At Least 50% of Human-Specific HERV-K (HML-2) Long Terminal Repeats Serve In Vivo as Active Promoters for Host Nonrepetitive DNA Transcription

Retroelements (REs) occupy up to 30 to 40% of vertebrate genomes (21, 36, 48) and are suggested to be potent agents of genomic instability. They cause numerous host DNA rearrangements due to recombination events (19), by transduction of 5′ (22) or 3′ (37) RE flanking sequences into new genomic loci, by creating pseudogenes (14), or by causing RNA recombination (5, 10). Recently expanded gene classes in the human genome, such as those involved in immunity or responses to external stimuli, have transcripts enriched in REs, suggesting a significant role of REs in the diversification and evolution of mammalian genes (45). As mobile carriers of transcriptional regulatory modules, REs can affect host gene expression (27, 31, 42), thus probably taking part in speciation processes (25). In particular, REs might be at least partly responsible for phenotypic differences between Homo sapiens and its closest relatives, Pan paniscus and Pan troglodytes chimpanzees (11, 43). Likely candidates for such a role are endogenous retroviral long terminal repeats (LTRs) (12). Their structure harbors functional enhancers (40), promoters, and polyadenylation signals (43) normally used for retroviral gene expression. However, it was recently demonstrated that LTRs may drive the transcription of adjacent host genomic sequences (26, 45). In the human genome, LTRs may either flank endogenous retroviral “bodies” or exist in the form of solitary LTRs, arisen most probably due to homologous recombination between two identical retroviral LTRs (20, 34) (Fig. ​(Fig.11). FIG. 1. Schematic representation of solitary (left) and proviral (right) LTR expression. The transcription driven from 5′-proviral LTRs results in mRNAs of viral genes, whereas the expression of either solitary or 3′-proviral LTRs results in the ... In attempts to identify factors that might be involved in human-chimpanzee evolutionary divergence, we have focused our research on promoter activities of the HERV-K (HML-2) family of human endogenous retroviruses, the only retroviral group known to contain human-specific members (9, 35). Human-specific HERV-K (HML-2) LTRs share significant sequence identity and form a well-defined cluster (named the HS family) in a phylogenetic tree (9, 35). The members of this family, who have retained their transcriptional activity (4, 15, 16, 47), were found to be tissue-specifically methylated (23, 24, 28) and probably still keep some infectious potential (13, 17, 39, 44). The HS family is thought to be the most biologically active retroviral family in human cells. Several individual HS LTRs are polymorphic in human populations (3, 30, 33), which suggests their very recent integration. In the human genome, the HS family is represented by 156 mostly (∼86%) human-specific LTR sequences. The HS family members can be parts of full-sized HERV-K (HML-2) proviruses (12% of individual HS representatives), truncated proviruses (5%), or solitary LTRs (83%). Recently, we developed a new technique, termed genomic repeat expression monitoring (GREM), for experimental genome-wide identification of promoter-active repetitive elements (7). The technique is based on hybridization of repeat 3′-flanking genomic DNA to pools of total cDNA 5′-terminal parts, followed by selective PCR amplification of the genomic DNA-cDNA heteroduplexes. The resulting library of cDNA/genomic DNA hybrids can be used as a source of tags for individual transcriptionally active repeats. GREM was shown to be adequate for tasks of both quantitative and qualitative analyses of promoter activity. In model experiments, we used GREM to create the first genome-wide map of HS elements that display promoter activity in the testis. Here we utilized GREM for the first comprehensive comparison of HS element promoter activities in healthy human tissue (testicular parenchyma) and in the corresponding cancer (seminoma) from the same patient. We found that at least 50% of HS LTRs were promoter active, and we mapped 20 new functional human-specific promoters. The transcription of many HS LTRs was up- or downregulated in the seminoma. The promoter strengths differed greatly among individual HS elements, and their transcript levels ranged from ∼3 to ∼0.001% of the marker beta-actin gene transcript level. We showed that the main factors affecting the LTR promoter activity were the LTR type (5′ proviral, 3′ proviral, or solitary) and location relative to genes.