Human LINE retrotransposons generate processed pseudogenes.

Long interspersed elements (LINEs) are endogenous mobile genetic elements that have dispersed and accumulated in the genomes of higher eukaryotes via germline transposition, with up to 100,000 copies in mammalian genomes. In humans, LINEs are the major source of insertional mutagenesis, being involved in both germinal and somatic mutant phenotypes. Here we show that the human LINE retrotransposons, which transpose through the reverse transcription of their own transcript, can also mobilize transcribed DNA not associated with a LINE sequence by a process involving the diversion of the LINE enzymatic machinery by the corresponding mRNA transcripts. This results in the 'retroposition' of the transcribed gene and the formation of new copies that disclose features characteristic of the widespread and naturally occurring processed pseudogenes: loss of intron and promoter, acquisition of a poly(A) 3' end and presence of target-site duplications of varying length. We further show-by introducing deletions within either coding sequence of the human LINE-that both ORFs are necessary for the formation of the processed pseudogenes, and that retroviral-like elements are not able to produce similar structures in the same assay. Our results strengthen the unique versatility of LINEs as genome modellers.

Taming of transposable elements by homology-dependent gene silencing.

Transposable elements can invade virgin genomes within a few generations, after which the elements are 'tamed' and retain only limited transpositional activity. Introduction of the I element, a transposon similar to mammalian LINE elements, into Drosophila melanogaster genomes devoid of such elements initially results in high-frequency transposition of the incoming transposon, high mutation rate, chromosomal nondisjunction and female sterility, a syndrome referred to as hybrid dysgenesis (for review, see refs 2-4); a related syndrome has also been described in mammals. High-frequency transposition is transient, as the number of I elements reaches a finite value and transposition ceases after approximately ten generations. It has been proposed that the I elements encode a factor that negatively regulates their own transcription, but evidence for such a mechanism is lacking. Using the hybrid dysgenesis syndrome in Drosophila as a model, we show here that transpositional activity of the I element can be repressed by prior introduction of transgenes expressing a small internal region of the I element. This autoregulation presents features characteristic of homology-dependent gene silencing, a process known as cosuppression. Repression does not require any translatable sequence, its severity correlates with transgene copy number and it develops in a generation-dependent manner via germline transmission of a silencing effector in females only. These results demonstrate that transposable elements are prone to and can be tamed by homology-dependent gene silencing, a process that may have emerged during the course of evolution as a specific defense mechanism against these elements.

Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta is restricted to villous cytotrophoblastic cells.

Recently, the expression of a human endogenous retrovirus HERV-FRD, able to encode a fusogenic envelope protein (syncytin 2), has been observed in human placenta. The aim of the present study was to localize the expression of syncytin 2 in first trimester placenta. In addition, we investigated the presence of HERV-FRD transcripts during the in vitro differentiation of isolated villous and extravillous trophoblastic cells from first trimester chorionic villi. Using a monoclonal antibody specifically raised against the HERV-FRD Env protein, syncytin 2 was immunolocalized only in the villous trophoblast of the chorionic villi, at the level of cytotrophoblastic cells. Interestingly, immunostaining was not observed in all cells but only in some of them, and was detected, more frequently, at the membrane level at the interface between the cytotrophoblastic cells and syncytiotrophoblast. Labeling was observed neither in the syncytiotrophoblast nor in the mesenchymal core of the villi nor in the extravillous trophoblast. In vitro detection of HERV-FRD transcripts was restricted to villous trophoblastic cells and decreased significantly with time in culture. These results suggest that syncytin 2 might play a role in human trophoblastic cell fusion.

Germ line-specific expression of intracisternal A-particle retrotransposons in transgenic mice.

Intracisternal A-particle (IAP) sequences are endogenous retrovirus-like mobile elements, or retrotransposons, present at 1,000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. To analyze the in vivo pattern of expression of these retrovirus-like elements, we constructed several independent transgenic mice with either a complete IAP element marked with an intron or with the IAP promoter, or long terminal repeat (LTR), alone controlling the expression of a lacZ reporter gene with a nuclear localization signal. For all transgenic lines analyzed, IAP expression as determined by reverse transcription-PCR analysis was found to be essentially restricted to the male germ line. Furthermore, in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of all organs disclosed specific beta-galactosidase-positive blue cells only within the testis, found as patches along the seminiferous tubules and often organized as assemblies of 2, 4, 8, or 16 cells. Histochemical analyses of tissues from 13.5-day-old embryos to adults demonstrated that this LTR activity is restricted to gonocytes and premeiotic undifferentiated spermatogonia. Finally, analysis of the methylation status of both transgenes and endogenous IAP LTRs demonstrated identical patterns, with methylation in somatic tissues and hypomethylation in the testis. Transgenic mice therefore reveal an intrinsic, highly restricted IAP expression which had escaped detection in previous global Northern (RNA) blot analyses and with possible strong biological relevance, as IAP activation specifically within the germ line might be a way to generate diversity at the evolutionary level without being deleterious to individuals.

