Lucia Piacentini

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.

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 (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.

Chromosomal distribution of heterochromatin protein 1 (HP1) in Drosophila: a cytological map of euchromatic HP1 binding sites.

The Heterochromatin Protein 1 (HP1) is a conserved protein which is best known for its strong association with the heterochromatin of Drosophila melanogaster. We previously demonstrated that another important property of HP1 is its localization to the telomeres of Drosophila, a feature that reflects its critical function as a telomere capping protein. Here we report our analysis of the euchromatic sites to which HP1 localizes. Using an anti-HP1 antibody, we compared immunostaining patterns on polytene chromosomes of the Ore-R wild type laboratory strain and four different natural populations. HP1 was found to accumulate at specific euchromatic sites, with a subset of the sites conserved among strains. These sites do not appear to be defined by an enrichment of known repetitive DNAs. Comparisons of HP1 patterns among several Drosophila species revealed that association with specific euchromatic regions, heterochromatin and telomeres is a conserved characteristic of HP1. Based on these results, we argue that HP1 serves a broader function than typically postulated. In addition to its role in heterochromatin assembly and telomere stability, we propose that HP1 plays an important role in regulating the expression of many different euchromatic regions.

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.

The trithorax group and Pc group proteins are differentially involved in heterochromatin formation in Drosophila.

In Drosophila, the Polycomb group and trithorax group proteins play a critical role in controlling the expression states of homeotic gene complexes during development. The common view is that these two classes of proteins bind to the homeotic complexes and regulate transcription at the level of chromatin. In the present work, we tested the involvement of both groups in mitotic heterochromatin formation in Drosophila. Using specific antibodies, we show that some of the tested Pc-G proteins are present in heterochromatin, while all the tested trx-G proteins localize to specific regions of heterochromatin in both mitotic chromosomes and interphase nuclei. We also observed that mutations in trx-G genes are recessive enhancers of position-effect variegation and are able to repress the transcription of heterochromatic genes. These results strongly suggest that trx-G proteins, along with some Pc-G proteins, play an active role in heterochromatin formation in Drosophila.

Yeti, an essential Drosophila melanogaster gene, encodes a protein required for chromatin organization

ABSTRACT The evolutionarily conserved family of Bucentaur (BCNT) proteins exhibits a widespread distribution in animal and plants, yet its biological role remains largely unknown. Using Drosophila melanogaster as a model organism, we investigated the in vivo role of the Drosophila BCNT member called YETI. We report that loss of YETI causes lethality before pupation and defects in higher-order chromatin organization, as evidenced by severe impairment in the association of histone H2A.V, nucleosomal histones and epigenetic marks with polytene chromosomes. We also find that YETI binds to polytene chromosomes through its conserved BCNT domain and interacts with the histone variant H2A.V, HP1a and Domino-A (DOM-A), the ATPase subunit of the DOM/Tip60 chromatin remodeling complex. Furthermore, we identify YETI as a downstream target of the Drosophila DOM-A. On the basis of these results, we propose that YETI interacts with H2A.V-exchanging machinery, as a chaperone or as a new subunit of the DOM/Tip60 remodeling complex, and acts to regulate the accumulation of H2A.V at chromatin sites. Overall, our findings suggest an unanticipated role of YETI protein in chromatin organization and provide, for the first time, mechanistic clues on how BCNT proteins control development in multicellular organisms.

Canalization by Selection of De Novo-Induced Mutations

Waddington elaborated the concepts of “canalization and assimilation” to explain how an apparently somatic, stress-induced variant... 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.

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.

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.