Charakterisierung und Regulation des humanen mad1-Promotors
The transcriptionfactor Mad1 is a potential tumorsuppressor. As a member of the Myc/Max/Madnetwork it antagonizes the transforming ability of the protooncogene c-myc. It was shown, that Mad1 can inhibit the transformation of REFs by c-myc/Ras through blockade of the G1/S-transition. Moreover it was shown, that Mad1 is an inhibitor of apoptosis. The members of the Myc/Max/Madnetwork are transcriptonfactors and regulate gene-expression after binding to specific DNA-sequences and then recruiting chromatin-modifying factors. In principle c-myc is described as an activator of transcription while mad1 is described as an inhibitor of transcription. Mad1 is an essential factor in the development of myeloid cells and loss of Mad1-function results in delayed differentiation of granulocytes and monocytes. Accordingly to this it fits, that mad1 gets upregulated after TPA- or GCSF-stimulation of U937-cells. Therefore it is interesting to know how Mad1 itself is transcriptionally regulated, to get more insight into crucial cellular processes like proliferation and differentiation. What are essential extracellular factors and what different intracellular signaltransduction-pathways are involved? In this study a human DNA-fragment was isolated which has mad1-promoter-function and contains a set of regulatory elements like binding sites for transcriptionfactors. This promoter-fragment is located only a few basepairs upstream of the translation-start-site. Alignement of this human sequence with DNA-sequences of mouse and rat reveales a box with nearly 70% homology and binding sites for transcriptionfactors important for myeloid development and differentiation. The mad1-promoter is very GC-rich and contains no TATA-box. In this study one transcription-start-site of the mad1-gene was identified. It is located at the 3´-end of the homologous promoter region. The influence of chromatin and its modifications on transcription is known. So it is important to investigate the nucleosomal structure of cis-acting DNA-elements like promoters and/or enhancers. In this study nucleosomal mapping of the mad1-promoter in U937-cells was done. It reveales that exactly the previous identified homologous region is nucleosome-free. This fits to other results, which show, that free access to regulatory DNA is a prereqiusite for transcription initiation. Unfortunately no GCSF- or TPA-effect on the nucleosomal structure was observed. The homology-box of the mad1- promoter is nucleosome-free in proliferating and/or differentiating U937-cells. To investigate the influence of different transcriptionfactors on Mad1-expression in detail, the Mad1- promoter was cloned as a reportergene-construct and several deletion-mutants were constructed. In following reportergene-assays c-myb, PU.1 and C/EBP Alpha, Beta and Epsilon were identified as positive regulators of mad1-transcription in RK13-cells in vitro. In the case of c-myb, cotransfection of GCSF/R and stimulation with GCSF results in a twofold increase of luciferase-activity. STAT1 and STAT3 exhibit only low but GCSF-dependent transcriptional potential. The ChIP-method (Chromatin-Immunpräzipitation) is very sensitive and specific in investigating protein/DNA-interactions in vivo. Using ChIP binding of RNA-Pol II and PU.1 to the human mad1- promoter was detected. This confirms some in vitro results. Additionally with the ChIP-method also histone-acetylation at the 3´-side of the homology-box was observed. But for other transcriptionfactors like STATs or C/EBPs no DNA-binding to the mad1-promoter was detected. Also no GCSFdependent recruitment of factors or histone-modification. In conclusion the results obtained in this study prove the essential role of the identified DNA-region in regulation of mad1-transcription and reveales the nucleosomal structure of this mad1-promoter. Also potential transcriptionfactors involved in upregulating mad1-expression were detected. But to completely understand the regulation of mad1-expression further investigation like timedependent and cofactor-dependent ChIP- and Reportergene-assays are needed.
info:eu-repo/classification/ddc/610, Medizin, Transkription, Chromatin, Nukleosomen, Hämatopoese, GCSF/R, Signaltransduktion
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