Transcription enhancers as major determinants of SV40 polyomavirus growth efficiency and host cell tropism.

The non-coding control region (NCCR) of polyomaviruses includes the promoters for early and late genes, a transcription enhancer and the origin of DNA replication. Particularly virulent variants of the human pathogens BKPyV and JCPyV, as well as of simian virus 40 (SV40), occur in vitro and in vivo. These strains often harbour rearrangements in their NCCR, typically deletions of some DNA segment(s) and/or duplications of others. Using an SV40-based model system we provide evidence that duplications of enhancer elements, whether from SV40 itself or from the related BKPyV and JCPyV, increase early gene transcription and replicative capacity. SV40 harbouring subsegments of the strong cytomegalovirus (HCMV) enhancer replicated better than the common 'wild-type' SV40 in the human cell lines HEK293 and U2OS. In conclusion, replacing the SV40 enhancer with heterologous enhancers can profoundly influence SV40's infective capacity, underscoring the potential of small DNA viruses to overcome cell type and species barriers.

Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome.

Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome.

Short-lived mammals (shrew, mouse) have a less robust metal-responsive transcription factor than humans and bats.

Non-essential "heavy" metals such as cadmium tend to accumulate in an organism and thus are a particular threat for long-lived animals. Here we show that two unrelated, short-lived groups of mammals (rodents and shrews, separated by 100 Mio years of evolution) each have independently acquired mutations in their metal-responsive transcription factor (MTF-1) in a domain relevant for robust transcriptional induction by zinc and cadmium. While key amino acids are mutated in rodents, in shrews an entire exon is skipped. Rodents and especially shrews are unique regarding the alterations of this region. To investigate the biological relevance of these alterations, MTF-1s from the common shrew (Sorex araneus), the mouse, humans and a bat (Myotis blythii), were tested by cotransfection with a reporter gene into cells lacking MTF-1. Whereas shrews only live for 1.5-2.5 years, bats, although living on a very similar insect diet, have a lifespan of several decades. We find that bat MTF-1 is similarly metal-responsive as its human counterpart, while shrew MTF-1 is less responsive, similar to mouse MTF-1. We propose that in comparison to most other mammals, the short-lived shrews and rodents can afford a "lower-quality" system for heavy metal homeostasis and detoxification.

Emergence of infectious simian virus 40 whose AT tract in the replication origin/early promoter region is substituted by cellular or viral DNAs.

In simian virus 40 (SV40) and several other polyomaviruses, the TATA box of the early promoter is embedded in an AT tract that is also an essential part of the replication origin. We generated an 'AT trap', an SV40 genome lacking the AT tract and unable to grow in CV-1 monkey cells. Co-transfection of the AT trap with oligonucleotides containing AT tracts of human polyomaviruses, a poly(A : T) tract or variants of the SV40 WT sequence all restored infectious virus. In a transfection of the AT trap without a suitable oligonucleotide, an AT-rich segment was incorporated, stemming either from bovine (calf serum) or monkey (host cell) DNA. Similarly, when cells were grown with human serum, a human DNA segment was captured by SV40 to substitute for the missing AT stretch. We conclude that the virus is quite opportunistic in accepting heterologous substitutes, and that even low-abundance DNA from serum can be incorporated into the viral genome.

A recent evolutionary change affects a regulatory element in the human FOXP2 gene.

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

The legless lizard Anguis fragilis (slow worm) has a potent metal-responsive transcription factor 1 (MTF-1).

The metal-responsive transcription factor-1 (MTF-1) is a key regulator of heavy metal homeostasis and detoxification. Here we characterize the first MTF-1 from a reptile, the slow worm Anguis fragilis. The slow worm, or blind worm, is a legless lizard also known for its long lifespan of up to several decades. Anguis MTF-1 performs well and matches the strong zinc and cadmium response of its human ortholog, clearly surpassing the activity of rodent MTF-1s. Some amino acid positions critical for metal response are the same in humans and slow worm but not in rodent MTF-1. This points to a divergent evolution of rodent MTF-1, and we speculate that rodents can afford a less sophisticated metal handling than humans and (some) reptiles.

A conserved cysteine cluster, essential for transcriptional activity, mediates homodimerization of human metal-responsive transcription factor-1 (MTF-1).

