| Abstract |
The basic helix-loop-helix (bHLH) family of proteins plays important roles in many developmental processes in eukaryotes, including the determination of the Central and Peripheral Nervous System (CNS and PNS, respectively) in metazoa. Early neurogenesis in Drosophila is characterized by the expression of the genes of the Achaete-Scute Complex (AS-C), which encode bHLH transcriptional activators that can heterodimerize with the ubiquitous bHLH protein daughterless (Da), and activate transcription of target genes by direct binding onto specific DNA sequences, the so called EA boxes. Expression of AS-C is initially observed in groups of cells called proneural clusters. In later stages however, AS-C expression is limited to one or few cells in each cluster, which will differentiate to become neuroblasts. The factors responsible for the limitation of AS-C expression are encoded by the Enhancer of split complex [E(spl)-C], which contains, among others, bHLH transcriptional repressors, that have the ability to bind as homodimers to specific DNA sequences, such as the EB, EC and N boxes. The bHLH E(spl) genes are transcriptionally activated by the Notch (N) signaling pathway and they act to repress neural fate in the cells of the proneural clusters that receive the N signal both in the CNS and the PNS of the fly. The antagonistic action of the two groups of proteins has been shown to be based on direct physical interactions, so that the E(spl) proteins are recruited on their targets via physical association with the activator complexes which are bound on DNA, without the need of direct DNA binding by E(spl) themselves. Their targets include the AS-C genes (which are positively autoregulated), and possibly other neuron-specific genes that are common targets for both groups. Nevertheless, the confirmed ability of E(spl) to bind DNA and the presence of conserved binding sites in the regulatory regions of their target genes prompted us to look more carefully at the binding preferences of E(spl) homodimers and heterodimers. Then, we asked whether particular binding sites on natural enhancers can affect recruitment on DNA and transcriptional repression conferred by E(spl). Finally, using appropriate artificial enhancers we tried to define the actual role of direct DNA binding versus recruitment by activators for transcriptional repression by E(spl). Initially, we confirmed that the optimal binding site for E(spl) is the EB and not the EC box. However, we noticed that the seven proteins do not share the same affinity for DNA binding, that is some homodimers bind better than others on the same sites. We also showed that particular heterodimers can have altered DNA binding affinity with respect to homodimers and calculated the dissociation constant for some of the complexes. The dimerization among the seven proteins, which is an important prerequisite for DNA binding, seemed to depend both on the HLH domain and the Orange domain, which is also conserved in the family. The in vivo results also showed that the seven proteins can bind on the same EB and EC sites, albeit not with the same strength. Moreover, we demonstrated that the trinuclotide sequences surrounding the core EB/C site, previously shown to be important for binding, can dramatically affect their access onto DNA. Finally, we showed that direct DNA binding by E(spl) can work in cooperation with recruitment by activators, as transcriptional repression by E(spl) is enhanced when both mechanisms are employed. In fact, physical interactions between E(spl) and Da/AS-C seemed to stabilize their binding on DNA. The above results support a model, where transcriptional repression by E(spl) depends on their differential ability to homo- and heterodimerize, but also on particular preferences for interaction with both DNA and Da/AS-C activators. Access to regulatory elements of target genes is achieved by both direct DNA binding and recruitment by activators, depending on the particular members of the two groups of proteins expressed but also on the presence and relative positions of binding sites for both complexes. The two mechanisms can contribute to a sharp, accurate transcriptional response, under conditions where the amount and, perhaps, the activity of the regulatory proteins involved continiously fluctuates, as a result of integration of signals from multiple signaling pathways.
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In vitro και in vivo μελέτες της πρόσδεσης των πρωτεϊνών bHLH E(SPL) σε στόχους DNA