A truncated form of the human CAF-1 p150 subunit impairs the maintenance of transcriptional gene silencing in mammalian cells.

Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA. In S. cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacDelta mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin. Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner. This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state. Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies. We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5' truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells. Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes. These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin.

Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor.

By transfection experiments, we previously identified a 72-bp enhancer sequence within the Drosophila copia retrotransposon which is involved in the control of the transcription level of this mobile element in cells in culture. Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells further demonstrated specific interactions of at least two nuclear factors with this enhancer sequence. Using this sequence as a probe for the screening of an expression cDNA library that we constructed from DH-33 cells RNA, we have isolated a cDNA clone encoding a 110-kDa protein with features common to those of known transcription factors; these include a two-zinc-finger motif at the C terminus, three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain which shares homology with the Bric-à-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element was demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses revealed a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance. Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D.melanogaster, with a repressor effect in the central nervous systems of the embryos.

Members of the SRY family regulate the human LINE retrotransposons.

LINEs are endogenous mobile genetic elements which have dispersed and accumulated in the genomes of most higher eukaryotes via germline transposition, with up to 100 000 copies for the human LINE-1 (L1H) sequences. Although severely repressed in most normal tissues, L1H is still functional, with evidence for both germline and somatic-essentially in tumors-transpositions. Yet, no transcription factor that could regulate their transcription and be responsible for their transposition has hitherto been described. Here we show that factors belonging to the family of the testis-determining factor gene SRY (the SOX family) can modulate L1H promoter activity over a 10-fold range in a transient transfection assay using a luciferase reporter gene. These effects depend on two functional SRY binding sites which can be identified within the L1H promoter via mobility shift assays. Induction of endogenous L1Hs upon ectopic expression of the SOX11 transcription factor is further demonstrated, thus strengthening the physiological relevance of these new-and highly dispersed-target sites for the otherwise unclassical transcription factors of the SRY family.

Expression of intracisternal A-particle retrotransposons in primary tumors of oncogene-expressing transgenic mice.

Intracisternal A-Particle (IAP) sequences are endogenous retrovirus-like mobile elements, present at 1000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. The in vivo pattern of expression of these potentially mutagenic elements had previously been analysed in normal mice, and we have now investigated their expression in transgenic mice carrying different oncogenes (e.g. c-myc, v-Ha-ras, SV40 T-antigen) under tissue-specific promoters and disclosing tumors within the brain, the mammary or salivary glands, or the lymphoid organs. Northern blot analysis of IAP expression within the resulting tumors demonstrates a lack of significant and/or systematic effect of v-Ha-ras and SV40 T-antigen expression, but a systematic IAP induction in the myc-induced tumors. In this case, however, analysis of double transgenic mice obtained by crossing the tumor-prone mice with previously described transgenic mice carrying IAP reporter genes did not provide any evidence for induction of the IAP transgenes, therefore strongly suggesting that c-myc expression had an effect on only a limited number of IAP sequences--most probably depending on their position and/or methylation state. These results strengthen the importance of in vivo studies for a correct appraisal of complex biological processes, and moderate previous conclusions derived from in vitro analyses on the general activation of IAPs by oncogenes and on the role of these transposable elements in tumorigenesis.

IAP retrotransposons in the mouse liver as reporters of ageing.

IAP are endogenous retrovirus-like elements present at a thousand copies in the murine genome. They can modulate the level of expression of the tagged genes into which they have inserted, and conversely their activity could be influenced by the level of activity of the genes and/or DNA sequences into which they are embedded. In this report, we have analysed by Northern blots the pattern of expression of the IAP-related transcripts in the organs of young and ageing mice. We show that IAP transcripts of unexpected size (namely 10 kb and 6 kb) are induced in the liver of ageing mice from all inbred and hybrid strains tested. These transcripts are not detected in young mice, and their intensity disclose variations depending on the strain, as those observed for the two canonical 7.2 and 5.4 kb IAP transcripts. It is suggested that these age-dependent IAP transcripts originate from unique sites within the mouse genome that are 'tagged' by an IAP sequence, which would be sensitive both to strain-dependent cellular factors acting at the level of all IAPs, and to an age-dependent liver-specific cellular factor and/or DNA state, responsible for the position-dependent effect. These age-dependent transcripts should allow the identification of putative genes or factors of 'senescence'.

