CIS-acting elements controlling the expression of the human GLI3 gene

Limb development is a complex mechanism involving many molecular factors and signaling pathways, which orchestrate during embryogenesis the transformation of the limb bud to the complete extremities. Posterior-anterior patterning is directed by the sonic hedgehog signal molecule, which acts upon transcriptional activity of target genes via the GLI transcription factors. Time, location, and amount of the transcription of GLI-genes are of critical importance. Mutation affecting the availability of the appropriate amounts of these factors in limb bud cells as well as mutations impairing their function can result in developmental defects and tumorigenesis. To contribute to the detection and functional characterization of cis-acting regulatory elements for GLI3 and their potential relevance for pathogenity three questions were adressed in this thesis. · How is the expression of human GLI3 regulated? · Do GLI2 and GLI3 share similar regulatory elements? · Are mutations in GLI3 regulatory elements involved in pathogenity? Towards this end, 24 patients with limb defects classified as potential GLI3 morphopathies were screened for mutations. 20 cases, which cannot be attributed to a mutation in coding sequences of the gene, are candidates to be searched for mutations in cis-regulatory elements. The transcriptional control of GLI3 gene expression involves promoter as well as cis-acting sequences, such as enhancers. A minimal promoter was defined and tested functionally. Two initiator sites were identified by using templates from placenta and skeletal muscle. By mutagenesis, sequence elements involved in control of GLI3 expression were identified. Functional studies in transgenic mice suggest that GLI2 and GLI3 might have greatly overlapping expression domains. The 5’ transcribed region of human GLI2 was extended by about 1 kb of noncoding DNA, however, sequence comparison of human and murine GLI2 or GLI3 did not detect homologies of regulatory elements. Evolutionary genomic sequence comparisons were applied to guide the search for highly conserved non-coding elements, which might represent cis-regulatory elements for GLI3. Three potential enhancer elements were tested for their regulatory capacity on reporter genes with foreign as well as the original GLI3 minimal promoter in transiently transfected cell cultures and in transgenic mice. Mutagenesis followed by tests for retention of their regulatory capacity in cellular reporter gene assays pinpointed particularly critical sites. Transcription factors that could be involved in GLI3 regulation, such as NFATp, await independent confirmation. In transgenic mouse embryos it was determined, that one of the potential enhancer elements directs an expression pattern, which follows part of the time course and the spatial pattern of the endogenous mouse GLI3, in particular the brain, mandibles, nostrils and heart. The results obtained contribute to our understanding of the spatial and temporal control of the expression of GLI3, a key factor of the hedgehog signaling cascade and provide insight into the potential role of highly conserved non-coding sequence elements in the human genome.