Dixie L. Mager

Retroviral Elements and Their Hosts: Insertional Mutagenesis in the Mouse Germ Line

The inbred mouse is an invaluable model for human biology and disease. Nevertheless, when considering genetic mechanisms of variation and disease, it is important to appreciate the significant differences in the spectra of spontaneous mutations that distinguish these species. While insertions of transposable elements are responsible for only ~0.1% of de novo mutations in humans, the figure is 100-fold higher in the laboratory mouse. This striking difference is largely due to the ongoing activity of mouse endogenous retroviral elements. Here we briefly review mouse endogenous retroviruses (ERVs) and their influence on gene expression, analyze mechanisms of interaction between ERVs and the host cell, and summarize the variety of mutations caused by ERV insertions. The prevalence of mouse ERV activity indicates that the genome of the laboratory mouse is presently behind in the “arms race” against invasion.

Transposable Elements: An Abundant and Natural Source of Regulatory Sequences for Host Genes

The fact that transposable elements (TEs) can influence host gene expression was first recognized more than 50 years ago. However, since that time, TEs have been widely regarded as harmful genetic parasites-selfish elements that are rarely co-opted by the genome to serve a beneficial role. Here, we survey recent findings that relate to TE impact on host genes and remind the reader that TEs, in contrast to other noncoding parts of the genome, are uniquely suited to gene regulatory functions. We review recent studies that demonstrate the role of TEs in establishing and rewiring gene regulatory networks and discuss the overall ubiquity of exaptation. We suggest that although individuals within a population can be harmed by the deleterious effects of new TE insertions, the presence of TE sequences in a genome is of overall benefit to the population.

Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Induced pluripotent stem (iPS) cells may be of use in regenerative medicine. However, the low efficiency of reprogramming is a major impediment to the generation of patient-specific iPS cell lines. Here we report the first selection system for the isolation of human iPS cells. We developed the EOS (Early Transposon promoter and Oct-4 (Pou5f1) and Sox2 enhancers) lentiviral vector to specifically express in mouse and human embryonic stem cells but not in primary fibroblasts. The bicistronic EOS vector marked emerging mouse and human iPS cell colonies with EGFP, and we used puromycin selection to aid the isolation of iPS cell lines that expressed endogenous pluripotency markers. These lines differentiated into cell types from all three germ layers. Reporter expression was extinguished upon differentiation and therefore monitored the residual pluripotent cells that form teratomas. Finally, we used EOS selection to establish Rett syndrome–specific mouse and human iPS cell lines with known mutations in MECP2.

Endogenous Human Retroviruses

The genomes of all eukaryotes contain multiple copies of DNA sequences that are related to sequences found in infectious retroviruses (for review, see Coffin, 1984; Garfinkel, 1992). These elements are transmitted through the germ line as stable Mendelian genes, yet they exhibit structural and sequence similarities to infectious exogenous retroviruses. It is these similarities that have led investigators to speculate that endogenous retroviruses are remnants of prior infections with exogenous retroviral agents and, with evolutionary time, changes have occurred to make them no longer infectious or pathogenic. These speculations have been supported with experimental studies that show that the genomes of infectious, exogenous retroviruses can integrate into the host chromosome and be inherited through the germ line. Since retroviruses are thought to have evolved from retrotransposons (Temin, 1980, 1992), it is also possible that some endogenous retrovirus-related sequences are actually precursors of infectious forms. In either case, once they are part of the host genome, these proviruses can serve as a pool of genetic material that exogenous viruses can use to produce variants with altered host specificities and phenotypes; they can encode gene products that compete for or complement in trans retrovirus function(s); and they can, themselves, act as insertional mutagens to change the regulation of host genes.

Endogenous retroviral LTRs as promoters for human genes: A critical assessment

Gene regulatory changes are thought to be major factors driving species evolution, with creation of new regulatory regions likely being instrumental in contributing to diversity among vertebrates. There is growing appreciation for the role of transposable elements (TEs) in gene regulation and, indeed, laboratory investigations have confirmed many specific examples of mammalian genes regulated by promoters donated by endogenous retroviruses (ERVs) or other TEs. Bioinformatics studies have revealed hundreds of additional instances where this is likely to be the case. Since the long terminal repeats (LTRs) of retroviruses naturally contain abundant transcriptional regulatory signals, roles for ERV LTRs in regulating mammalian genes are eminently plausible. Moreover, it seems reasonable that exaptation of an LTR regulatory module provides opportunities for evolution of new gene regulatory patterns. In this Review we summarize known examples of LTRs that function as human gene alternative promoters, as well as the evidence that LTR exaptation has resulted in a pattern of novel gene expression significantly different from the pattern before LTR insertion or from that of gene orthologs lacking the LTR. Available data suggest that, while new expression patterns can arise as a result of LTR usage, this situation is relatively rare and is largely restricted to the placenta. In many cases, the LTR appears to be a minor, alternative promoter with an expression pattern similar to that of the native promoter(s) and hence likely exerts a subtle overall effect on gene expression. We discuss these findings and offer evolutionary models to explain these trends.

DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG‐rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a−/− cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1α binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially ‘rescues’ the DNA methylation defect observed in G9a−/− cells. Taken together, these observations reveal that H3K9me3 and HP1α recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.

