Barry M. Honda

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.

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.

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 di- and 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.

O -linked N -acetylglucosamine transferase (OGT) reversibly modifies serine and threonine residues of many intracellular proteins with a single β- O -linked N -acetylglucosamine residue ( O -GlcNAc), and has been implicated in insulin signaling, neurodegenerative disease, cellular stress response, and other important processes in mammals. OGT also glycosylates RNA polymerase II and various transcription factors, which suggests that it might be directly involved in transcriptional regulation. We report here that the Drosophila OGT is encoded by the Polycomb group (PcG) gene, super sex combs ( sxc ). Furthermore, major sites of O -GlcNAc modification on polytene chromosomes correspond to PcG protein binding sites. Our results thus suggest a direct role for O -linked glycosylation by OGT in PcG-mediated epigenetic gene silencing, which is important in developmental regulation, stem cell maintenance, genomic imprinting, and cancer. In addition, we observe rescue of sxc lethality by a human Ogt cDNA transgene; thus Drosophila may provide an ideal model to study important functional roles of OGT in mammals.

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.

In this review, we combine the results of our published and unpublished work with the published results of other laboratories to provide an updated map of the centromeric heterochromatin of chromosome 3 in Drosophila melanogaster. To date, we can identify more than 20 genes (defined DNA sequences with well-characterized functions and (or) defined genetic complementation groups), including at least 16 essential loci. With the ongoing emergence of data from genetic, cytological, and genome sequencing studies, we anticipate continued, substantial progress towards understanding the function, structure, and evolution of centric heterochromatin.Key words: heterochromatin, Drosophila, cytogenetics, genomics.

Heterochromatin comprises a transcriptionally repressive chromosome compartment in the eukaryotic nucleus; this is exemplified by the silencing effect it has on euchromatic genes that have been relocated nearby, a phenomenon known as position-effect variegation (PEV), first demonstrated in Drosophila melanogaster. However, the expression of essential heterochromatic genes within these apparently repressive regions of the genome presents a paradox, an understanding of which could provide key insights into the effects of chromatin structure on gene expression. To date, very few of these resident heterochromatic genes have been characterized to any extent, and their expression and regulation remain poorly understood. Here we report the cloning and characterization of two proximal heterochromatic genes in D. melanogaster, located deep within the centric heterochromatin of the left arm of chromosome 3. One of these genes, RpL15, is uncharacteristically small, is highly expressed, and encodes an essential ribosomal protein. Its expression appears to be compromised in a genetic background deficient for heterochromatin protein 1 (HP1), a protein associated with gene silencing in these regions. The second gene in this study, Dbp80, is very large and also appears to show a transcriptional dependence upon HP1; however, it does not correspond to any known lethal complementation group and is likely to be a nonessential gene.

Eight different amber suppressor tRNA (suptRNA) mutations in the nematode Caenorhabditis elegans have been isolated; all are derived from members of the tRNA^Trp gene family (K. Kondo, B. Makovec, R. H. Waterston, and J. Hodgkin, J. Mol. Biol. 215:7–19, 1990). Genetic assays of suppressor activity suggested that individual tRNA genes were differentially expressed, probably in a tissue- or developmental stage-specific manner. We have now examined the expression of representative members of this gene family both in vitro, using transcription in embryonic cell extracts, and in vivo, by assaying suppression of an amber-mutated lacZ reporter gene in animals carrying different suptRNA mutations. Individual wild-type tRNA^Trp genes and their amber-suppressing counterparts appear to be transcribed and processed identically in vitro, suggesting that the behavior of suptRNAs should reflect wild-type tRNA expression. The levels of transcription of different suptRNA genes closely parallel the extent of genetic suppression in vivo. The results suggest that differential expression of tRNA genes is most likely at the transcriptional rather than the posttranscriptional level and that 5′ flanking sequences play a role in vitro, and probably in vivo as well. Using suppression of a lacZ(Am) reporter gene as a more direct assay of suptRNA activity in individual cell types, we have again observed differential expression which correlates with genetic and in vitro transcription results. This provides a model system to more extensively study the basis for differential expression of this tRNA gene family.