Funktionelle Charaktersierung der Arginin Methyltransferase PRMT6

Das primäre Ziel der vorliegenden Arbeit bestand in der Untersuchung der Histonmethyltransferaseaktivität von PRMT6. Trotz der Tatsache, dass PRMT6 im Zellkern lokalisiert ist und für einige andere PRMTs Histonmethylierung gefunden wurde, blieb die Argininmethylierung in Histonen durch PRMT6 bish...

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Bibliographic Details
Main Author: Dawin Michael Hyllus
Contributors: Lars-Oliver Essen (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2009
Molekularbiologie und Tumorforschung
Subjects:
MLL
Online Access:PDF Full Text
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Table of Contents: The primary goal of this work was to characterise the histone methyltransferase activity of PRMT6. Despite the fact that PRMT6 was found to be mainly nuclear localised and that several PRMT members exhibit activity towards histones, the histone-methylation by PRMT6 has not been investigated so far. In this work PRMT6 was found to methylate histones H3, H2A and H4 in vitro. Free histones were substrates of PRMT6, whereas nucleosomes could not be methylated by PRMT6 in vitro. The methylation sites were localized in the N-termini of histones and identified as R2 in histone H3 and R3 in histones H4 and H2A, respectively. The characterisation of the methylation mechanism resulted in a catalytic preference of PRMT6 for monomethylated arginines. This indicates that PRMT6 is mainly responsible for the dimethylation of arginines. Considering the methylation of R2 in histone H3 other histone modifications were found to exhibit an influence on the in vitro activity of PRMT6. While methylation of K4 and K9 in H3 inhibited PRMT6 activity, methylation of K27 increased PRMT6 activity. Subsequently, the investigation of H3R2 dimethylation function in the context of the histone code was the next goal of this work. This should be achieved by identifying potential binding partners (effector proteins) for this modification. Peptide pulldowns with synthetically modified histone H3-pepides were used to search for differentially interacting proteins. This technique did not result in the identification of specific interaction partners for H3R2 dimethylation. A supposed functional relationship between PRMT6 and the PRC2-complex as well as between their corresponding modifications R2 and K27 could not be confirmed using peptide pulldowns. Recent studies considering the genome-wide localisation of R2 di- and the K4 trimethylation suggested a negative cross-talk between these histone marks. Additionally, PRMT6 overexpression experiments demonstrated a repressive effect of PRMT6 on the expression of distinct HoxA genes and c-myc targets. The regulation of c-myc gene expression by the Wnt-pathway was used to further characterise the regulation mechanism of PRMT6. RNAi-mediated PRMT6-knockdown exhibited no effect of PRMT6 on the gene expression of c-myc in this context. Alternatively, the negative cross-talk between R2Me2 and K4Me3 should be described on the mechanistic level. Therefore, the in vitro influence of R2 dimethylation on K4 trimethyltransferase MLL activity as well as on histone binding affinity of MLL-complex subunit WDR5 was investigated. The R2 dimethylation exerted a strong inhibitory effect on MLL activity and on WDR5 binding to the histone H3-tail. The negative effect of R2 methylation on K4 methylation could be verified in vivo using chromatin immunoprecipitation on the HoxA2 promoter in PRMT6 overexpressing cells. Exogenous PRMT6 levels resulted in increased PRMT6 and R2 dimethylation levels at the promoter and decreased K4 trimethylation as well as reduced recruitment of MLL and WDR5. These promoter events were described in a model of neuronal cell differentiation, which confirmed the model for the repressive function of PRMT6- mediated R2 dimethylation in gene regulation. Together, the data from the present work identify PRMT6 as a novel histone methyltransferase, which dimethylates R2 in histone H3. Furthermore, the specific gene regulation mechanism for PRMT6 was characterised in the context of the histone code, where H3R2Me2 regulates H3K4Me3-mediated expression of certain genes.