Analysen zur Funktion von Mehmet, einem neuen Interaktionspartner des Transkriptionsfaktors AP-2-alpha, sowie Charakterisierung von Mehmet in Drosophila melanogaster
AP-2 transcription factors represent a family of five closely related and evolutionarily conserved DNA-binding proteins termed AP-2alpha, AP-2beta, AP-2gamma, AP-2delta and AP-2epsilon. They are characterized by an unique modular structure consisting of an aminoterminal transcriptional activation domain and a complex helix span helix motif for dimerization and DNA binding. AP-2 proteins have been suggested to regulate a variety of target genes, embryonic development, apoptosis and differentiation. Here we report the characterization of Mehmet, a potential new AP-2 interacting protein, isolated by Far Western screening. The cDNA encodes a 1306 aa protein with a molecular mass of apparently 150 kD. Sequence analysis revealed 3 main domains: The N-terminal region shows homology to different yeast-proteins (Y-Box), followed by an acidic region. Homology with 5‘ cytosine DNA methyltransferase and Drosophila trithorax protein was found in the central section, causing the name Mehmet (protein containing MECP-1 homology region similar to DNA-methyltransferase and mammalian trithorax proteins). The C-terminal domain is homologue to F-box- and leucin motives. Expression analysis revealed a 4.4 kb Mehmet transcript in liver and kidney, correlating to the expression pattern of AP-2alpha, and expression was also shown in brain, testis, spleen and heart. To confirm interaction between AP-2 and Mehmet in vitro and in vivo we performed GST pull down ­­­­­­­­­­­– and IP experiments. AP-2alpha, AP-2beta and AP-2gamma were tested and showed a positive result in GST-pull down assay used for the confirmation of AP-2/Mehmet interaction. Immunofluorescence microscopy revealed a translocation of Mehmet within the cell from cytoplasm into the nucleus. To understand the function we established stable transfectants with inducibly Mehmet expression. Potential degradation of AP-2alpha by Mehmet was researched and constant level of AP-2alpha in Western-Blot analysis proved that Mehmet has no influence on the stability of AP-2alpha. Proliferation and apoptosis assays demonstrated no functional interference for inducible expression of Mehmet. Data search analysis identified a highly conserved Mehmet-gene in Drosophila melanogaster (dMehmet). Expression Analysis of dMehmet by Northern Blot, RT-PCR and in situ hybridisation confirm a persistent expression pattern of dMehmet during Drosophila development.
info:eu-repo/classification/ddc/570, Taufliege, Transkriptionsfaktor, cDNS, Wechselwirkung, Biowissenschaften, Biologie, Mehmet, Transkriptionsfaktor AP-2alpha, Drosophila melanogaster
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