The Hsp70 chaperone is a major player in stress-induced transposable element activation.

Previous studies have shown that heat shock stress may activate transposable elements (TEs) in Drosophila and other organisms. Such an effect depends on the disruption of a chaperone complex that is normally involved in biogenesis of Piwi-interacting RNAs (piRNAs), the largest class of germline-enriched small noncoding RNAs implicated in the epigenetic silencing of TEs. However, a satisfying picture of how chaperones could be involved in repressing TEs in germ cells is still unknown. Here we show that, in Drosophila, heat shock stress increases the expression of TEs at a posttranscriptional level by affecting piRNA biogenesis through the action of the inducible chaperone Hsp70. We found that stress-induced TE activation is triggered by an interaction of Hsp70 with the Hsc70-Hsp90 complex and other factors all involved in piRNA biogenesis in both ovaries and testes. Such interaction induces a displacement of all such factors to the lysosomes, resulting in a functional collapse of piRNA biogenesis. This mechanism has clear evolutionary implications. In the presence of drastic environmental changes, Hsp70 plays a key dual role in increasing both the survival probability of individuals and the genetic variability in their germ cells. The consequent increase of genetic variation in a population potentiates evolutionary plasticity and evolvability.

Stress-induced strain and brain region-specific activation of LINE-1 transposons in adult mice.

Transposable elements (TEs) are conserved mobile genetic elements that are highly abundant in most eukaryotic genomes. Although the exact function of TEs is still largely unknown, it is increasingly clear that they are significantly modulated in response to stress in a wide range of organisms, either directly or indirectly through regulation of epigenetic silencing. We investigated the effect of repeated restraint stress (2 h a day, for 5 d) on transcription levels of LINE-1 (L1) retrotransposon in the brain of inbred BALB/c, DBA/2, C57BL/6N, and outbred CD1 mice. Repeated restraint stress induced strain and brain region-specific modulation of L1 activity. We observed a significant derepression of L1 transcription in the hippocampus (HIPP) of BALB/c mice and a significant downregulation in the hippocampus of C57BL/6N mice. No significant change in L1 expression was found in the other strains and brain regions. These findings indicate in mice the control of transposons expression as an additional mechanism in stress-induced pathophysiological responses, demonstrating that their regulation is highly dependent on the strain genetic background and the brain region. Lay summary Hippocampal expression of the transposon L1 is significantly altered by repeated restraint stress in mice. L1 modulation is not only region specific, but also strain dependent, suggesting that the genetic background is an important determinant of L1 response to environmental stimuli.

Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons.

The canalization concept describes the resistance of a developmental process to phenotypic variation, regardless of genetic and environmental perturbations, owing to the existence of buffering mechanisms. Severe perturbations, which overcome such buffering mechanisms, produce altered phenotypes that can be heritable and can themselves be canalized by a genetic assimilation process. An important implication of this concept is that the buffering mechanism could be genetically controlled. Recent studies on Hsp90, a protein involved in several cellular processes and development pathways, indicate that it is a possible molecular mechanism for canalization and genetic assimilation. In both flies and plants, mutations in the Hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden genetic variability. Thus, Hsp90 chaperone machinery may be an evolutionarily conserved buffering mechanism of phenotypic variance, which provides the genetic material for natural selection. Here we offer an additional, perhaps alternative, explanation for proposals of a concrete mechanism underlying canalization. We show that, in Drosophila, functional alterations of Hsp90 affect the Piwi-interacting RNA (piRNA; a class of germ-line-specific small RNAs) silencing mechanism leading to transposon activation and the induction of morphological mutants. This indicates that Hsp90 mutations can generate new variation by transposon-mediated 'canonical' mutagenesis.

Transposons, environmental changes, and heritable induced phenotypic variability.

