Maria Berloco

The ISWI Chromatin-Remodeling Protein Is Required for Gene Expression and the Maintenance of Higher Order Chromatin Structure In Vivo

Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.

The Heterochromatin Protein 1 Prevents Telomere Fusions in Drosophila

HP1 (Heterochromatin protein 1) is a conserved, non-histone chromosomal protein that is best known for its preferential binding to pericentric heterochromatin and its role in position effect variegation in Drosophila. Using immunolocalization, we show that HP1 is a constant feature of the telomeres of interphase polytene and mitotic chromosomes. This localization does not require the presence of telomeric retrotransposons, since HP1 is also detected at the ends of terminally deleted chromosomes that lack these elements. Importantly, larvae expressing reduced or mutant versions of HP1 exhibit aberrant chromosome associations and multiple telomeric fusions in neuroblast cells, imaginal disks, and male meiotic cells. Taken together, these results provide evidence that HP1 plays a functional role in mediating normal telomere behavior in Drosophila.

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.

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.

Heterochromatin protein 1 binds transgene arrays

Abstract. Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation.

The maternal effect gene, abnormal oocyte (abo), of Drosophila melanogaster encodes a specific negative regulator of histones

The abnormal oocyte (abo) gene of Drosophila melanogaster is a peculiar maternal effect gene whose mutations cause a maternal-effect lethality that can be rescued by specific regions of heterochromatin during early embryogenesis. Here we show that abo encodes an evolutionary conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. We also show a significant increase of histone transcripts in eggs of abo mutant mothers and a partial rescue of the abo maternal-effect defect by deficiencies of the histone gene cluster. On the basis of these results, we suggest that the Abo protein functions specifically as a negative regulator of histone transcription and propose a molecular model to account for the ability of heterochromatin to partially rescue the abo maternal-effect defect. Our model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant abo mothers and that a balanced pool of histones is critical for normal embryogenesis in Drosophila.

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.

Differential expression of the Drosophila BX-C in polytene chromosomes in cells of larval fat bodies: a cytological approach to identifying in vivo targets of the homeotic Ubx, Abd-A and Abd-B proteins.

We have analyzed the expression of homeotic Bithorax Complex proteins in the fat bodies of Drosophila larvae by staining with specific antibodies. We have found that these proteins are differentially expressed along the anteroposterior (AP) axis of the fat body, with patterns parallel to those previously characterized for the larval and adult epidermis. As fat body nuclei have polytene chromosomes, we were able to identify the BX-C locus and show that it assumes a strongly puffed conformation in cells actively expressing the genes of the BX-C. Immunostaining of these polytene chromosomes provided the resolution to cytologically map binding sites of the three proteins: Ubx, Abd-A and Abd-B. The results of this work provide a system with which to study the positioning of chromatin regulatory proteins in either a repressed and/or active BXC at the cytological level. In addition, the results of this work provide a map of homeotic target loci and thus constitute the basis for a systematic identification of genes that are direct in vivo targets of the BX-C genes.

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.

Interaction systems between heterochromatin and euchromatin inDrosophila melanogaster

The constitutive heterochromatin is still one of the major unsolved problems in genetics. InDrosophila melanogaster three genetic systems involving specific interactions between heterochromatic and euchromatic genetic elements are known: the Segregation Distortion, thecrystal-Stellate and theabo-ABO systems. The genetic and molecular analysis of each system will allow the identification of all the components and the elucidation of the mechanisms underlying their interactions. The results of this analysis should provide insights into the biological significance of heterochromatin and into the evolutionary forces that result in the maintainance and stability of this enigmatic genetic material.