Philippe Perot

Microarray-Based Sketches of the HERV Transcriptome Landscape

Human endogenous retroviruses (HERVs) are spread throughout the genome and their long terminal repeats (LTRs) constitute a wide collection of putative regulatory sequences. Phylogenetic similarities and the profusion of integration sites, two inherent characteristics of transposable elements, make it difficult to study individual locus expression in a large-scale approach, and historically apart from some placental and testis-regulated elements, it was generally accepted that HERVs are silent due to epigenetic control. Herein, we have introduced a generic method aiming to optimally characterize individual loci associated with 25-mer probes by minimizing cross-hybridization risks. We therefore set up a microarray dedicated to a collection of 5,573 HERVs that can reasonably be assigned to a unique genomic position. We obtained a first view of the HERV transcriptome by using a composite panel of 40 normal and 39 tumor samples. The experiment showed that almost one third of the HERV repertoire is indeed transcribed. The HERV transcriptome follows tropism rules, is sensitive to the state of differentiation and, unexpectedly, seems not to correlate with the age of the HERV families. The probeset definition within the U3 and U5 regions was used to assign a function to some LTRs (i.e. promoter or polyA) and revealed that (i) autonomous active LTRs are broadly subjected to operational determinism (ii) the cellular gene density is substantially higher in the surrounding environment of active LTRs compared to silent LTRs and (iii) the configuration of neighboring cellular genes differs between active and silent LTRs, showing an approximately 8 kb zone upstream of promoter LTRs characterized by a drastic reduction in sense cellular genes. These gathered observations are discussed in terms of virus/host adaptive strategies, and together with the methods and tools developed for this purpose, this work paves the way for further HERV transcriptome projects.

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes

Background Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data. Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed. Results CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5′ LTR often presented active histone marks. Conclusion These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.

A Comparative Portrait of Retroviral Fusogens and Syncytins

The strongest candidates for developmentally regulated cellular fusogens in mammals are Syncytins which contribute to cell–cell fusion leading to placental syncytiotrophoblast in higher primates, rodents, lagomorphs and sheeps. They consist of domesticated endogenous retroviral envelope glycoproteins (Env) whose fusion properties depend on the initial recognition of a specific receptor. In order to clearly understand Syncytins characteristics, we will first illustrate molecular details characterizing the maturation of class I fusion proteins by introducing envelope-driven fusion in an infectious context, i.e. virus cell fusion, exemplifying each step that lead to functional virions with the most relevant model such as HIV-1 lentivirus or MLV and type D interference group retroviruses. In a second part, we will comparatively present the current knowledge concerning Syncytins and the associated three levels of complexity. First, the placenta is probably more variable in structure than any of the mammalian organs. Second, Syncytins recognize specific and highly function-divergent/unrelated receptors. Third, some Syncytins were shown to exhibit other functions than fusion, such as proliferation, immunomodulation, receptor interference and anti-apoptotic properties. We will conclude by a brief overview of the consequences of Syncytin expression outside of its privileged tissue.

From Viruses to Genes: Syncytins

The content of 5–90 million years old retroviruses and even older retrotransposons of animal genomes and the wide variety of modern retroviruses infecting the same range of species suggest that these elements can be assimilated to shuttle across evolution. A snapshot taken a few decades ago showed us the capture of cellular proto-oncogenes by infectious elements, representing the dark side of the communication between the worlds of viruses and animals. Another snapshot we took more recently shows multiple captures by animal genomes of envelope genes originating from infectious retroviruses, illustrating a phenomenon of convergent evolution. This could be seen as the bright side of these relations as those envelopes were shown to be involved in the earlier steps of human development, i.e. fusion of placental syncytiotrophoblastic layer, therefore they were dubbed Syncytins. Sequencing of more and more animal genomes allowed comparative genomic analyses that revealed how these envelopes have been domesticated in human, mouse, goat, rabbit, etc. More generally, we illustrate in this chapter how close are the viral and animal genome worlds and, focusing mainly on the hominoid ERVWE1 locus encoding Synctin-1, how the different proviruses encoding Syncytins have been domesticated to achieve placental functions. Influence of the chromosomal integration context, the epigenetic control and the splicing strategy upon transcription, and protein maturation processes as well will be discussed in order to illustrate what makes these nowadays genes different from their ancestral infectious counterpart. The price to pay for this beneficial invasion will be illustrated by the possible implications of Syncytin-1 in a wide range of diseases. Last, the apparent stringency of placental regulation will await to be challenged as regard to the evidence of expression in other physiological fusogenic contexts such as myoblasts and osteoclasts.

A comprehensive hybridization model allows whole HERV transcriptome profiling using high density microarray

BackgroundHuman endogenous retroviruses (HERVs) have received much attention for their implications in the etiology of many human diseases and their profound effect on evolution. Notably, recent studies have highlighted associations between HERVs expression and cancers (Yu et al., Int J Mol Med 32, 2013), autoimmunity (Balada et al., Int Rev Immunol 29:351–370, 2010) and neurological (Christensen, J Neuroimmune Pharmacol 5:326–335, 2010) conditions. Their repetitive nature makes their study particularly challenging, where expression studies have largely focused on individual loci (De Parseval et al., J Virol 77:10414–10422, 2003) or general trends within families (Forsman et al., J Virol Methods 129:16–30, 2005; Seifarth et al., J Virol 79:341–352, 2005; Pichon et al., Nucleic Acids Res 34:e46, 2006).MethodsTo refine our understanding of HERVs activity, we introduce here a new microarray, HERV-V3. This work was made possible by the careful detection and annotation of genomic HERV/MaLR sequences as well as the development of a new hybridization model, allowing the optimization of probe performances and the control of cross-reactions.ResultsHERV-V3 offers an almost complete coverage of HERVs and their ancestors (mammalian apparent LTR-retrotransposons, MaLRs) at the locus level along with four other repertoires (active LINE-1 elements, lncRNA, a selection of 1559 human genes and common infectious viruses). We demonstrate that HERV-V3 analytical performances are comparable with commercial Affymetrix arrays, and that for a selection of tissue/pathological specific loci, the patterns of expression measured on HERV-V3 is consistent with those reported in the literature.ConclusionsGiven its large HERVs/MaLRs coverage and additional repertoires, HERV-V3 opens the door to multiple applications such as enhancers and alternative promoters identification, biomarkers identification as well as the characterization of genes and HERVs/MaLRs modulation caused by viral infection.