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Final Report Abstract
Methylated cytosines (mC) as well as their three recently discovered oxidized derivatives in genomic DNA of mammalian cells play many important roles in organism development and diseases. The discovery of Ten-Eleven-Translocation (TET) family proteins which enzymatically oxidises mC, gave a new breath into epigenetic research. Understanding a mechanism of active DNA demethylation as well as its targeting is biologically important. It is also important to know the mechanism and players for proper medical intervention. In our group we have established a stable isotope dilution, LC-MS/MS based technique, which allows for precise quantification of all the above mentioned cytosine modification in genomic DNA of different cells and tissues to study Tet dependent DNA demethylation in vivo or in cells. Project 1: Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation. Lars Schomacher, Michael Musheev, Dandan Han (AG Prof. Dr. Christof Niehrs, Institut für Molekulare Biologie (IMB) gGmbH, Mainz) In the TET-TDG demethylation pathway, methylated cytosine is iteratively oxidized by TET dioxygenases and unmodified cytosine is restored via thymine DNA glycosylase (TDG) and subsequent base excision repair factors. Our in vitro studies demonstrate that NEIL1 and NEIL2 DNA glycosylases themselves cannot excise any of the mC oxidation derivatives, instead they replace TDG on the abasic site, to further process it. That stimulates TDG substrate turnover, accelerating the whole DNA demethylation process. Using the LC/MS technique we corroborated the finding in vivo using genomic DNA of Xenopus embryos. Neil 2 overexpression alone has no significant effect on 5’-formyl-dC (5fC) and 5’-carboxy-dC (5caC). Upon TDG depletion, levels of 5fC and 5caC increase, since they cannot be excised from DNA. As the model predicts, NEIL2 overexpression partially rescues TDG activity and lowers levels of both 5fC and 5caC. Additionally a double depletion of TDG and NEIL cause an additive negative effect and further increases 5fC and 5caC levels in gDNA. Project 2: GADD45a physically and functionally interacts with TET1. Sabine Kienhöfer, Andrea Schäfer, Michael Musheev. (AG Prof. Dr. Christof Niehrs, Institut für Molekulare Biologie (IMB) gGmbH, Mainz) Different mechanisms of active DNA demethylation have been established. For example, Growth Arrest and DNA Damage 45-(GADD45) and TET proteins act in active DNA demethylation but their functional relationship is unresolved. Here we show that GADD45a physically interacts - and functionally cooperates with TET1 in methylcytosine (mC) processing. In reporter demethylation GADD45a requires endogenous TET1 and conversely TET1 requires GADD45a. On GADD45a target genes TET1 hyperinduces 5-hydroxymethylcytosine (hmC) in the presence of GADD45a, while 5-formyl-(fC) and 5-carboxylcytosine (caC) are reduced. Likewise, in global analysis GADD45a positively regulates TET1 mediated mC oxidation and enhances fC/caC removal. Our data suggest a dual function of GADD45a in oxidative DNA demethylation, to promote directly or indirectly TET1 activity and to enhance subsequent fC/caC removal.
Publications
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A transient ischemic environment induces reversible compaction of chromatin. Genome Biology, 16, 246. 2015
Kirmes, I.; Szczurek, A.; Prakash, K.; Charapitsa, I.; Heiser, C.; Musheev, M.; Schock, F.; Fornalczyk, K.; Ma, D.; Birk, U.; Cremer, C.; Reid, G.
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DAZL regulates Tet1 translation in murine embryonic stem cells. EMBO Reports, 16, 791-802. 2015
Welling, M.; Chen, H.H.; Muñoz, J.; Musheev, M.U.; Kester, L.; Junker, J.P.; Mischerikow, N.; Arbab, M.; Kuijk, E.; Silberstein, L.; Kharchenko, P.V.; Geens, M.; Niehrs, C.; van de Velde, H.; van Oudenaarden, A.; Heck, A.J.; Geijsen, N.
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GADD45a physically and functionally interacts with TET1. Differentiation, 90, 59-68. 2015
Kienhöfer, S.; Musheev, M.U.; Stapf, U.; Helm, M.; Schomacher, L.; Niehrs, C.; Schäfer, A.
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Octreotide-Mediated Tumor-Targeted Drug Delivery via a Cleavable Doxorubicin-Peptide Conjugate. Molecular Pharmaceutics, 12, 4290– 4300. 2015
Lelle, M.; Kaloyanova, S.; Freidel, C.; Theodoropoulou, M.; Musheev, M.; Niehrs, C.; Stalla, G.; Peneva, K.
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Enhancers reside in a unique epigenetic environment during early zebrafish development. Genome Biology, , 17, 146. 2016
Kaaij, L.J.; Mokry, M.; Zhou, M.; Musheev, M.; Geeven, G.; Melquiond, A.S.; de Jesus Domingues, A.M.; de Laat, W; Niehrs, C.; Smith, A.D.; Ketting, R.F.
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Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation. Nature Structural Molecular Biology, 23, 116-24. 2016
Schomacher, L.; Han, D.; Musheev, M.U.; Arab, K.; Kienhöfer, S.; von Seggern, A.; Niehrs, C.
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Overcoming drug resistance by cell-penetrating peptidemediated delivery of a doxorubicin dimer with high DNA-binding affinity. European Journal of Medicinal Chemistry. 130, 336-345. 2017
Lelle, M.; Freidel ,C.; Kaloyanova, S.; Tabujew, I.; Schramm, A.; Musheev, M.; Niehrs, C.; Müllen, K.; Peneva, K.
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RNA fate determination through cotranscriptional adenosine methylation and microprocessor binding. Nature Structural Molecular Biology, 24, 561-569. 2017
Knuckles, P.; Carl, S.H.; Musheev, M.; Niehrs, C.; Wenger, A.; Bühler, M.
