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The insulin signalling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. The human insulin receptor gene ( INSR) is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene ( InR ) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ~1.5 kbp segments, but we lack an understanding of the potential detailed mechanisms of their regulation. We characterized the substructure of these cis-regulatory elements in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. By identifying specific locations of activators and repressors within 300 bp subelements, we show that some previously identified enhancers consist of relatively compact clusters of activators, while others have a distributed architecture not amenable to further reduction. In addition, these assays uncovered a long-range repressive action of unliganded EcR. The complex transcriptional circuitry likely endows InR with a highly flexible and tissue-specific response to tune insulin signalling. Further studies will provide insights to demonstrate the impact of natural variation in this gene's regulation, applicable to human genetic studies.

The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or non-phosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (ΔIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. ΔIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1ΔIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.

The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, we examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. Here, we report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. We further show that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G(1)-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer.

Transcriptional corepressors play complex roles in developmental gene regulation. These proteins control transcription by recruiting diverse chromatin-modifying enzymes, but it is not known whether corepressor activities are finely regulated in different developmental settings or whether their basic activities are identical in most contexts. The evolutionarily conserved C-terminal binding protein (CtBP) is recruited by a variety of transcription factors that play crucial roles in development and disease. CtBP contains a central NAD(H) binding core domain that is homologous to D2 hydroxy acid dehydrogenase enzymes, as well as an unstructured C-terminal domain. NAD(H) binding is important for CtBP function, but the significance of its intrinsic dehydrogenase activity, as well as that of the unstructured C terminus, is poorly understood. To clarify the biological relevance of these features, we established genetic rescue assays to determine how different forms of CtBP function in the context of Drosophila melanogaster development. The mutant phenotypes and specific gene regulatory effects indicate that both the catalytic site of CtBP and the C-terminal extension play important, if nonessential roles in development. Our results indicate that the structural and enzymatic features of CtBP, previously thought to be dispensable for overall transcriptional control, are critical for modulating this protein's activity in diverse developmental settings.

Quantitative assessment of gene regulation is critical for mathematical modeling of transcriptional systems for systems biology efforts. Enhancers, also termed cis-regulatory modules (CRMs), are the primary mediators of transcriptional regulation in higher eukaryotes; transcription factors binding to CRMs dictate the likelihood and frequency of promoter activation. To provide a suitable platform for in-depth CRM analysis, we adapted a targeted integration vector to compare action of basal promoters with diverse combination of TATA, Inr and DPE motifs, as well as a set of 3'-UTRs representative of those used in different reporter vectors. This "Honda" series of reporter gene vectors was activated by a regulatory element binding Dorsal and Twist activators suitable for transcription in the early Drosophila embryo. The diverse promoters functioned in a similar manner with minor quantitative differences, consistent with a lack of enhancer-promoter specificity. Constructs bearing SV40 3'-UTR sequences appeared to produce somewhat higher levels of mRNA. Confocal laser scanning microscopy revealed that the mRNA distribution produced by these constructs was punctate; this pattern appears to be dependent on 5'-UTR sequences, as an optimized vector including an alternate 5'-UTR produced a more even distribution, which may be preferable for quantitative imaging. This set of Honda vectors contains convenient sites for modification of basal promoter, 3' UTR, and enhancer, and will be useful for analysis of CRMs and quantitative studies of gene expression.

The structure of chromatin in eukaryotes exerts significant influences on many DNA related processes, including transcription, replication, recombination and repair. A useful tool for mapping chromatin structure is micrococcal nuclease (MNase), which induces double-strand breaks within nucleosome linker regions, and with more extensive digestion, single-strand nicks within the nucleosome itself. Many studies, carried out largely with microbes and cell cultures, have used MNase to determine the positions of nucleosomes within a region of DNA to identify dynamic changes induced during gene regulation. To measure similar processes in a developmental context, we turned to a tractable model system, the Drosophila embryo. Here we describe a protocol that enables MNase mapping of the enhancer chromatin structure in the embryo, and show how it can be used to identify structural changes on a cis-regulatory element targeted by the Knirps repressor.

Transcriptional repressor proteins play key roles in the control of gene expression in development. For the Drosophila embryo, the following two functional classes of repressors have been described: short-range repressors such as Knirps that locally inhibit the activity of enhancers and long-range repressors such as Hairy that can dominantly inhibit distal elements. Several long-range repressors interact with Groucho, a conserved corepressor that is homologous to mammalian TLE proteins. Groucho interacts with histone deacetylases and histone proteins, suggesting that it may effect repression by means of chromatin modification; however, it is not known how long-range effects are mediated. Using embryo chromatin immunoprecipitation, we have analyzed a Hairy-repressible gene in the embryo during activation and repression. When inactivated, repressors, activators, and coactivators cooccupy the promoter, suggesting that repression is not accomplished by the displacement of activators or coactivators. Strikingly, the Groucho corepressor is found to be recruited to the transcribed region of the gene, contacting a region of several kilobases, concomitant with a loss of histone H3 and H4 acetylation. Groucho has been shown to form higher-order complexes in vitro; thus, our observations suggest that long-range effects may be mediated by a "spreading" mechanism, modifying chromatin over extensive regions to inhibit transcription.

Bioinformatics analysis of transcriptional control is guided by knowledge of the characteristics of cis-regulatory regions or enhancers. Features such as clustering of binding sites and co-occurrence of binding sites have aided enhancer identification, but quantitative predictions of enhancer function are not yet generally feasible. To facilitate the analysis of regulatory sequences in Drosophila melanogaster, we identified quantitative parameters that affect the activity of short-range transcriptional repressors, proteins that play key roles in development. In addition to the previously noted distance dependence, repression is strongly influenced by the stoichiometry, affinity, spacing, and arrangement of activator binding sites. Repression is insensitive to the type of activation domain, suggesting that short-range repression may primarily affect activators at the level of DNA binding. The activity of several short-range, but not long-range, repressors is circumscribed by the same quantitative parameters. This cis-regulatory "grammar" may aid the identification of enhancers regulated by short-range repressors and facilitate bioinformatic prediction of the functional output of transcriptional regulatory sequences.

Transcriptional repressors often employ multiple activities, but the molecular mechanisms and physiological relevance of this functional diversity remain obscure. The Drosophila melanogaster Knirps repressor uses CtBP corepressor-dependent and -independent pathways. To separately analyze the components of Knirps repression activity, we elucidated the specific repression properties of CtBP and of Knirps subdomains. Like Knirps, CtBP represses adjacent transcriptional activators; but unlike Knirps, CtBP is unable to repress basal promoter elements. We determined that the ability of CtBP to recapitulate only a subset of Knirps activities is due to a quantitative, rather than qualitative, deficiency in repression activity. The CtBP-dependent portion of Knirps synergizes with the CtBP-independent repression activity to potently repress promoter elements from enhancer- or promoter-proximal positions. This result indicates that multiple repression activities are combined to exceed critical thresholds on target genes. CtBP mutant proteins unable to bind NAD fail to interact with DNA-bound factors. We show that DNA-binding Gal4-CtBP fusion proteins also require NAD binding for activity, indicating that NAD plays a role in repression at a step subsequent to CtBP recruitment to the promoter.