The mechanisms of biological evolution have always been, and still are, the subject of intense debate and modeling. One of the main problems is how the genetic variability is produced and maintained in order to make the organisms adaptable to environmental changes and therefore capable of evolving. In recent years, it has been reported that, in flies and plants, mutations in Hsp90 gene are capable to induce, with a low frequency, many different developmental abnormalities depending on the genetic backgrounds. This has suggested that the reduction of Hsp90 amount makes different development pathways more sensitive to hidden genetic variability. This suggestion revitalized a classical debate around the original Waddington hypothesis of canalization and genetic assimilation making Hsp90 the prototype of morphological capacitor. Other data have also suggested a different mechanism that revitalizes another classic debate about the response of genome to physiological and environmental stress put forward by Barbara McClintock. That data demonstrated that Hsp90 is involved in repression of transposon activity by playing a significant role in piwi-interacting RNA (piRNAs)-dependent RNA interference (RNAi) silencing. The important implication is that the fixed phenotypic abnormalities observed in Hsp90 mutants are probably related to de novo induced mutations by transposon activation. In this case, Hsp90 could be considered as a mutator. In the present theoretical paper, we discuss several possible implications about environmental stress, transposon, and evolution offering also a support to the concept of evolvability.

HP1: a functionally multifaceted protein.

HP1 (heterochromatin protein 1) is a nonhistone chromosomal protein first discovered in Drosophila melanogaster because of its association with heterochromatin. Numerous studies have shown that such a protein plays a role in heterochromatin formation and gene silencing in many organisms, including fungi and animals. Cytogenetic and molecular studies, performed in Drosophila and other organisms, have revealed that HP1 associates with heterochromatin, telomeres and multiple euchromatic sites. There is increasing evidence that the different locations of HP1 are related to multiple different functions. In fact, recent work has shown that HP1 has a role not only in heterochromatin formation and gene silencing, but also in telomere stability and in positive regulation of gene expression.

Heterochromatin protein 1 (HP1a) positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila.

Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

Cytological heterogeneity of heterochromatin among 10 sequenced Drosophila species.

In Drosophila chromosomal rearrangements can be maintained and are associated with karyotypic variability among populations from different geographic localities. The abundance of variability in gene arrangements among chromosomal arms is even greater when comparing more distantly related species and the study of these chromosomal changes has provided insights into the evolutionary history of species in the genus. In addition, the sequencing of genomes of several Drosophila species has offered the opportunity to establish the global pattern of genomic evolution, at both genetic and chromosomal level. The combined approaches of comparative analysis of syntenic blocks and direct physical maps on polytene chromosomes have elucidated changes in the orientation of genomic sequences and the difference between heterochromatic and euchromatic regions. Unfortunately, the centromeric heterochromatic regions cannot be studied using the cytological maps of polytene chromosomes because they are underreplicated and therefore reside in the chromocenter. In Drosophila melanogaster, a cytological map of the heterochromatin has been elaborated using mitotic chromosomes from larval neuroblasts. In the current work, we have expanded on that mapping by producing cytological maps of the mitotic heterochromatin in an additional 10 sequenced Drosophila species. These maps highlight 2 apparently different paths, for the evolution of the pericentric heterochromatin between the subgenera Sophophora and Drosophila. One path leads toward a progressive complexity of the pericentric heterochromatin (Sophophora) and the other toward a progressive simplification (Drosophila). These maps are also useful for a better understanding how karyotypes have been altered by chromosome arm reshuffling during evolution.

Characterization of Gfat1 (zeppelin) and Gfat2, Essential Paralogous Genes Which Encode the Enzymes That Catalyze the Rate-Limiting Step in the Hexosamine Biosynthetic Pathway in Drosophila melanogaster.

The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. We show here that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. We also describe the isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345), and demonstrate that ubiquitous expression of Gfat1+ or Gfat2+ transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 to be located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. We have also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.

Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin.

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.

Heterochromatin protein 1 interacts with 5'UTR of transposable element ZAM in a sequence-specific fashion.

The realization of cross talks between transposable elements of class I and their host genome involves non-histonic chromatin proteins. These interactions have been widely analyzed through the characterization of the gypsy retrotransposon leader region, which holds a particularly strong insulator element, and the proteins required for its function, Su(Hw), Mod(mdg4), and Cp190. Here we provide evidence that a similar interaction should occur between ZAM, a gypsy-like element, and HP1, one of the most extensively studied chromatin proteins. We first assayed the existence of this binding using the yeast cells one-hybrid system and then we verified it in vivo by ChIP assay. In order to characterize the interaction between HP1 and the ZAM 5' untranslated region we performed a series of gel shift analyses. Our observations confirm an HP1 co-operative DNA-binding and display for the first time the HP1 DNA target motif that, we hypothesize, should be one of its nucleation sites.

