The canalization concept¹ 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^2,3,4,5, 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^6,7. 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 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) 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. Heterochromatin Protein 1 (HP1a) is a very well known prototype protein of a general model for heterochromatin formation and epigenetic gene silencing in different species including humans. Here, we report our experiments showing that HP1a is also required for the positive regulation of more than one hundred euchromatic genes by its association with the corresponding RNA transcripts and by its interaction with heterogeneous nuclear ribonucleoproteins (hnRNPs) belonging to different classes. Importantly, we also found that all the tested hnRNP proteins bind to the heterochromatin and are dominant suppressors of position effect variegation, thus suggesting they also have a role in heterochromatin organization. Taken together, our data show novel and important functions, not only for HP1a, but also for hnRNPs, which were previously believed to participate only in RNA processing. These results shed new light on the epigenetic mechanisms of gene silencing and gene expression. They also establish a link between RNA transcript metabolism and heterochromatin formation and change several aspects of the canonical views about these apparently different processes.

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.