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.

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. Here, we review the main epigenetic mechanisms of centromeres’ adaptability to preserve their functional stability, with particular reference to neocentromeres and holocentromeres. The centromere position can shift in response to altered chromosome structures, but how and why neocentromeres appear in a given chromosome region are still open questions. Models of neocentromere formation developed during the last few years will be hereby discussed. Moreover, we will discuss the evolutionary significance of diffuse centromeres (holocentromeres) in organisms such as nematodes. Despite the differences in DNA sequences, protein composition and centromere size, all of these diverse centromere structures promote efficient chromosome segregation, balancing genome stability and adaptability, and ensuring faithful genome inheritance at each cellular generation.

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.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and intellectual disability. Mutations in over 30 genes, nearly half linked to centrosome biogenesis or microtubule (MT) dynamics, highlight spindle defects in disease aetiology, yet these alone do not fully explain MCPH. Here, we show that the Drosophila orthologs of ASPM/MCPH5 (asp) and CENPJ/MCPH6 (Sas4) contribute to safeguard nuclear architecture and chromatin organization during brain development. Loss of either gene perturbs MT organization and centromere clustering, leading to reduced Lamin and HP1α levels, and to deformed nuclear lamina. Mutants also display a global reduction in heterochromatin-associated histone marks, H3K9me2/3 and H3K27me3, along with an increase in the euchromatin-associated mark H3K4me3 and elevated DNA damage with delayed repair. Notably, inhibiting demethylases with methylstat restores H3K9me3 and nuclear morphology. These findings suggest a previously unreported role for centrosome proteins in regulating chromatin organization, providing new insights into the mechanisms underlying MCPH pathogenesis.

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 (per⁰) of the clock gene period (per) in Drosophila melanogaster to ask whether PER may play a role during normal brain development. In third-instar larvae, we have observed that the absence of functional per results in increased genotoxic stress compared to wild-type controls. We have detected increased double-strand DNA breaks in the central nervous system and chromosome aberrations in dividing neuronal precursor cells. We have demonstrated that reactive oxygen species (ROS) are causal to the genotoxic effect and that expression of PER in glia is necessary and sufficient to suppress such a phenotype. Finally, we have shown that the absence of PER may result in less condensed chromatin, which contributes to DNA damage.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by reduced brain size and non-progressive intellectual disability. Mutations in over 30 genes have been linked to MCPH. Nearly a half of the genes identified by these mutations encode proteins involved in centrosome biogenesis or microtubule (MT) dynamics, suggesting a central role for mitotic spindle organization and division plane orientation in disease aetiology. However, it has been suggested that disruptions in spindle positioning alone are not sufficient to lead to microcephaly. Here, we investigate the contribution of the Drosophila orthologs of ASPM / MCPH5 ( asp ) and CENPJ/MCPH6 ( Sas4 ) to nuclear architecture, chromatin organization, and genome stability. We show that loss of either Sas4 or Asp leads to aberrant microtubule architecture, mislocalization of the LINC complex, and deformation of the nuclear lamina. These defects are accompanied by reduced levels of both lamin and HP1α and impaired centromere clustering in interphase cells. Sas4 and asp mutants also exhibit a global reduction in heterochromatin-associated histone marks (H3K9me2/3 and H3K27me3) and increased levels of the euchromatin-associated mark H3K4me3. Remarkably, treatment with Methylstat, a demethylase inhibitor, reduced nuclear invaginations by partially restoring H3K9me3 levels. Additionally, Sas4 or Asp depletion leads to DNA damage, increased sensitivity to genotoxic stress, and delayed DNA repair. Together, these findings reveal a previously underappreciated role for Asp and Sas4 in preserving nuclear architecture and chromatin integrity, offering new insight into the pathogenesis of MCPH. GRAPHICAL