LINEs, SINEs and processed pseudogenes: parasitic strategies for genome modeling.

Two major classes of retrotransposons have invaded eukaryotic genomes: the LTR retrotransposons closely resembling the proviral integrated form of infectious retroviruses, and the non-LTR retrotransposons including the widespread, autonomous LINE elements. Here, we review the modeling effects of the latter class of elements, which are the most active in humans, and whose enzymatic machinery is subverted to generate a large series of "secondary" retroelements. These include the processed pseudogenes, naturally present in all eukaryotic genomes possessing non-LTR retroelements, and the very successful SINE elements such as the human Alu sequences which have evolved refined parasitic strategies to efficiently bypass the original "protectionist" cis-preference of LINEs for their own retrotransposition.

Human endogenous retroviruses: from infectious elements to human genes.

Mammalian genomes contain a heavy load (42% in humans) of retroelements, which are mobile sequences requiring reverse transcription for their replicative transposition. A significant proportion of these elements is of retroviral origin, with thousands of sequences resembling the integrated form of infectious retroviruses, with two LTRs bordering internal regions homologous to the gag, prt, pol, and env genes. These elements, named endogenous retroviruses (ERVs), are most probably the proviral remnants of ancestral germ-line infections by active retroviruses, which have thereafter been transmitted in a Mendelian manner. The complete sequencing of the human genome now allows a comprehensive survey of human ERVs (HERVs), which can be grouped according to sequence homologies into approximately 80 distinct families, each containing a few to several hundred elements. As reviewed here, strong similarities between HERVs and present-day retroviruses can be inferred from phylogenetic analyses on the reverse transcriptase (RT) domain of the pol gene or the transmembrane subunit (TM) of the env gene, which disclose interspersion of both classes of elements and suggest a common history and shared ancestors. Similarities are also observed at the functional levels, since despite the fact that most HERVs have accumulated mutations, deletions, and/or truncations, several elements still possess some of the functions of retroviruses, with evidence for viral-like particle formation, and occurrence of envelope proteins allowing cell-cell fusion and even conferring infectivity to pseudotypes. Along this line, a genomewide screening for human retroviral genes with coding capacity has revealed 16 fully coding envelope genes. These genes are transcribed in several healthy tissues including the placenta, three of them at a very high level. Besides their impact in modelling the genome, HERVs thus appear to contain still active genes, which most probably have been subverted by the host for its benefit and should be considered as bona fide human genes. Some of their characteristic features and possible physiological roles, as well as potential pathological effects inherited from their retroviral ancestors are also reviewed.

Cosuppression of I transposon activity in Drosophila by I-containing sense and antisense transgenes.

We have previously shown that the activity of functional I elements introduced into Drosophila devoid of such elements can be repressed by transgenes containing an internal nontranslatable part of the I element itself and that this repressing effect presents features characteristic of homology-dependent gene silencing or cosuppression. Here we show that transgenes containing a fragment of the I element in antisense orientation induce I-element silencing with the same characteristic features as the corresponding sense construct: namely, repression takes several generations to be fully established, with similar rates for sense and antisense constructs, and it is only maternally transmitted, with reversal of the effect through paternal transmission. We also show that transcription of the transgenes is necessary to produce the silencing effect and that repression can be maintained for at least one generation following elimination of the transgenes, thus strongly suggesting that a transgene product and not the transgene per se is the essential intermediate in the silencing effect. The data presented strongly support models in which the repressing effect of antisense transcripts involves the same mechanisms as cosuppression by sense constructs and emphasize the role of symmetrically acting nucleic acid structures in mediating repression.

Identification of four families of yCCR4- and Mg2+-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding.

BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. RESULTS: Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

ERV-3 envelope expression and congenital heart block: what does a physiological knockout teach us.