Metal-responsive transcription factor-1 (MTF-1) is a zinc finger protein that activates transcription in response to heavy metals such as Zn(II), Cd(II) and Cu(I) and is also involved in the response to hypoxia and oxidative stress. MTF-1 recognizes a specific DNA sequence motif termed the metal response element (MRE), located in the promoter/enhancer region of its target genes. The functional domains of MTF-1 include, besides the DNA-binding and activation domains and signals for subcellular localization (NLS and NES), a cysteine cluster 632CQCQCAC638 located near the C-terminus. Here we show that this cysteine cluster mediates homodimerization of human MTF-1, and that dimer formation in vivo is important for basal and especially metal-induced transcriptional activity. Neither nuclear translocation nor DNA binding is impaired in a mutant protein in which these cysteines are replaced by alanines. Although zinc supplementation induces MTF-1 dependent transcription it does not per se enhance dimerization, implying that actual zinc sensing is mediated by another domain. By contrast copper, which on its own activates MTF-1 only weakly in the cell lines tested, stabilizes the dimer by inducing intermolecular disulfide bond formation and synergizes with zinc to boost MTF-1 dependent transcription.

Simian virus 40 strains with novel properties generated by replacing the viral enhancer with synthetic oligonucleotides.

Typical enhancers of viral or cellular genes are approximately 100 to 400 bp long and contain several transcription factor binding sites. Previously, we have shown that simian virus 40 (SV40) genomic DNA that lacks its own enhancer can be used as an "enhancer trap" since it reacquires infectivity upon incorporation of heterologous enhancers. Here, we show that SV40 infectivity can be restored with synthetic enhancers that are assembled by the host cell. We found that several oligonucleotides, cotransfected with enhancerless SV40 DNA into host cells, were incorporated into the viral genome via cellular DNA end joining. The oligonucleotides tested included metal response elements (MREs), the binding sites for the transcription factor MTF-1, which induces gene activity in response to heavy metals. These recombinant SV40 strains showed preferential growth on cells overloaded with zinc or cadmium. We also cotransfected enhancerless SV40 DNA with oligonucleotides corresponding to enhancer motifs of human and mouse cytomegalovirus (HCMV and MCMV, respectively). In contrast to SV40 wild type, the viruses with cytomegalovirus-derived patchwork enhancers strongly expressed T-antigen in human HEK293 cells, accompanied by viral DNA replication. Occasionally, we also observed the assembly of functional viral genomes by incorporation of fragments of bovine DNA, an ingredient of the fetal calf serum in the medium. These fragments contained, among other sites, binding sites for AP-1 and CREB transcription factors. Taken together, our studies show that viruses with novel properties can be generated by intracellular incorporation of synthetic enhancer DNA motifs.

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals.

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural 'linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regulatory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manner.

The Drosophila copper transporter Ctr1C functions in male fertility.

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.

Toxicity of Alzheimer's disease-associated Aß peptide is ameliorated in a Drosophila model by tight control of zinc and copper availability.

Amyloid plaques consisting of aggregated Aß peptide are a hallmark of Alzheimer's disease. Among the different forms of Aß, the one of 42aa length (Aß42) is most aggregation-prone and also the most neurotoxic. We find that eye-specific expression of human Aß42 in Drosophila results in a degeneration of eye structures that progresses with age. Dietary supplements of zinc or copper ions exacerbate eye damage. Positive effects are seen with zinc/copper chelators, or with elevated expression of MTF-1, a transcription factor with a key role in metal homeostasis and detoxification, or with human or fly transgenes encoding metallothioneins, metal scavenger proteins. These results show that a tight control of zinc and copper availability can minimize cellular damage associated with Aß42 expression.

Characterization of MtnE, the fifth metallothionein member in Drosophila.

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight metal-binding proteins which occur in almost all forms of life. They bind physiological metals, such as zinc and copper, as well as nonessential, toxic heavy metals, such as cadmium, mercury, and silver. MT expression is regulated at the transcriptional level by metal-regulatory transcription factor 1 (MTF-1), which binds to the metal-response elements (MREs) in the enhancer/promoter regions of MT genes. Drosophila was thought to have four MT genes, namely, MtnA, MtnB, MtnC, and MtnD. Here we characterize a new fifth member of Drosophila MT gene family, coding for metallothionein E (MtnE). The MtnE transcription unit is located head-to-head with the one of MtnD. The intervening sequence contains four MREs which bind, with different affinities, to MTF-1. Both of the divergently transcribed MT genes are completely dependent on MTF-1, whereby MtnE is consistently more strongly transcribed. MtnE expression is induced in response to heavy metals, notably copper, mercury, and silver, and is upregulated in a genetic background where the other four MTs are missing.

