Article

Arginine methylation provides epigenetic transcription memory for retinoid-induced differentiation in myeloid cells.

Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Nagyerdei krt. 98, Debrecen H-4012, Hungary.
Molecular and Cellular Biology (Impact Factor: 5.04). 08/2005; 25(13):5648-63. DOI: 10.1128/MCB.25.13.5648-5663.2005
Source: PubMed

ABSTRACT Cellular differentiation is governed by changes in gene expression, but at the same time, a cell's identity needs to be maintained through multiple cell divisions during maturation. In myeloid cell lines, retinoids induce gene expression and a well-characterized two-step lineage-specific differentiation. To identify mechanisms that contribute to cellular transcriptional memory, we analyzed the epigenetic changes taking place on regulatory regions of tissue transglutaminase, a gene whose expression is tightly linked to retinoid-induced differentiation. Here we report that the induction of an intermediary or "primed" state of myeloid differentiation is associated with increased H4 arginine 3 and decreased H3 lysine 4 methylation. These modifications occur before transcription and appear to prime the chromatin for subsequent hormone-regulated transcription. Moreover, inhibition of methyltransferase activity, pre-acetylation, or activation of the enzyme PAD4 attenuated retinoid-regulated gene expression, while overexpression of PRMT1, a methyltransferase, enhanced retinoid responsiveness. Taken together, our results suggest that H4 arginine 3 methylation is a bona fide positive epigenetic marker and regulator of transcriptional responsiveness as well as a signal integration mechanism during cell differentiation and, as such, may provide epigenetic memory.

