In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. We report here that an essential locus within chromosome 3L centric heterochromatin encodes the previously uncharacterized Drosophila melanogaster ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). Our results suggest that dSet1 acts as a "global" or general H3K4 diand trimethyl HMT in Drosophila. Levels of H3K4 di-and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/ or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). We present biochemical evidence that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence that these members are functionally required for H3K4 methylation. Taken together, our results suggest that dSet1 is responsible for the bulk of H3K4 di-and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans.

The cohesin complex is a key player in regulating cell division. Cohesin proteins SMC1, SMC3, Rad21, and stromalin (SA), along with associated proteins Nipped-B, Pds5, and EcoI, maintain sister chromatid cohesion before segregation to daughter cells during anaphase. Recent chromatin immunoprecipitation (ChIP) data reveal extensive overlap of Nipped-B and cohesin components with RNA polymerase II binding at active genes in Drosophila. These and other data strongly suggest a role for cohesion in transcription; however, there is no clear evidence for any specific mechanisms by which cohesin and associated proteins regulate transcription. We report here a link between cohesin components and trithorax group (trxG) function, thus implicating these proteins in transcription activation and/or elongation. We show that the Drosophila Rad21 protein is encoded by verthandi (vtd), a member of the trxG gene family that is also involved in regulating the hedgehog (hh) gene. In addition, mutations in the associated protein Nipped-B show similar trxG activity i.e., like vtd, they act as dominant suppressors of Pc and hh Mrt without impairing cell division. Our results provide a framework to further investigate how cohesin and associated components might regulate transcription.Hedgehog ͉ heterochromatin ͉ Nipped-B ͉ Polycomb ͉ cohesion I n eukaryotic mitosis, accurate chromosome segregation requires paired sister chromatids to attach to opposite spindle poles. Sister chromatids are held together by the cohesin protein complex, which consists of four core subunits, Rad21/SCC1, stromalin (SA) and structural maintenance of chromosome (SMC) proteins SMC1 and SMC3. A widely accepted model postulates that cohesin forms a ring-like structure via interaction of the N-and C-termini of Rad21 with a SMC1/SMC3 heterodimer. With the participation of SCC2/Nipped-B, SCC4, EcoI/Ctf7, and Pds5 proteins, sister-chromatid cohesion is maintained until the onset of mitosis. Cleavage of Rad21 and the resulting removal of cohesin then allow separation of sister chromatids in anaphase (see refs. 1-3 for reviews). Mutation of genes encoding these subunits leads to errors in chromosome segregation and aneuploidy, which are hallmarks of cancer and a leading cause of birth defects in humans (4).Given the highly conserved role for cohesin in sister chromatid cohesion, it was unexpected to discover that cohesin and associated proteins might also play a distinct, independent role in regulating gene expression. Reduction in Nipped-B expression in Drosophila affects expression of the cut and Ultrabithorax genes (5-7), and mutations in the human orthologue, NIPBL, result in Cornelia de Lange Syndrome (8). In zebrafish, mutations in rad21 or Smc3 affect embryonic runx gene transcription in heterozygous mutant animals without compromising cell division, suggesting that these proteins may have functions in transcription that are distinct from a mitotic role (9). Recently, Misulovin et al. observed extensive overlap of Nipped-B and cohesin components with RNA polymer...

A large portion of the Drosophila melanogaster genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools. Here we present an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). We have generated and characterized a number of new, overlapping deficiencies (Dfs) which remove regions of 3L Het. These Dfs were critically important reagents in our subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. We also identified putative essential gene sequences in 3L Het by using RNA interference to target candidate gene sequences. We report that at least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in Drosophila , and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.

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.

Abstract:In Drosophila melanogaster, the borders between pericentric heterochromatin and euchromatin on the major chromosome arms have been defined in various ways, including chromatin-specific histone modifications, the binding patterns of heterochromatin-enriched chromosomal proteins, and various cytogenetic techniques. Elucidation of the genetic properties that independently define the different chromatin states associated with heterochromatin and euchromatin should help refine the boundary. Since meiotic recombination is present in euchromatin, but absent in heterochromatin, it constitutes a key genetic property that can be observed transitioning between chromatin states. Using P element insertion lines marked with a su(Hw) insulated mini-white gene, meiotic recombination was found to transition in a region consistent with the H3K9me2 transition observed in ovaries.Key words: heterochromatin, euchromatic-heterochromatic border, meiotic recombination, Drosophila.Résumé : Chez le Drosophila melanogaster, les frontières entre l'hétérochromatine péricentrique et l'euchromatine sur les bras chromosomiques majeurs ont été définies de plusieurs façons, incluant les modifications d'histones spécifiques du type de chromatine, la liaison de protéines chromosomiques enrichies provenant de l'hétérochromatine, ainsi que diverses techniques cytogénétiques. L'élucidation des propriétés génétiques qui définissent indépendamment les différents états de la chromatine associés avec l'hétérochromatine et l'euchromatine devrait aider à raffiner le positionnement de ces frontières. Comme la recombinaison méiotique est présente chez l'euchromatine, mais absente chez l'hétérochromatine, elle constitue une propriété génétique clé que l'on peut observer lors de la transition entre états de la chromatine. Au moyen de lignées d'insertions de l'élément P marquées avec un gène mini-white isolé su(Hw), les auteurs ont observé que le changement observé dans la recombinaison méiotique au sein d'une région était conforme à la transition observée au sein des ovaires pour la méthylation H3K9me2. [Traduit par la Rédaction] Mots-clés : hétérochromatine, frontières l'hétérochromatine et l'euchromatine, recombinaison méiotique, Drosophila.