 amino acids 1338–1735, with a his
-resistant gene cassette. DT40 cell cultures, electroporation and subsequent selection, RT-PCR analysis, cell growth determination, colony formation assay were performed as described 
. For γ-ray irradiation, 137
Cs gammacell 40 exactor (MDS Nordion) was used. One allele of chATAD5
gene was targeted as evidenced by Southern blot (Figure S1B
). Semi-quantitative RT-PCR analysis showed that ATAD5
mRNA levels were decreased to nearly half of the wild type level (Figure S2C
). Despite numerous attempts to disrupt the second allele, we were unable to recover homozygous chATAD5
null DT40 cells. In striking contrast to the targeting efficiency of the first chATAD5 allele ~10% (3/29), no double positive clones were obtained from 139 transfectants. Thus, we speculate that complete loss of the chATAD5
gene might be lethal.
Pulsed-Field Gel Electrophoresis
MEFs were treated with 0.01% MMS for one hour and then allowed to recover for 16 hours. ~6×105 cells were imbedded into Pulsed-Field Certified Agarose (Bio-Rad) for a final agarose concentration of 0.75%. Agarose plugs were then digested in proteinase K reaction buffer (100 mM EDTA, pH 8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, and 1 mg/ml Proteinase K) at 50°C overnight and washed 4 times in wash buffer (20 mM Tris, pH 8.0, 50 mM EDTA). The plugs were loaded onto a 1% Pulsed-Field Certified Agarose gel (Bio-Rad). Separation was performed on a CHEF-DR III pulsed-field electrophoresis system (Bio-Rad; 100° field angle, 1200 s switch time, 2 V/cm, 14°C) for 72 hours. The gel was stained with ethidium bromide.
Array-Based Comparative Genome Hybridization (CGH) Analysis
Genomic DNA isolated from normal and tumor tissues from the Atad5m/+ mice, using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), was used for array-CGH array analysis assisted by the NHGRI Genomics core. Data analysis was performed by the NHGRI Bioinformatics core. DNAs were hybridized to Whole Mouse Genome CGH Microarrays (105K) (Agilent Technologies) according to the manufacturer's protocol. After hybridization, arrays were washed and scanned. The scanned array images were analyzed and data was extracted using Agilent Feature Extraction (FE) Software with Linear Lowess normalization and background subtraction. Agilent's DNA Analytics Software (Version 4.0.85) was used to identify chromosomal imbalances. The altered chromosomal regions and breakpoints were detected using ADM-1 with threshold 6.0.
Tissue Microarray Analysis, Pathway Analysis, and Integration of CGH Data with Expression Analysis
Expression profiling was accomplished using the Agilent High Throughput Array (HTA) GeneChip system. All the necessary enzymes and reagents were purchased from the SABiosciences unless specified below. Total RNA was isolated from normal and tumor tissues by guanidinium thiocyanate-phenol-chorofrom extraction using TRIzol reagent (Invitrogen) following the manufacturer's protocol. 600 ng of total RNA from 4 (2 wild-type and 2 tumor) samples were reverse transcribed into cDNA using T7
primer containing the poly T sequence and the promoter for T7 RNA polymerase. After second strand synthesis, double-stranded cDNA was used as a template to synthesize complementary RNA (cRNA) in a modified “Eberwine” type RNA amplification process. During this amplification process, cRNA was labeled with aminoallyl-UTP (Sigma). 8 µg of aminoallyl-cRNA from each sample was conjugated with Cy3-NHS ester (GE Healthcare) to generate Cy3-labelled cRNA. 1.65 µg Cy3-labelled cRNA generated was fragmented by the cRNA fragmentation reaction and then applied for hybridization. Hybridization was done at 65°C for 17 hours with 4 rpm shaking. The Whole Mouse Genome Oligo Microarray assembly was then dissembled in the hybridization wash buffer 1 at room temperature. The microarray slides were washed with the fresh hybridization wash buffer 1 for 1 minute, followed with the hybridization with the wash buffer 2 for one minute at 37°C, with acetonitrile for 1 minute at room temperature, and with the stabilization and drying solution for 30 seconds. The dried microarray slide was scanned into a TIFF image file on an Agilent microarray scanner at 5 µm resolution using default settings. Microarray intensity data were extracted from the TIFF image using the Agilent Feature Extraction Software 9.1.3. The Feature Extraction Software also processed the signal intensities from Cy3 channel for each microarray. The processed data were then imported into GeneSpring GX 10 (Agilent Technologies) software and normalized using the standard settings for Agilent microarray including per chip global normalization. Global normalization was performed by dividing each measurement by the 50th
percentile of all measurements in that sample. The percentile was calculated using only genes marked as present. Data for genes that were absent in all samples and all the control probes were excluded from analyses. Paired t-test were performed to identify a list of statistically significant (P<0.05) differentially expressed genes between normal and tumor samples with a cut-off criteria of expression differences greater than or equal to 2-fold. Each differentially expressed gene was classified according to its Gene Ontology (GO), in which genes are organized into hierarchical categories based on biological process, molecular function and cellular compartment. GO term analysis was performed using Genespring GX 10 software. To determine the functional relationships among the identified genes, differentially expressed genes were imported into Ingenuity Pathway Analysis software (IPA) (http://www.ingenuity.com
). IPA contains the most literature knowledge of biological interactions among the gene products. This web-based software generated a set of molecular network based on interaction between uploaded genes and all other genes present in the knowledge base. In order to compare the measurements obtained from CGH array analysis and microarray expression data, the genes that were present in regions of amplification and deletion detected by array-CGH were chosen to determine their correlation in microarray expression. The goal of this analysis was to detect genes where a copy number change is correlated with the change in expression. Multiple public expression database, Oncomine (https://www.oncomine.org/resource/login.html
) was manually interrogated for mouse Atad5
and its aliases. Only studies comparing normal against tumor tissues were queried for this gene.
