HERV expression heat-map visualization. Figure shows a heat-map visualization based on the expression of the 100 most significant probesets for all samples and obtained by hierarchical clustering of probeset expression. The hierarchical clustering is represented by the dendrogram on the left-hand side, and is based on the correlation distance between the probeset expression profiles. The color assigned to each point in the heat-map grid is based on the level of expression (log2 scale) of one probeset (rows) in one sample (columns). Red indicates a high expression and green indicates a low expression. Br, breast; Co, colon; Lu, lung; Ov, ovary; Pro, prostate; Ut, uterus; Te, testis; Pl, placenta; N, normal tissue; T, tumoral tissue.

ERVWE1 locus splicing strategy in tumoral testis. (A) ERVWE1 genomic structure and transcripts. The transcription start site at the U3 promoter/R boundary of the 5′ LTR is represented by an arrow, and the splice donor site (DS) and the splice acceptor sites (AS1 and AS2) leading to the two major transcripts are indicated. ERVWE1-specific sequence signature is located by a star. The use of DS in combination with AS1 gives rise to an 8-kb transcript. mRNA (8-kb) contains gag, pol and env regions, and does not code for protein. The use of SD in combination with AS2 gives rise to a 3.1-kb transcript. The 3.1-kb mRNA contains the env ORF exclusively, and is responsible for the production of the Syncytin-1 protein. (B) ERVWE1 probesets. Location of the probesets designed for the detection of ERVWE1 overall transcription (envW_at) and Synyctin-1-specific mRNA (sp.ervwe1_at) are indicated by boxes under ERVWE1 mRNAs illustration. sp.ervwe1_at is composed of probes located at the SD/SA2 junction and probes located at the locus signature (boxes with star into). Position of each probe within each probeset is detailed below, and for each probeset, signal intensity in normal (light gray) and in related tumoral (dark gray) testis, and in placenta (black) samples are illustrated probe-by-probe. (C) qRT-PCR quantification of ERVWE1-spliced Syncytin-1 transcript in normal and tumoral testis. Syncytin-1-specific mRNA (sp.ervwe1) was evaluated by qRT-PCR in four pairs of normal and tumoral related testicular samples. Expressions are normalized with the expression of G6PD housekeeping gene having an averaged number of 1000 copies per 5 ng of total RNA in 250 cells. An arbitrary cutoff at 250 is indicated by a dotted line, mimicking one copy per cell. The star indicates the pair sample used in the microarray experiment.

U3 promoter global methylation comparison in normal versus tumoral testis. (A) Global methylation comparison in the differentially expressed HERV-W LTR. (B) Global methylation comparison in the HERV control LTR. Comparison of CpG methylation status in normal (TN4) and tumoral (TT4) testicular cancer sample of the U3 part of the six differentially expressed HERV-W LTRs (column A) and six HERV LTR controls, namely, three of the HERV-W family and one of each of the HERV-E, HERV-H and HERV-FRD families (column B). All U3 parts are represented and CpG positions are noted by gray circles; CAAT boxes are noted in light gray; and TATAA boxes in darker gray. Highly mutated CAAT and TATAA boxes, in comparison with the reference sequence, are not represented (e.g. in the HERV-E.PTN LTR). Methylated CpG are represented in solid circles and unmethylated CpG are in open circles, undetermined methylation status is in gray circles. Identified SNPs are represented by gray crosses. The percentage of U3 global methylation is indicated for each LTR. For ERVWE1 locus, both the 5′ LTR U3 region and the upstream placental enhancer (TSE) are represented, and the percentage of methylation is indicated for each region.

Identification of HERV-W loci specifically up-regulated in testicular tumor. (A) Structure and genomic position of the identified HERV-W loci. For all the identified loci, the HERV genomic structures are detailed: U3, R and U5 are the functional regions of the LTRs; gag, pol and env are the proviral genes; and Δenv indicates that the envelope gene is deleted. The family type of 5′ and 3′ parts of the HW13386 chimeric locus are indicated. (B) Fold change between normal and tumoral counterpart. Differential RNA expression between normal and tumoral counterpart is indicated for each tissue (testis, uterus, prostate, lung, breast, ovary and colon) for the six overexpressed loci (HW08822, HW13386, HW22795, HW33438, HW38223 and ERVWE1/Wenv). The RNA samples were similar to those used in the microarray experiments. The qRT-PCR results were normalized with G6PD housekeeping gene expression, which had an averaged copy number of 1000.

Identification of autonomous versus non-autonomous LTR activity. (A) Microarray data on U3 versus U5 activity. The dichotomic expressions observed between the U3 and U5 regions of the LTR are represented probe-by-probe for the three identified loci with U3 and U5 parts present on the microarray. Dark gray represents tumoral-testis intensity of the probes, and light gray shows the normal-testis intensity. The positions of all probes along the LTR are schematized below by boxes. The putative initiation site is indicated by an arrow. (B) 5′-RACE identification of autonomous transcriptional induction for the locus HW08822. Sequence alignment of ERVWE1 5′LTR, HW08822 5′LTR and clones obtained by 5′ RACE are represented. The transcriptional initiation site of ERVWE1 is indicated by an arrow as well as the initiation site of HW08822 observed in the three clones. TT1/TN1 match pair was the sample used as in microarray experiment. (C) U3- and U5-specific qRT-PCR s in four testicular normal/tumoral pairs. Expressions of the loci identified as overexpressed in the testicular tumors are represented by dendrograms. Expressions in LTR U3 part are in hatched gray boxes and expressions in LTR U5 part are in filled boxes. Expressions in normal RNA are in light gray and those in tumoral RNA are in dark gray. Expressions are normalized with the expression of G6PD housekeeping gene, which had an averaged copy number of 1000. The values for 1000 copies of G6PD gene are represented by a dotted line. The normal/tumoral sample pair used in the microarray experiment is Testis 1 (*).