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.

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.

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.

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.

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.