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Available from: Laszlo Nagy, Jul 03, 2015
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Im Laufe der Untersuchung wurden jedoch einige Hinweise gefunden, die der Argininmethylierung von STAT1 oder von STAT3 widersprechen. Erstens konnte nicht gezeigt werden, dass die anti-Methylarginin-Antikörper STAT1 und STAT3 spezifisch präzipitieren. Zweitens wurde gezeigt, dass die Methyltransferaseinhibitoren, MTA und MDA, die Phosphorylierung von STAT1 und STAT3 zwar deutlich beeinflussen, MDA allerdings zusätzlich auch die p38- und Erk-Signalkaskaden, die bekanntermaßen auch mit dem Jak/STAT-Weg wechselwirken. Drittens wurde gezeigt, dass der Austausch von Arg31 gegen Lys zur Destabilisierung von STAT1 und von STAT3 führt. Dies impliziert eine wichtige strukturelle Rolle von Arg31. Schließlich wurde gezeigt, dass STAT1 und STAT3 durch PRMT1 in vitro nicht methyliert und Koexpression von PRMT1 keinen signifikanten Einfluss auf die STAT1-vermittelte Aktivierung eines Reportergens hat. Zusammenfassend geben die vorliegenden Ergebnisse somit keine Hinweise auf eine Argininmethylierung von STAT1 und STAT3. Bei der Jak/STAT-Signaltransduktion spielen Feedback-Inhibitoren der SOCS-Familie eine wichtige Rolle. Im zweiten Teil dieser Arbeit wurde die Zytokin-resistente Melanomzelllinie 1286 untersucht, um die molekularen Mechanismen ihrer Zytokinresistenz besser zu verstehen. Obwohl die relevanten Rezeptoren gp130 und OSMR an der Zelloberfläche dieser Zellen exprimiert wurden, führte Zytokin-Stimulation kaum zur Aktivierung von Jak1, STAT3 oder STAT1. Interessanterweise konnte in den resistenten 1286-Zellen eine hohe konstitutive SOCS3-Protein-Expression nachgewiesen werden. Die Unterdrückung der SOCS3-Expression durch RNA-Interferenz stellte die Zytokin-Sentitivität gegen IL-6-Typ-Zytokine wieder her bzw. erhöhte sie. Die SOCS3-siRNA behandelten Zellen zeigten eine starke STAT3-Phosphorylierung und eine noch stärkere STAT1-Phosphorylierung. Außerdem wurden die 1286-Zellen durch die SOCS3-Suppression empfindlich für anti-proliferative Effekte von IL-6 und OSM, aber nicht für diejenigen von IFN-alpha. In Zytokin-sensitiven WM239-Zellen konnte ebenfalls gezeigt werden, dass die Unterdrückung von induzierbarem SOCS3 die Zytokin-Sensitivität erhöht. Folglich stellt die SOCS3-Expression einen Wachstumsvorteil für diese Melanomzellen dar. Konstitutiv exprimierte SOCS3-mRNA, allerdings in geringeren Spiegeln als in 1286-Zellen, wurde auch in neun weiteren humanen Melanomzelllinien sowie in normalen humanen Melanozyten gefunden, wobei die SOCS3-Protein-Expression in diesen Zellen jedoch nicht oder nur gering nachweisbar war. Allerdings konnte in einer in-situ-Analyse von Melanomschnitten in drei von zehn Proben eine Immunreaktivität gegen SOCS3 detektiert werden, was bedeuten könnte, dass SOCS3 – zumindest in einem Teil der Tumorzellen - auch in vivo eine Rolle in der IL-6-Resistenz spielen könnte. Cytokines play an important role in the growth regulation of melanoma cells. Whereas the growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, melanoma cells of advanced tumor stages have often been reported to be “multi-cytokine resistant”. Thus, the aim of this study was to investigate the molecular mechanisms underlying cytokine resistance of melanoma cells. Transcription factors of the STAT family are crucial in signal transduction of cytokines. Previously, it has been reported that methylation of STAT1 on a conserved arginine residue (Arg31) is important for STAT1 function and loss of this modification was proposed to be involved in interferon resistance of cancer cells. Since STAT3 is involved in growth inhibition of melanoma cells, in the first part of this thesis I thus investigated the arginine methylation of STAT3 and STAT1. Here I provide several independent lines of evidence that did not support the occurrence of arginine methylation of STAT1 or STAT3. First, it was shown that the anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Second, it was shown that the methylation inhibitors, MTA and MDA, had profound and rapid effects on phosphorylation of STAT1 and STAT3, but MDA additionally also affected the p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Third, it was shown that mutation of Arg31 to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg31. Finally, the in vitro methylation assay using purified catalytically active protein arginine methyltransferases (PRMT1, -2, -3, -4, and -6) demonstrated that STAT proteins are not methylated, and cotransfection of PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, the data presented in this thesis suggest the absence of arginine methylation of STAT1 and STAT3. The Jak/STAT signaling pathway is subject to feedback inhibition by members of the suppressors of cytokine signaling (SOCS) family. In the second part of this thesis, I analysed the melanoma cell line 1286, resistant towards the growth-inhibitory effects of IL-6 and OSM, to better understand the mechanisms underlying cytokine resistance. Although the relevant receptors gp130 and OSMR are expressed at the cell surface of these cells, cytokine stimulation hardly led to activation of Jak1, STAT3 and STAT1. I found a high level constitutive expression of SOCS3 that did not further increase after cytokine treatment. Importantly, upon suppression of SOCS3 by short interfering RNA, cells became susceptible towards OSM and IL-6: they showed an enhanced STAT3 phosphorylation and a dramatically increased STAT1 phosphorylation. Moreover, suppression of SOCS3 rendered 1286 cells sensitive to the anti-proliferative action of IL-6 and OSM, but not of IFN-alpha. Interestingly, SOCS3-siRNA treatment also increased the growth-inhibitory effect in cytokine-sensitive WM239 cells expressing SOCS3 in an inducible way. Thus, SOCS3 expression confers a growth advantage to these cell lines. Constitutive SOCS3 mRNA expression, although at lower levels than in 1286 cells, was found in nine additional human melanoma cell lines and in normal human melanocytes while at protein level SOCS3 expression was marginal at best. However, in situ analysis of human melanoma specimens revealed SOCS3 immunoreactivity in 3 out of 10 samples suggesting that in vivo SOCS3 may possibly play a role in IL-6 resistance in at least a fraction of tumors.