Distorted copper homeostasis with decreased sensitivity to cisplatin upon chaperone Atox1 deletion in Drosophila.

Copper is an integral part of a number of proteins and thus an essential trace metal. However, free copper ions can be highly toxic and every organism has to carefully control its bioavailability. Eukaryotes contain three copper chaperones; Atx1p/Atox1 which delivers copper to ATP7 transporters located in the trans-Golgi network, Cox17 which provides copper to the mitochondrial cytochrome c oxidase, and CCS which is a copper chaperone for superoxide dismutase 1. Here we describe the knockout phenotype of the Drosophila homolog of mammalian Atox1 (ATX1 in yeast). Atox1-/- flies develop normally, though at reduced numbers, and the eclosing flies are fertile. However, the mutants are unable to develop on low-copper food. Furthermore, the intestinal copper importer Ctr1B, which is regulated by copper demand, fails to be induced upon copper starvation in Atox1-/- larvae. At the same time, intestinal metallothionein is upregulated. This phenotype, which resembles the one of the ATP7 mutant, is best explained by intestinal copper accumulation, combined with insufficient delivery to the rest of the body. In addition, compared to controls, Drosophila Atox1 mutants are relatively insensitive to the anticancer drug cisplatin, a compound which is also imported via Ctr1 copper transporters and was recently found to bind mammalian Atox1.

Role of amyloid-beta glycine 33 in oligomerization, toxicity, and neuronal plasticity.

The aggregation of the amyloid-beta (Abeta) peptide plays a pivotal role in the pathogenesis of Alzheimer's disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of Abeta there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of Abeta. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic Abeta sequence is important for the aggregation dynamics of the peptide. Abeta peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than Abeta(42) wild type (WT), in vitro and in vivo. Also, whereas Abeta(42) WT is known to inhibit LTP, Abeta(42) G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of Abeta on the molecular level and suggest that G33 is the key amino acid in the toxic activity of Abeta. Thus, a specific toxic conformation of Abeta exists, which represents a promising target for therapeutic interventions.

Extended lifespan of Drosophila parkin mutants through sequestration of redox-active metals and enhancement of anti-oxidative pathways.

The mechanisms underlying neuron death in Parkinson's disease are unknown, but both genetic defects and environmental factors are implicated in its pathogenesis. Mutations in the parkin gene lead to autosomal recessive juvenile Parkinsonism (AR-JP). Here we report that compared to control flies, Drosophila lacking parkin show significantly reduced lifespan but no difference in dopamine neuron numbers when raised on food supplemented with environmental pesticides or mitochondrial toxins. Moreover, chelation of redox-active metals, anti-oxidants and overexpression of superoxide dismutase 1 all significantly reversed the reduced longevity of parkin-deficient flies. Finally, parkin deficiency exacerbated the rough eye phenotype of Drosophila caused by overexpression of the copper importer B (Ctr1B). Taken together, our results demonstrate an important function of parkin in the protection against redox-active metals and pesticides implicated in the etiology of Parkinson's disease. They also corroborate that oxidative stress, perhaps as a consequence of mitochondrial dysfunction, is a major determinant of morbidity in parkin mutant flies.

Human copper transporter Ctr1 is functional in Drosophila, revealing a high degree of conservation between mammals and insects.

Living cells have to carefully control the intracellular concentration of trace metals, especially of copper, which is at the same time essential but owing to its redox activity can also facilitate generation of reactive oxygen species. Mammals have two related copper transporters, Ctr1 and Ctr2, with Ctr1 playing the major role. The fruit fly Drosophila has three family members, termed Ctr1A, Ctr1B, and Ctr1C. Ctr1A is expressed throughout development, and a null mutation causes lethality at an early stage. Ctr1B ensures efficient copper uptake in the intestinal tract, whereas Ctr1C is mainly expressed in male gonads. Ectopic expression of Ctr1 transporters in Drosophila causes toxic effects due to excessive copper uptake. Here, we compare the effects of human Ctr1 (hCtr1) with those of the Drosophila homologs Ctr1A and Ctr1B in two overexpression assays. Whereas the overexpression of Drosophila Ctr1A and Ctr1B results in strong phenotypes, expression of hCtr1 causes only a very mild phenotype, indicating a low copper-import efficiency in the Drosophila system. However, this can be boosted by coexpressing the human copper chaperone CCS. Surprisingly, hCtr1 complements a lethal Ctr1A mutation at least as well as Ctr1A and Ctr1B transgenes. These findings reveal a high level of conservation between the mammalian and insect Ctr1-type copper importers, and they also demonstrate that the Drosophila Ctr1 proteins are functionally interchangeable.