Heterochromatic genes in Drosophila: a comparative analysis of two genes.

Centromeric heterochromatin comprises approximately 30% of the Drosophila melanogaster genome, forming a transcriptionally repressive environment that silences euchromatic genes juxtaposed nearby. Surprisingly, there are genes naturally resident in heterochromatin, which appear to require this environment for optimal activity. Here we report an evolutionary analysis of two genes, Dbp80 and RpL15, which are adjacent in proximal 3L heterochromatin of D. melanogaster. DmDbp80 is typical of previously described heterochromatic genes: large, with repetitive sequences in its many introns. In contrast, DmRpL15 is uncharacteristically small. The orthologs of these genes were examined in D. pseudoobscura and D. virilis. In situ hybridization and whole-genome assembly analysis show that these genes are adjacent, but not centromeric in the genome of D. pseudoobscura, while they are located on different chromosomal elements in D. virilis. Dbp80 gene organization differs dramatically among these species, while RpL15 structure is conserved. A bioinformatic analysis in five additional Drosophila species demonstrates active repositioning of these genes both within and between chromosomal elements. This study shows that Dbp80 and RpL15 can function in contrasting chromatin contexts on an evolutionary timescale. The complex history of these genes also provides unique insight into the dynamic nature of genome evolution.

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element.

It was described earlier that the Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, was reported to be a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction was proposed to contribute to the functional regulation of the bithorax complex (BX-C). Here, we show that mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. Here, we report that GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes and that mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function.

Canalization by Selection of de Novo Induced Mutations.

One of the most fascinating scientific problems, and a subject of intense debate, is that of the mechanisms of biological evolution. In this context, Waddington elaborated the concepts of "canalization and assimilation" to explain how an apparently somatic variant induced by stress could become heritable through the germline in Drosophila He resolved this seemingly Lamarckian phenomenon by positing the existence of cryptic mutations that can be expressed and selected under stress. To investigate the relevance of such mechanisms, we performed experiments following the Waddington procedure, then isolated and fixed three phenotypic variants along with another induced mutation that was not preceded by any phenocopy. All the fixed mutations we looked at were actually generated de novo by DNA deletions or transposon insertions, highlighting a novel mechanism for the assimilation process. Our study shows that heat-shock stress produces both phenotypic variants and germline mutations, and suggests an alternative explanation to that of Waddington for the apparent assimilation of an acquired character. The selection of the variants, under stress, for a number of generations allows for the coselection of newly induced corresponding germline mutations, making the phenotypic variants appear heritable.

Chromosome Healing Is Promoted by the Telomere Cap Component Hiphop in Drosophila.

The addition of a new telomere onto a chromosome break, a process termed healing, has been studied extensively in organisms that utilize telomerase to maintain their telomeres. In comparison, relatively little is known about how new telomeres are constructed on broken chromosomes in organisms that do not use telomerase. Chromosome healing was studied in somatic and germline cells of Drosophila melanogaster, a nontelomerase species. We observed, for the first time, that broken chromosomes can be healed in somatic cells. In addition, overexpression of the telomere cap component Hiphop increased the survival of somatic cells with broken chromosomes, while the cap component HP1 did not, and overexpression of the cap protein HOAP decreased their survival. In the male germline, Hiphop overexpression greatly increased the transmission of healed chromosomes. These results indicate that Hiphop can stimulate healing of a chromosome break. We suggest that this reflects a unique function of Hiphop: it is capable of seeding formation of a new telomeric cap on a chromosome end that lacks a telomere.

Trans-splicing of the mod(mdg4) complex locus is conserved between the distantly related species Drosophila melanogaster and D. virilis.

The modifier of mdg4, mod(mdg4), locus in Drosophila melanogaster represents a new type of complex gene in which functional diversity is resolved by mRNA trans-splicing. A protein family of >30 transcriptional regulators, which are supposed to be involved in higher-order chromatin structure, is encoded by both DNA strands of this locus. Mutations in mod(mdg4) have been identified independently in a number of genetic screens involving position-effect variegation, modulation of chromatin insulators, apoptosis, pathfinding of nerve cells, and chromosome pairing, indicating pleiotropic effects. The unusual gene structure and mRNA trans-splicing are evolutionary conserved in the distantly related species Drosophila virilis. Chimeric mod(mdg4) transcripts encoded from nonhomologous chromosomes containing the splice donor from D. virilis and the acceptor from D. melanogaster are produced in transgenic flies. We demonstrate that a significant amount of protein can be produced from these chimeric mRNAs. The evolutionary and functional conservation of mod(mdg4) and mRNA trans-splicing in both Drosophila species is furthermore demonstrated by the ability of D. virilis mod(mdg4) transgenes to rescue recessive lethality of mod(mdg4) mutant alleles in D. melanogaster.

