Comment[ArrayExpressAccession] E-GEOD-56450 MAGE-TAB Version 1.1 Public Release Date 2014-06-01 Investigation Title Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines (ChIP-seq data). Comment[Submitted Name] Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines (ChIP-seq data). Experiment Description Background: Germ Cell Cancers (GCC), originating from Primordial Germ Cells /gonocytes, are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS, stem cell component: embryonal carcinoma (EC)). Somatic mutations are rarely found in GCC. It has been proposed that disruption of the epigenetic constitution, either primarily or secondary (e.g. environmental influences), is involved in cancer, and specifically in GCC. Results: This study aims at identifying epigenetic footprints of SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in GCC and related disruption of germ cell maturation. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data was acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP sequencing (activating histone modifications (H3K4me3, H3K27ac)). The data show that known germ cell markers are not only present and differentiating between SE and NS at the expression level, but also in the epigenetic landscape. Conclusion: The overall similarity between TCam-2 / NCCIT supports an erased embryonic gem cell arrested in early gonadal development as common origin. Subtle difference in the (integrated) epigenetic and expression profiles indicated TCam-2 to exhibit a more germ cell like profile (enrichment Androgen regulation) while NCCIT proved more pluripotent. The results provide insight into an integrated analysis of the functional genome in GCC cell lines. Two wildtype germ cell cancer (type II germ cell tumor) cell lines were analyzed. TCam-2 (representative for the seminomatous subtype of germ cell cancer) , [1, 2]) and NCCIT (representative of the non-seminomatous (embryonal carcinoma) subtype of germ cell cancer, [3]). Of each cell line two biological replicates were included. Genomic positions reported are based on the GRch37/hg19 assembly. 1. Mizuno, Y., et al., [Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2)]. Nihon Hinyokika Gakkai Zasshi, 1993. 84(7): p. 1211-8. 2. de Jong, J., et al., Further characterization of the first seminoma cell line TCam-2. Genes Chromosomes Cancer, 2008. 47(3): p. 185-96. 3. Teshima, S., et al., Four new human germ cell tumor cell lines. Lab Invest, 1988. 59(3): p. 328-36. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Rijlaarsdam van der Zwan Rijlaarsdam Rossello Notini de Boer Watkins Gillis Dorssers White Looijenga Person First Name Martin Yvonne Martin Fernando Amanda Suzan D Ad Lambert Stefan Leendert Person Mid Initials Anne G A J J N J C J H Person Email m.a.rijlaarsdam@gmail.com Person Affiliation Erasmus MC Person Phone 0031645408508 Person Address Pathology, Erasmus MC, Wytemaweg 80, Rotterdam, Netherlands Person Roles submitter Protocol Name P-GSE56450-3 P-GSE56450-2 P-GSE56450-1 Protocol Description Image acquisition, bead processing, quality metrics and base calling was performed using SOLiDM-bM-^DM-" Instrument Control Software and SOLiDM-bM-^DM-" Experiment Track System (http://www.lifetechnologies.com/au/en/home/technical-resources/software-downloads/solid-software.html) The ChIP assay was performed according to the low cell number ChIP protocol from Diagenode (Liege, Belgium), with minor modifications. In brief, 1x10e6 cells were cross-linked for eight minutes by addition of formaldehyde to a final concentration of 1%, followed by neutralization with 1.25M glycine. The cells were then lysed, and chromatin was sheared to ~500 bp fragments using the Covaris sonicator under the following conditions; duty cycle 20%, peak incident power 200 watts, cycles/burst 200, time 5min, temperature 4M-aM-5M-^RC. Protein A-coated Dynabeads (Invitrogen) were incubated with 7 M-5g of the following antibodies: H3K4me3 (Diagenode pAb-003-050) or H3K27ac (Abcam Ab4729). The beads were combined with chromatin from 1x106 cells overnight on a rotating wheel. The immunobeads were washed, and DNA was purified using the iPure DNA purification kit (Diagenode AL-100-0100) according to manufacturerM-bM-^@M-^Ys instructions. DNA fragments were sheared a second time using a Covaris sonicator (duty cycle 10%, peak incidence power 175 watts, cycles/burst 200, time 5 minutes, temperature 4M-aM-5M-^RC). Average sequence length was 300nt. According to the manufacturer's instructions Cells (wild type) were cultured in DMEM medium (Life Technologies, #31966-021) containing 10% fetal calf serum (FCS, Hyclone) in T75 cm2 flasks to 75-90% confluence. Protocol Type normalization data transformation protocol nucleic acid library construction protocol growth protocol Experimental Factor Name CELL LINE HISTOLOGICAL_SUBTYPE_PRIMARY TUMOR ANATOMICAL LOCALISATION ANTIBODY Experimental Factor Type cell line histological_subtype_primary tumor anatomical localisation antibody Publication Title Seminoma and embryonal carcinoma footprints identified by analysis of integrated genome-wide epigenetic and expression profiles of germ cell cancer cell lines. Publication Author List van der Zwan YG, Rijlaarsdam MA, Rossello FJ, Notini AJ, de Boer S, Watkins DN, Gillis AJ, Dorssers LC, White SJ, Looijenga LH PubMed ID 24887064 Publication DOI 10.1371/journal.pone.0098330 Comment[SecondaryAccession] GSE56450 Comment[GEOReleaseDate] 2014-06-01 Comment[ArrayExpressSubmissionDate] 2014-04-02 Comment[GEOLastUpdateDate] 2014-07-01 Comment[AEExperimentType] ChIP-seq Comment[SecondaryAccession] SRP040804 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1213182-SRR1213185 SDRF File E-GEOD-56450.sdrf.txt