Identifikation und Charakterisierung neuer Interaktionspartner und Zielgene der Proteinargininmethyltransferase 4

As a transcriptional coactivator, proteine arginine methyltransferases 4 (PRMT4) is a crucial regulator of gene expression. At the one hand, the enzyme feeds the histone code by the methylation of arginines at Histone H3, and on the other hand, PRMT4 interacts with other coactivators to enhance transcription in a cooperative manner. So far, a synergy between PRMT4 and another PRMT, PRMT1, has been described only for a small number of transcription factors. In the first part of this work, it was shown that PRMT1 and PRMT4 cooperatively regulate the putative STAT5-target genes CITED2 and KRT8. It was demonstrated that treatment of HeLa cells with the cytokine IL-4 causes phosphorylation and therefore activation of STAT5- and STAT6-signalling as well as transcriptional activation of CITED2 and KRT8. The transcriptional induction of CITED2 was strongly dependent on the transcription factor STAT5 as well as the coactivators PRMT1 and PRMT4. Chromatin immunoprecipitation (ChIP) analysis unveiled direct recruitment of STAT5, PRMT1 and PRMT4 at the promoter of CITED2 following IL-4 stimulation. Moreover, the transcriptional induction of CITED2 coincides with the arginine methylation of histones typical for both PRMTs. These findings prove PRMT1 and PRMT4 as direct regulators of CITED2 on the level of the promoter. As PRMT1 and PRMT4 interact with STAT5 in an IL-4-dependent manner, STAT5 probably mediates the target gene specificity of the PRMTs for CITED2. These data reveal for the first time a direct cooperative coactivating function of PRMT1 and PRMT4 in JAK-STAT-signalling. PRMT4 exert its function as transcriptional coactivator by interacting with other proteins. Gelfiltration analysis carried out in this work implicated PRMT4 reside within higher molecular weight complexes. The aim of the second part of this work was to identify novel interaction partners of PRMT4 to uncover unknown functions and signalling pathways of the enzyme. Using ion-exchange chromatography and subsequent affinity purification enabled the separation of endogenous PRMT4-containing complexes and the identification of the proteins Mi-2 and ALIX as novel interaction partners of PRMT4 by mass spectrometry. These interactions have been verified by independent interaction assays. It turned out, that the Mi-2 related protein Mi-2 is also able to interact with PRMT4. As member of the protein family of the ATP-dependent chromatin remodelers, Mi-2 (Mi-2/) plays a key role in transcriptional regulation, implicating a common function with PRMT4 in gene expression. Association of PRMT4 with the NuRD repressor complex, containing the integral components Mi-2 or Mi-2, could be excluded by coimmunoprecipitations (CoIPs). Using the transformed chicken macrophage cell line HD11, a synergy between PRMT4 and both Mi-2 proteins in the activation of c-Myb dependent transcription of the mim-1 and Lysozyme gene has been observed by cotransfection assays. RNAi-mediated depletions as well as ChIP analysis in the human erythroleukemic cell line K562 demonstrate a positive and direct regulation of the c-Myb target genes c-MYC and CDC7 by PRMT4 and Mi-2. Studies carried out to investigate the biological relevance of PRMT4 and Mi-2 in hematopoietic cells showed a correlation with c-Myb mediated effects on cell proliferation, cell cycle and erythroid differentiation. The results described here, together with the finding that c-Myb interacts with PRMT4 and Mi-2, show PRMT4 and Mi-2 as cooperative coactivators of c-Myb. This data indicates that PRMT4 and Mi-2 are regulators of c-Myb mediated functions, and therefore have an impact on erythroid differentiation.