Charakterisierung der Repressorfunktion von PRMT6 und deren Rolle bei der Differenzierung, Proliferation und Seneszenz

PRMT6 belongs to the enzyme family of protein arginine methyltransferases and was identified in 2002 by sequence homology search in the human genome using the conserved catalytic domain. The enzyme is predominantly localized in the nucleus. Although some PRMT6 substrates have been described, the precise function of this enzyme still remains elusive. The aim of this work was the identification and characterization of novel chromatin functions of PRMT6. At first the in vivo existence and relevance of the H3R2 dimethylation catalyzed by PRMT6 was verified. It was shown that the PRMT6-mediated H3R2 dimethylation counteracts the active histone modification H3K4me3 and thereby represses the expression of a subset of HOXA genes and c-Myc target genes. In the following the underlying mechanism of this antagonism between H3R2me2 and H3K4me3 was uncovered at the HOXA2 gene. PRMT6-mediated H3R2 dimethylation inhibits H3K4 trimethylation by preventing the chromatin recruitment of the components MLL1 and WDR5 of the H3K4 methyltransferase complex. This repressive function of PRMT6 was also found to be relevant for HOXA2 gene expression during neuronal differentiation in the NT2/D1 cell model. In the further course of this work a potential positive cross-talk between PRMT6 and polycomb group proteins was studied. PRMT6 was shown to interact with a subset of PcG proteins and similar to PcGs to be involved in the maintenance of the specific expression pattern of the entire HOXA gene locus during neuronal differentiation. Similar to the depletion of PcG proteins, depletion of PRMT6 was accompanied by hyperactivation of the anterior-localized HOXA genes and derepression of the posterior-localized HOXA genes following ATRA-induced differentiation of NT2/D1 cells. This deregulation of the HOXA genes upon loss of PRMT6 coincided with enhanced enrichment of the active histone modification H3K4me3 and decreased amounts of the repressive histone modification H3K27me3. In summary, these findings identify PRMT6 as a novel transcriptional repressor depending on its H3R2 methyltransferase activity and reveal as molecular mechanism of this repression a cross-talk between histone arginine methylation and lysine methylation. Since PcG proteins have been linked to the regulation of proliferation and senescence by repressing the INK4B-ARF-INK4A locus, it was investigated next whether PRMT6 would similarly control the INK4B-ARF-INK4A expression. The loss of PRMT6 in human embryonic fibroblasts resulted in a proliferation block, which was characterized by an accumulation of G1-phase cells. Moreover, depletion of PRMT6 caused cellular senescence indicated by beta-galactosidase positive cells and led to the upregulation of the CDK-inhibitors p16/INK4A as well as CDKN1A (p21/CIP1). In agreement with these results, the clonogenic growth potential of tumor cells was reduced in PRMT6-depleted cells. In conclusion, these data suggest that PRMT6 is necessary for the proliferation of both human diploid fibroblasts and tumor cells and inhibits cellular senescence as indicated here by controlling the expression of important cell cycle regulators. Finally, an unbiased screen for novel interaction partners of PRMT6 was established using overexpression of TAP (tandem affinity purification)-tagged PRMT6. Subsequent to affinity purification of exogenous PRMT6, co-purifying proteins were identified by mass spectrometry and analyzed by the database STRING and literature search to uncover published connections between the putative interactors, arginine methylation and PRMT6. These results will serve as a starting point for future research extending our knowledge on further nuclear functions of PRMT6.