Untersuchung der Rolle von PRMT1 bei der Estrogenrezeptor vermittelten Genaktivierung und Aufreinigung endogener PRMT1- und PRMT4- haltiger Proteinkomplexe

Die Modifikation der N-Termini von Histonen trägt zur Regulierung der Expression von Genen bei. Einzelmodifikationen oder Kombinationen regulieren den Verpackungsstatus des Chromatins. So Methylieren z. B. die Protein Arginin Methyltransferasen (PRMT) 1, 4 und 5 Arginine der Histone H2A, H3 und H4....

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Bibliographic Details
Main Author: Wagner, Sabine Astrid
Contributors: Bauer, Uta Maria (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2006
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Gene expression can be regulated by modifications of the N-termini of histones. Single modifications or combinations of modifications determine the condensation status of chromatin. Thus the arginines of histones H2A, H3 and H4 are methylated by protein arginine methyltransferases (PRMTs) 1, 4 and 5. Aim of this work was to unravel the role of the asymmetric dimethylation of arginine 3 on histone H4, catalysed by PRMT1, within transcriptional regulation. Furthermore a strategy to purify endogenous protein complexes containing PRMT4 was established. Histone H4 peptides (aa 1 – 15), eihter unmodified or asymmetrically dimethylated on arginine 3, were used in pull-down assays to identify differentially binding proteins from nuclear extracts. MALDI TOF analysis revealed that TAF-Iβ, part of the INHAT complex which inhibits histone acetylation, selectively binds to unmethylated peptides. In this work it could be demonstrated that TAF-Iβ binds to the uninduced PRMT1-dependend pS2 promoter and leaves it after induction of gene expression by estradiol. Nevertheless arginine 3 methylation seems not to be the crucial event for TAF-Iβ to leave the promoter in vivo. Successful reduction of TAF-Iβ by using specific siRNA caused a premature hyperacetylation of the pS2 promoter. This leads to an inhibition of arginine 3 methylation and probably further modifications resulting in insufficient transcription of the gene. Analyses of further estrogen inducible genes revealed that transcription of another PRMT1 dependend gene, lactoferrin, is also impaired by an untimely hyperacetylation of the promoter region. However, transcription of lactoferrin was not regulated by TAF-Iβ implying the existence of other proteins saving promoters from an early acetylation. In contrast hyperacetylation was sufficient for transcription of PRMT1 independend genes even without addion of hormone. Taken together, this thesis defines prerequisites for efficient recruitment of PRMT1 and following arginine 3 methylation to estrogen inducible genes. A second project in the course of this dissertation was to establish a strategy to purify endogenous protein complexes containing PRMT1 or PRMT4 by using ion-exchange chromatography. Determination of the sizes of PRMT1 or PRMT4 containing complexes with size exclusion chromatography suggest the existence of multiprotein complexes. A following anaylsis of the composition of such complexes will give insight into functions of PRMT1 and PRMT4 and their recruitment to their target promoters.