Nucleic Acid Isolation
Genomic DNA was isolated from macrodissected tissue with greater than 70% tumor purity using the Puregene kit (Qiagen). Total RNA was isolated with TRIzol (Invitrogen), according to the manufacturer's instructions. cDNA was synthesized using the Super Script III First Strand Synthesis System (Invitrogen).
Human Cell Line Culture and Transfection
Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (Hyclone), 100 U/mL penicillin G and 100 µg/mL streptomycin. Transfections were carried out using Lipofectamine2000 (Invitrogen) and cells were incubated for 48 h prior to harvesting. For RNA interference, transfection was performed two times with an interval of 24 h.
DNA Constructs and siRNA
Full-length ATAD5 cDNA was cloned into p3XFLAG CMV10 expression vector and used as a template for site-directed mutagenesis with the QuickChange Site-Directed Mutagenesis kit (Stratagene). Construct integrity was verified by nucleotide sequencing. siRNAs (Dharmacon) to target the 3′ untranslated region (Utr) of ATAD5 were: 5′- GUAUAUUUCUCGAUGUAC A-3′ (sense) and 5′- UGUACAUCGAGAAAUAUACUU -3′ (antisense).
Immunoprecipitation and Immunoblotting
Triton X-100 soluble and insoluble fractions were isolated using a modified version of the method described by Kannouche et al 
. For immunoprecipitation, proteins were pre-cleared with protein G Sepharose beads (GE Healthcare), incubated with specific antibodies, and then the complex recognized by antibodies was precipitated with protein G Sepharose beads. Proteins or immunoprecipitated samples were resolved on NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) and transferred to PVDF membranes (Bio-Rad), followed by immunoblotting.
Quantitative Reverse Transcription and Real-Time PCR
Total RNAs (500 ng), prepared using TRIzol (Invitrogen) were subjected to qRT-PCR using the SuperScriptIII Platinum Two Step qRT-PCR kit (Invitrogen) and a 7300 Real Time PCR system (Applied Biosystems). Primer sequences used for qRT-PCR were: ATAD5, 5′-CGG AGA CGA AGA AAG CAA AG-3′ (sense) and 5′-CAA TGA GAA ACA AGG GCA GA-3′ (antisense), β-actin, 5′-GCT CGT CGT CGA CAA CGG CTC-3′ (sense) and 5′-CAA ACA TGA TCT GGG TCA TCT TCT C-3′ (antisense). β-actin expression served as an internal control.
PCR Amplification of ATAD5 from Clinical Samples
PCR was performed in a total volume of 10 µl containing 5 ng genomic DNA, 0.16 µM sense primer, 0.16 µM antisense primer, and Immomix (Bioline). PCR amplification was performed on MBS Satellite 384 Thermalcyclers (Thermo Scientific). Cycling conditions were 95°C for 3 min followed by 40 cycles of 95°C for 15 s; 60°C for 15 s; 72°C, 1 min; followed by a final extension of 72°C for 5 min.
Nucleotide Sequencing of ATAD5 from Clinical Samples
Sequence trace quality was assessed with the base-calling program, Phred 
. All traces were included in the subsequent analysis, since deletion-insertion polymorphisms can mimic poor quality data from a Phred-quality measure, but may contain valid sequence data. All sequences for a given primer pair were assembled using Consed 
; overlapping amplimers were assembled separately to allow independent cross validation of calls in overlapping regions. Sequence variants, including single-nucleotide differences and short (<100 base pair) insertions and deletions, were identified using PolyPhred v6.11 
and DIPDetector, an insertion-deletion (indel) detector optimized for improved sensitivity in finding insertions and deletions from aligned trace data. Since a heterozygous insertion or deletion produces traces that are superpositions of two alleles of different length, DIPDetector is able to use Phred's primary and secondary peak predictions at each base position to calculate an autocorrelation function of the trace with itself shifted by different potential insertion or deletion sizes. A maximum in the value of this function with respect to shift size indicates a potential indel. To improve accuracy, DIPDetector also examines the trace alignments for homozygous insertions and deletions, and uses these homozygous genotypes to refine its prediction for the insertion or deletion position, resulting in greater accuracy of positions when compared to PolyPhred. Human genome assembly hg18 (NCBI Build 36.1) was used as the reference sequence. Variant positions were cross-referenced to dbSNP (Build 129) entries to identify known polymorphisms. To determine whether novel variants were somatic mutations or germline polymorphisms, the appropriate tumor DNA and matched normal DNA were re-amplified in an independent PCR followed by sequence analysis of the variant position.
Laser Capture Microdissection and RT-PCR
Frozen tissue sections (8 µM) of OCT-embedded primary tumors were prepared at American HistoLabs Inc., (Gaithersburg MD), and were H&E stained immediately prior to microdissection. Approximately 4000–5000 laser pulses were performed using the following infrared laser parameters: laser spot size 7.5 µm, power 70 mW, duration 950 µs. Each tissue section and the subsequent dissection was reviewed by a staff pathologist. RNA was isolated from LCM samples using the Arcturus Picopure RNA isolation kit (MDS Analytical Technologies). RNA (50–200 ng) was converted to cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). RT-PCR primers were designed to span intronic boundaries to avoid amplification of any contaminating genomic DNA. Primer sequences (5′-3′) were: CTT TTT GAG GAG GTT GAT G (sense) and GTC CAA GTC AGT GTC AAA TAG ATC AC (antisense). Purified RT-PCR products were subjected to bidirectional sequencing to determine whether there was monoallelic or biallelic expression of mutant ATAD5 alleles.