The Ly-49 family: genes, proteins and recognition of class I MHC

Summary: The Ly‐49 family consists of at least nine members, of which Ly‐49A and C have been found to be NK‐cell inhibitory receptors specific for class I MHC. The functions of other Ly‐49 molecules are still unclear. Further analysis of Ly‐49 is complicated by the cross‐reactivities of some anti‐Ly‐49 anybodies initially thought to be specific for individual Ly‐49 molecules. Studies on the role of Ly‐49 in hybrid resistance as well as on allelic exclusion are also complicated by our recent finding that a novel Ly‐49CB6 gene is the likely allelic form of Ly‐49CMLB AS opposed to a previously reported highly related but distinct gene in B6 mice. In cell binding assays, only Ly‐49A and C show significant binding to class I MHC, Ly‐49A and C also bind some polysaccharides, and carbohydrates on class I MHC seem to be important for its binding to Ly‐49, However, this interaction involves NOT only the carbohydrate recognition domain of Ly‐49 but also a part of the stalk region, suggesting that both caxbohydrates and peptide backbone of class I MHC may be recognized by Ly‐49. It is likely that additional Ly‐49 mole cells yet to be identified function as NK‐inhibitory receptors specific for class I MHC.

Keeping active endogenous retroviral-like elements in check : the epigenetic perspective

Endogenous retrovirus-like elements, or ERVs, are an abundant component of all eukaryotic genomes. Their transcriptional and retrotranspositional activities have great potential for deleterious effects on gene expression. Consequences of such activity may include germline mutagenesis and cancerous transformation. As a result, mammalian genomes have evolved means of counteracting ERV transcription and mobilization. In this review, we discuss epigenetic mechanisms of ERV and LTR retrotransposon control during mouse development, focusing on involvement of DNA methylation, histone modifications, small RNAs and their interaction with one another. We also address relevance of research performed in the mouse system to human and challenges associated with studying repetitive families. (Part of a multi-author review).Abstract.Endogenous retrovirus-like elements, or ERVs, are an abundant component of all eukaryotic genomes. Their transcriptional and retrotranspositional activities have great potential for deleterious effects on gene expression. Consequences of such activity may include germline mutagenesis and cancerous transformation. As a result, mammalian genomes have evolved means of counteracting ERV transcription and mobilization. In this review, we discuss epigenetic mechanisms of ERV and LTR retrotransposon control during mouse development, focusing on involvement of DNA methylation, histone modifications, small RNAs and their interaction with one another. We also address relevance of research performed in the mouse system to human and challenges associated with studying repetitive families. (Part of a Multi-author Review)

An endogenous retroviral long terminal repeat is the dominant promoter for human β1,3-galactosyltransferase 5 in the colon

LTRs of endogenous retroviruses are known to affect expression of several human genes, typically as a relatively minor alternative promoter. Here, we report that an endogenous retrovirus LTR acts as one of at least two alternative promoters for the human β1,3-galactosyltransferase 5 gene, involved in type 1 Lewis antigen synthesis, and show that the LTR promoter is most active in the gastrointestinal tract and mammary gland. Indeed, the LTR is the dominant promoter in the colon, indicating that this ancient retroviral element has a major impact on gene expression. Using colorectal cancer cell lines and electrophoretic mobility-shift assays, we found that hepatocyte nuclear factor 1 (HNF-1) binds a site within the retroviral promoter and that expression of HNF-1 and interaction with its binding site correlated with promoter activation. We conclude that HNF-1 is at least partially responsible for the tissue-specific activation of the LTR promoter of human β1,3-galactosyltransferase 5. We demonstrate that this tissue-specific transcription factor is implicated in the activation of an LTR gene promoter.

Repeated Recruitment of LTR Retrotransposons as Promoters by the Anti-Apoptotic Locus NAIP during Mammalian Evolution

Neuronal apoptosis inhibitory protein (NAIP, also known as BIRC1) is a member of the conserved inhibitor of apoptosis protein (IAP) family. Lineage-specific rearrangements and expansions of this locus have yielded different copy numbers among primates and rodents, with human retaining a single functional copy and mouse possessing several copies, depending on the strain. Roles for this gene in disease have been documented, but little is known about transcriptional regulation of NAIP. We show here that NAIP has multiple promoters sharing no similarity between human and rodents. Moreover, we demonstrate that multiple, domesticated long terminal repeats (LTRs) of endogenous retroviral elements provide NAIP promoter function in human, mouse, and rat. In human, an LTR serves as a tissue-specific promoter, active primarily in testis. However, in rodents, our evidence indicates that an ancestral LTR common to all rodent genes is the major, constitutive promoter for these genes, and that a second LTR found in two of the mouse genes is a minor promoter. Thus, independently acquired LTRs have assumed regulatory roles for orthologous genes, a remarkable evolutionary scenario. We also demonstrate that 5′ flanking regions of IAP family genes as a group, in both human and mouse are enriched for LTR insertions compared to average genes. We propose several potential explanations for these findings, including a hypothesis that recruitment of LTRs near NAIP or other IAP genes may represent a host-cell adaptation to modulate apoptotic responses.