Neuronal Progenitors Suffer Genotoxic Stress in the Clock Mutant .

The physiological role and the molecular architecture of the circadian clock in fully developed organisms are well established. Yet, we have a limited understanding of the function of the clock during ontogenesis. We have used a null mutant () of the clock gene () in to ask whether PER may play a role during normal brain development.

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.

Transposable Elements: Major Players in Shaping Genomic and Evolutionary Patterns.

Transposable elements (TEs) are ubiquitous genetic elements, able to jump from one location of the genome to another, in all organisms. For this reason, on the one hand, TEs can induce deleterious mutations, causing dysfunction, disease and even lethality in individuals. On the other hand, TEs can increase genetic variability, making populations better equipped to respond adaptively to environmental change.

Epigenetics as an Evolutionary Tool for Centromere Flexibility.

Centromeres are the complex structures responsible for the proper segregation of chromosomes during cell division. Structural or functional alterations of the centromere cause aneuploidies and other chromosomal aberrations that can induce cell death with consequences on health and survival of the organism as a whole. Because of their essential function in the cell, centromeres have evolved high flexibility and mechanisms of tolerance to preserve their function following stress, whether it is originating from within or outside the cell.

Understanding the Early Evolutionary Stages of a Tandem Drosophilamelanogaster-Specific Gene Family: A Structural and Functional Population Study.

Gene families underlie genetic innovation and phenotypic diversification. However, our understanding of the early genomic and functional evolution of tandemly arranged gene families remains incomplete as paralog sequence similarity hinders their accurate characterization. The Drosophila melanogaster-specific gene family Sdic is tandemly repeated and impacts sperm competition.

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 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.

A role of the Trx-G complex in Cid/CENP-A deposition at Drosophila melanogaster centromeres.

Centromeres are epigenetically determined chromatin structures that specify the assembly site of the kinetochore, the multiprotein machinery that binds microtubules and mediates chromosome segregation during mitosis and meiosis. The centromeric protein A (CENP-A) and its Drosophila orthologue centromere identifier (Cid) are H3 histone variants that replace the canonical H3 histone in centromeric nucleosomes of eukaryotes. CENP-A/Cid is required for recruitment of other centromere and kinetochore proteins and its deficiency disrupts chromosome segregation.

Heterochromatin protein 1 (HP1) is intrinsically required for post-transcriptional regulation of Drosophila Germline Stem Cell (GSC) maintenance.

A very important open question in stem cells regulation is how the fine balance between GSCs self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance.

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

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 , a nontelomerase species.

Canalization by Selection of 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 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.

Comparative Genomic Analyses Provide New Insights into the Evolutionary Dynamics of Heterochromatin in Drosophila.

The term heterochromatin has been long considered synonymous with gene silencing, but it is now clear that the presence of transcribed genes embedded in pericentromeric heterochromatin is a conserved feature in the evolution of eukaryotic genomes. Several studies have addressed the epigenetic changes that enable the expression of genes in pericentric heterochromatin, yet little is known about the evolutionary processes through which this has occurred. By combining genome annotation analysis and high-resolution cytology, we have identified and mapped 53 orthologs of D.

Expression of human Cfdp1 gene in Drosophila reveals new insights into the function of the evolutionarily conserved BCNT protein family.

The Bucentaur (BCNT) protein family is widely distributed in eukaryotes and is characterized by a highly conserved C-terminal domain. This family was identified two decades ago in ruminants, but its role(s) remained largely unknown. Investigating cellular functions and mechanism of action of BCNT proteins is challenging, because they have been implicated in human craniofacial development.

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.

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.

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

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.

The "Special" crystal-Stellate System in Drosophila melanogaster Reveals Mechanisms Underlying piRNA Pathway-Mediated Canalization.

The Stellate-made crystals formation in spermatocytes is the phenotypic manifestation of a disrupted crystal-Stellate interaction in testes of Drosophila melanogaster. Stellate silencing is achieved by the piRNA pathway, but many features still remain unknown. Here we outline the important role of the crystal-Stellate modifiers.

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.

Fluorescent in situ hybridization (FISH) of mitotic chromosomes from Drosophila larval brain.

The fluorescent in situ hybridization (FISH) technique permits fine mapping of both middle and highly repetitive DNA sequences along Drosophila melanogaster heterochromatin. Best results are obtained when this technique is coupled with DAPI staining and digital recording of fluorescent signals. For example, if digital images of the FISH signals and DAPI fluorescence are detected separately using a charge-coupled device (CCD) camera, they can then be pseudocolored and merged using suitable computer programs.

Chromosome banding of mitotic chromosomes from Drosophila larval brain.

The classical chromosome-banding techniques developed for mammalian chromosomes do not differentiate the euchromatic arms of Drosophila mitotic chromosomes. However, some of these techniques produce a sharp and highly reproducible banding of Drosophila heterochromatin. For example, the use of quinacrine-, Hoechst-, and N-banding differentiates Drosophila heterochromatin into 61 cytological entities, allowing precise localization of heterochromatic breakpoints.

Preparation and orcein staining of mitotic chromosomes from Drosophila larval brain.

In this protocol, larval brains from Drosophila are incubated in vitro with colchicine, treated with hypotonic solution, fixed, and squashed in aceto-orcein. This procedure provides a large number of well-spread metaphase figures (200-400 per brain) that can be analyzed for chromosome morphology, the presence of chromosome aberrations, and the degree of ploidy.

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.

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.

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.

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.

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.