Congenital heart block is a serious condition with significant mortality due in most cases to the transplacental transfer of autoantibodies from an otherwise asymptomatic mother. Although SSA/Ro and SSB/La autoantibodies have been implicated, attention has focused recently on autoantibodies to envelope proteins of endogenous retrovirus-3 (ERV-3). We have recently identified in 1% of the caucasian population a natural knock out of ERV-3 due to a premature stop mutation generating a severely truncated form of the protein [corrected]. If a pregnant female homozygous for the truncated form of the ERV-3 carries a foetus expressing the entire protein, the mother might be expected to acquire high titre immunity, while the foetus homozygous for the truncated form would not be expected to immunise its mother. In order to test whether this naturally occurring model could shed light on the pathogenesis of CHB, we determined the status of the ERV-3 stop polymorphism in 12 mothers of CHB infants [corrected]. The fact that none was homozygous for the stop mutation tends to rule out a role for the stop polymorphism of the mothers in the generation of the CHB disease, but does not exclude that other polymorphisms might be involved [corrected].

Allosteric effects of diprobutine on acetylcholine receptors.

The nicotinic effects of a novel antiparkinsonian compound, diprobutine were investigated on the acetylcholine receptor (AChR) from Torpedo marmorata electric organ and on rat brain membranes by a variety of techniques including stopped flow measurements. On the nicotinic AChR from Torpedo, diprobutine behaved as a typical noncompetitive blocker: it inhibited the agonist-regulated 22Na+ efflux from excitable microsacs; it shifted in the ms-s time-range the conformation of the AChR towards a high affinity state for agonists; it competed with [3H]PCP bound to its high affinity 'allosteric' site. On rat brain membrane, it displaced [3H]PCP bound to its high affinity site. The pharmacological properties of diprobutine are discussed in the context of its biochemical effects.

From ancestral infectious retroviruses to bona fide cellular genes: role of the captured syncytins in placentation.

During their replication, infectious retroviruses insert a reverse-transcribed cDNA copy of their genome, a "provirus", into the genome of their host. If the infected cell belongs to the germline, the integrated provirus can become "fixed" within the host genome as an endogenous retrovirus and be transmitted vertically to the progeny in a Mendelian fashion. Based on the numerous proviral sequences that are recovered within the genomic DNA of vertebrates--up to ten percent in the case of mammals--such events must have occurred repeatedly during the course of millions of years of evolution. Although most of the ancient proviral sequences have been disrupted, a few "endogenized" retroviral genes are conserved and still encode functional proteins. In this review, we focus on the recent discovery of genes derived from the envelope glycoprotein-encoding (env) genes of endogenous retroviruses that have been domesticated by mammals to carry out an essential function in placental development. They were called syncytins based on the membrane fusogenic capacity that they have kept from their parental env gene and which contributes to the formation of the placental fused cell layer called the syncytiotrophoblast, at the materno-fetal interface. Remarkably, the capture of syncytin or syncytin-like genes, sometimes as pairs, was found to have occurred independently from different endogenous retroviruses in diverse mammalian lineages such as primates--including humans--, muroids, leporids, carnivores, caviids, and ovis, between around 10 and 85 million years ago. Knocking out one or both mouse syncytin-A and -B genes provided evidence that they indeed play a critical role in placentation. We discuss the possibility that the immunosuppressive domain embedded within retroviral envelope glycoproteins and conserved in syncytin proteins, may be involved in the tolerance of the fetus by the maternal immune system. Finally, we speculate that the capture of a founding syncytin-like gene could have been instrumental in the dramatic transition from egg-laying to placental mammals.

A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha.

Syncytins are genes of retroviral origin that have been co-opted by mammalian hosts for a function in placentation. Two such genes have already been identified in simians, as well as two distinct, unrelated ones in Muridae and a fifth in the rabbit. Here we searched for similar genes in the guinea pig, which belongs to the Caviomorpha lineage within the Hystricognathi suborder of rodents and displays a placental structural organization with several characteristic features comparable to those of the human organ, including deep trophoblast invasion of maternal tissues. An in silico search for envelope (env) genes with full coding capacity identified a candidate gene that showed specific expression in the placenta, as revealed by RT-qPCR using RNAs from a large panel of tissues. This gene belongs to an endogenous retroviral element present at a single-copy in the guinea pig genome, still displaying a retroviral organization - with a degenerate gag and pol, but an intact env gene. In situ hybridization of guinea pig placenta sections demonstrated specific expression at the level of the invasive trophoblast-containing junctional zone, as observed in humans for syncytin-1 and consistent with a role in invasion of the maternal uterine tissues. The identified gene displays a conserved open reading frame in the Caviomorpha, consistent with an entry date >30 million years, and sequence analyses showed purifying selection of the gene. Conclusively, despite the absence of a demonstrated fusogenic activity, it is likely that the identified env gene - that we named syncytin-like env-Cav1 - exerts a physiological function possibly related to trophoblast invasion, in the course of caviomorph placentation.