The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins.

In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. Here, we have characterized the Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes. We show that Drosophila Ring corresponds to the Sex combs extra (Sce), a previously described PcG gene. We find that Ring/Sce is expressed and required throughout development and that the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of Ring/Sce. This phenotypic rescue is also obtained by ectopic expression of the murine Ring1/Ring1A, suggesting a functional conservation of the proteins during evolution. In addition, we find that Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which overlap those of other PcG products such as Polycomb, Posterior sex combs and Polyhomeotic, and 30% of which are unique. We also show that Ring/Sce interacts directly with PcG proteins, as it occurs in mammals.

Loss of Pol32 in Drosophila melanogaster causes chromosome instability and suppresses variegation.

Pol32 is an accessory subunit of the replicative DNA Polymerase δ and of the translesion Polymerase ζ. Pol32 is involved in DNA replication, recombination and repair. Pol32's participation in high- and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32rd1 and pol32rds in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutants suppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

Position effect variegation and viability are both sensitive to dosage of constitutive heterochromatin in Drosophila.

The dosage effect of Y-chromosome heterochromatin on suppression of position effect variegation (PEV) has long been well-known in Drosophila. The phenotypic effects of increasing the overall dosage of Y heterochromatin have also been demonstrated; hyperploidy of the Y chromosome produces male sterility and many somatic defects including variegation and abnormal legs and wings. This work addresses whether the suppression of position effect variegation (PEV) is a general feature of the heterochromatin (independent of the chromosome of origin) and whether a hyperdosage of heterochromatin can affect viability. The results show that the suppression of PEV is a general feature of any type of constitutive heterochromatin and that the intensity of suppression depends on its amount instead of some mappable factor on it. We also describe a clear dosage effect of Y heterochromatin on the viability of otherwise wild-type embryos and the modification of that effect by a specific gene mutation. Together, our results indicate that the correct balance between heterochromatin and euchromatin is essential for the normal genome expression and that this balance is genetically controlled.

Immunostaining of mitotic chromosomes from Drosophila larval brain.

Good mitotic chromosome preparations are essential for the immunolocalization of chromosomal proteins. Although methanol/acetic acid fixation techniques preserve chromosome morphology very well, they remove a substantial fraction of chromosomal proteins. We have developed fixation/immunostaining procedures, described here, that are suitable for the immunolocalization of proteinaceous components of metaphase chromosomes from larval Drosophila brain cells. These procedures result in good chromosomal quality with minimal removal of proteins.

Positive regulation of euchromatic gene expression by HP1.

HP1 is a conserved prototype protein that plays an essential role in heterochromatin formation and epigenetic gene silencing through its interaction with histone methyltransferase enzymes (HMTases) and the histone H3 at lysine 9 (H3-MeK9). HP1 is also involved in telomere capping and, more surprisingly, in positive regulation of gene expression. Recently, a wide expression analysis, using a RIP-chip assays (RNA-immunoprecipitation on microarrays), has shown that HP1 associates with the transcripts of more than one hundred euchromatic genes and interacts with the heterogeneous nuclear ribonucleoproteins (hnRNPs) that are known to be involved in RNA processing. By these results, HP1 seems to be part of a complex that stabilizes RNA transcripts. Though previously unsuspected, it was also found that HP1-interacting hnRNPs have a functional role in heterochromatin formation. These proteins bind heterochromatin and are dominant suppressors of position effect variegation. Taken together, the results in the paper by Piacentini et al. open a window on a possible new conceptual landscape in which similar epigenetic mechanisms could have a significant role, both in the metabolism of RNA transcripts and in heterochromatin formation, producing opposite functional effects. These data seem to establish a functional link between euchromatin and heterochromatin.