T-box proteins in development and pathogenesis
T-box transcription factors arose during metazoan evolution and control the development of epithelial and mesenchymal tissues. All Tbx proteins contains a highly conserved DNA binding domains of about 180 amino acids and exhibit essentially the same binding preference for an octameric DNA sequence. The human genome contains 17 TBX genes. Mutations in many of these cause inherited diseases, in which the devolpment of heart, limbs, and facial structures can be disturbed.
We are investigating in particular the Tbx2 subfamily of these transcription factors. In vertebrates, it encompasses four members (TBX2, 3, 4, and 5), in Drosophila it contains only one member (optomotor-blind, omb). The Tbx2 subfamily is also of relevance because TBX2 and TBX3 are overexpressed in many human tumors. Drosophila, aside from the many advantages it has as a model organism, facilitates investigation of the Tbx subfamily because genetic investigations are not hampered by redundancy. omb stretches over more than 150 kb, making it one of the largest genes in the fly genome. This size reflects its complex regulation. Correct expression of omb is essential for development of brain (in particular the optic lobes), limbs (in particular the wings), head (in particular the eyes), and abdominal integument.
1. Genetic and epigenetic regulation of omb
The entire omb locus was subcloned by us and others in enhancer reporter vectors. We investigate regulation and developmental relevance of individual enhancers. We search to understand whether the distribution of individual regulatory elements follows a recognizable logic. omb contains binding sites for Polycomb-group and trithorax-group proteins (PREs). This suggests that omb is under epigenetic control. This is in agreement with the finding that the omb promoter region mediates pairing sensitive silencing (PSS). Some enhancers in omb can act over more than hundred kb on the omb promoter but do not affect neighbouring promoters separated by only a few hundred bp. We try to identify the hypothetical boundary elements.
2. Tbx-controlled genes
The Drosophila genome contains about 250.000 potential Tbx binding elements (TBEs). We want to identify sequence features of factual TBEs by the identification of Tbx-controlled genes. A sufficiently large set of Tbx target genes will allow to determine whether and how TBEs for different paralogous Tbx proteins differ (methods: bioinformatics, transcriptome and ChIP-chip analysis, genetics, histology). The identification of target genes contributes to the mechanistic understanding of the various biological Tbx functions.
3. Tbx proteins in morphogenesis and maintenance of epithelial structures
Tbx function frequently is strongly dose-dependent (cf. haploinsufficiency of most human TBX genes). In the case of omb, both loss and overexpression can destabilize epithelia. Dorsocross (a Tbx6 subfamily gene) is necessary and sufficient for certain folding processes of the Drosophila wing.
4. TBX2/3 in metastasis
TBX2 and TBX3 are overexpressed in many human tumors. In the case of melanoma it has been demonstrated that TBX2/3 overexpression is critical for the metastatic behaviour of this cancer. Invasive behaviour can also be demonstrated in a fly model, when omb or human TBX2/3 are overexpressed. We employ a Drosophila model and human tissue culture to screen for TBX2/3 inhibitors.
5. Proliferation control by Omb
In different regions of a given tissue, Omb can either stimulate or repress proliferation (e.g. dorsal vs. ventral eye imginal disc, medial vs. lateral wing imaginal disc). We search to identify the molecular determinants that govern these behaviours.
6. Structure-function analysis of the Tbx proteins Omb and Org-1.
The DNA binding domain of Tbx proteins is highly conserved. Functional differences of paralogous proteins are mainly caused by flanking protein domains that are rich in repetitive sequences (poly-glutamine and poly-alanine). Homo-Q and homo-A stretches are of interest also with regard to their involvement in proteinopathies.
7. Evolution of regulation in the Tbx2 subfamily
omb is the sole member of the Tbx2 subfamily in insects. In vertebrates, this subfamily encompasses four members, that share similarity with omb in their expression pattern. Can this similarity be traced back to evolutionarily conserved regulatory elements?
In several species of Drosophila, the abdominal pigmentation is genetically polymorphic within a given species. Omb is necessary and sufficient for abdominal pigmentation in Drosophila melanogaster. By identifying the abdominal enhancers in omb, it can be analysed whether variation in omb is involved in abdominal pigmentation polymorphism.