Identifikation neuer Bindungspartner des c-MYC/MIZ-1–Netzwerks und Charakterisierung der Regulation der Transkription durch die c-MYC–Kofaktoren H2A.Z und MED24

Das Proto-Onkogen c-MYC kodiert für einen Transkriptionsfaktor, der die Expression von Zielgenen entweder aktiviert oder reprimiert. Die Repression wird bei Zielgenen wie P15INK4B und P21CIP1 durch Bindung an den ansonsten aktivierend wirkenden Transkriptionsfaktor MIZ-1...

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Bibliographic Details
Main Author: Adhikary, Till
Contributors: Bölker, Michael (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2009
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The proto-oncogene c-MYC encodes for a transcription factor capable of activating the expression of some while repressing the expression of other target genes. Repression of the target genes P15INK4B and P21CIP1 is mediated via binding to the transcription factor MIZ-1, which can otherwise activate the expression of these genes. After chromatographic enrichment of MIZ-1 and subsequent analysis via mass spectrometry, the putative binding partners Mi-2beta/CHD4 (chromodomain helicase DNA binding protein 4) and IRS4 (insulin receptor substrate 4) were identified, and interactions with c-MYC were shown by coimmunoprecipitation. In addition to Mi-2beta/CHD4, other components of the NuRD (nucleosome remodeling and histone deacetylation) were identified in the fractions analysed. The interaction with NuRD, which has been described as a repressor of transcription, suggest a possible mechanism of repression involving c-MYC and MIZ-1. IRS1, another member of the IRS family, was described as a transcription factor, suggesting the the regulation of transcription through an interaction of IRS4 with the c-MYC/MIZ-1 network. H2A.Z is a highly conserved variant of the canonical histone H2A, which is essential in mammals and can regulate transcription. In Drosophila melanogaster, the H2A.Z ortholog H2Av is exchanged by the Domino ATPase subunit of the Tip60 (Tat-interacting protein, 60 kDa) complex. In mammalian cells, c-MYC can recruit the Tip60 complex. H2A.Z was found to be recruited to target gene promoters in c-MYC-inducible 3T3-immortalised mouse fibroblasts and immediately thereafter being evicted from the promoters. This process occured close to the transcription start site and correlated with a significant increase of the expression of ccnd2 (cyclin D2), a c-MYC target gene. shRNA-mediated downregulation of H2A.Z expression in human osteosarcoma cells led to weaker activation and weaker repression of c-MYC target genes. The highly conserved Mediator complex forms a link between transcription factors and RNA polymerase II. In human cell lines, c-MYC interacts directly with the Mediator subunit MED24/TRAP100. Via shRNA-mediated downregulation of the expression of med24 and cdk8 (which encodes for another, facultative Mediator subunit) in c-MYC-inducible immortalised mouse fibroblasts, it could be shown that the interaction of c-MYC with Med24 and the presence of Cdk8 is essential for the activation and for the repression of virtually all c-MYC target genes. Also, reduced expression of med24 and cdk8 led to an increase of the expression of some, but not all c-MYC target genes examined. Furthermore, a c-MYC-dependent recruitment of Mediator subunits MED1 and CDK8 to the CCND2 promoter was shown in c-MYC-inducible human osteosarcoma cells. This suggests a role of the Mediator complex including its facultative CDK8 module in the activation of this promoter.