Drosophila bloom helicase maintains genome integrity by inhibiting recombination between divergent DNA sequences.

DNA double strand breaks (DSB) can be repaired either via a sequence independent joining of DNA ends or via homologous recombination. We established a detection system in Drosophila melanogaster to investigate the impact of sequence constraints on the usage of the homology based DSB repair via single strand annealing (SSA), which leads to recombination between direct repeats with concomitant loss of one repeat copy. First of all, we find the SSA frequency to be inversely proportional to the spacer length between the repeats, for spacers up to 2.4 kb in length. We further show that SSA between divergent repeats (homeologous SSA) is suppressed in cell cultures and in vivo in a sensitive manner, recognizing sequence divergences smaller than 0.5%. Finally, we demonstrate that the suppression of homeologous SSA depends on the Bloom helicase (Blm), encoded by the Drosophila gene mus309. Suppression of homeologous recombination is a novel function of Blm in ensuring genomic integrity, not described to date in mammalian systems. Unexpectedly, distinct from its function in Saccharomyces cerevisiae, the mismatch repair factor Msh2 encoded by spel1 does not suppress homeologous SSA in Drosophila.

Copper sensing function of Drosophila metal-responsive transcription factor-1 is mediated by a tetranuclear Cu(I) cluster.

Drosophila melanogaster MTF-1 (dMTF-1) is a copper-responsive transcriptional activator that mediates resistance to Cu, as well as Zn and Cd. Here, we characterize a novel cysteine-rich domain which is crucial for sensing excess intracellular copper by dMTF-1. Transgenic flies expressing mutant dMTF-1 containing alanine substitutions of two, four or six cysteine residues within the sequence (547)CNCTNCKCDQTKSCHGGDC(565) are significantly or completely impaired in their ability to protect flies from copper toxicity and fail to up-regulate MtnA (metallothionein) expression in response to excess Cu. In contrast, these flies exhibit wild-type survival in response to copper deprivation thus revealing that the cysteine cluster domain is required only for sensing Cu load by dMTF-1. Parallel studies show that the isolated cysteine cluster domain is required to protect a copper-sensitive S. cerevisiae ace1Delta strain from copper toxicity. Cu(I) ligation by a Cys-rich domain peptide fragment drives the cooperative assembly of a polydentate [Cu(4)-S(6)] cage structure, characterized by a core of trigonally S(3) coordinated Cu(I) ions bound by bridging thiolate ligands. While reminiscent of Cu(4)-L(6) (L = ligand) tetranuclear clusters in copper regulatory transcription factors of yeast, the absence of significant sequence homology is consistent with convergent evolution of a sensing strategy particularly well suited for Cu(I).

A family knockout of all four Drosophila metallothioneins reveals a central role in copper homeostasis and detoxification.

Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's "copper cells." The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.

The Hordeum bulbosum 25S-18S rDNA region: comparison with Hordeum vulgare and other Triticeae.

Hordeum vulgare and Hordeum bulbosum are two closely related barley species, which share a common H genome. H. vulgare has two nucleolar organizer regions (NORs), while the NOR of H. bulbosum is only one. We sequenced the 2.5 kb 25S-18S region in the rDNA of H. bulbosum and compared it to the same region in H. vulgare as well as to the other Triticeae. The region includes an intergenic spacer (IGS) with a number of subrepeats, a promoter, and an external transcribed spacer (5'ETS). The IGS of H. bulbosum downstream of 25S rRNA contains two 143-bp repeats and six 128-bp repeats. In contrast, the IGS in H. vulgare contains an array of seven 79-bp repeats and a varying number of 135-bp repeats. The 135-bp repeats in H. vulgare and the 128-bp repeats in H. bulbosum show similarity. Compared to H. vulgare, the 5'ETS of H. bulbosum is shorter. Additionally, the 5'ETS regions in H. bulbosum and H. vulgare diverged faster than in other Triticeae genera. Alignment of the Triticeae promoter sequences suggests that in Hordeum, as in diploid Triticum, transcription starts with guanine and not with adenine as it is in many other plants.