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.

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.

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

Centromeric heterochromatin comprises 30% of the Drosophila melanogaster genome, forming a transcriptionally repressive environment that silences euchromatic genes juxtaposed nearby. Surprisingly, there are genes naturally resident in heterochromatin, which appear to require this environment for optimal activity. Here we report an evolutionary analysis of two genes, Dbp80 and RpL15, which are adjacent in proximal 3L heterochromatin of D. melanogaster. DmDbp80 is typical of previously described heterochromatic genes: large, with repetitive sequences in its many introns. In contrast, DmRpL15 is uncharacteristically small. The orthologs of these genes were examined in D. pseudoobscura and D. virilis. In situ hybridization and whole-genome assembly analysis show that these genes are adjacent, but not centromeric in the genome of D. pseudoobscura, while they are located on different chromosomal elements in D. virilis. Dbp80 gene organization differs dramatically among these species, while RpL15 structure is conserved. A bioinformatic analysis in five additional Drosophila species demonstrates active repositioning of these genes both within and between chromosomal elements. This study shows that Dbp80 and RpL15 can function in contrasting chromatin contexts on an evolutionary timescale. The complex history of these genes also provides unique insight into the dynamic nature of genome evolution.

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.