Browsing by Author "Jurkowska, Renata Z."

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Open Access
Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2
(2018) Rajavelu, Arumugam; Lungu, Cristiana; Emperle, Max; Dukatz, Michael; Bröhm, Alexander; Broche, Julian; Hanelt, Ines; Parsa, Edris; Schiffers, Sarah; Karnik, Rahul; Meissner, Alexander; Carell, Thomas; Rathert, Philipp; Jurkowska, Renata Z.; Jeltsch, Albert
Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.
Open Access
EpiCRISPR targeted methylation of Arx gene initiates transient switch of mouse pancreatic alpha to insulin-producing cells
(2023) Đorđević, Marija; Stepper, Peter; Feuerstein-Akgoz, Clarissa; Gerhauser, Clarissa; Paunović, Verica; Tolić, Anja; Rajić, Jovana; Dinić, Svetlana; Uskoković, Aleksandra; Grdović, Nevena; Mihailović, Mirjana; Jurkowska, Renata Z.; Jurkowski, Tomasz P.; Jovanović, Jelena Arambašić; Vidaković, Melita
Beta cell dysfunction by loss of beta cell identity, dedifferentiation, and the presence of polyhormonal cells are main characteristics of diabetes. The straightforward strategy for curing diabetes implies reestablishment of pancreatic beta cell function by beta cell replacement therapy. Aristaless-related homeobox (Arx) gene encodes protein which plays an important role in the development of pancreatic alpha cells and is a main target for changing alpha cell identity. In this study we used CRISPR/dCas9-based epigenetic tools for targeted hypermethylation of Arx gene promoter and its subsequent suppression in mouse pancreatic αTC1-6 cell line. Bisulfite sequencing and methylation profiling revealed that the dCas9-Dnmt3a3L-KRAB single chain fusion constructs (EpiCRISPR) was the most efficient. Epigenetic silencing of Arx expression was accompanied by an increase in transcription of the insulin gene (Ins2) mRNA on 5th and 7th post-transfection day, quantified by both RT-qPCR and RNA-seq. Insulin production and secretion was determined by immunocytochemistry and ELISA assay, respectively. Eventually, we were able to induce switch of approximately 1% of transiently transfected cells which were able to produce 35% more insulin than Mock transfected alpha cells. In conclusion, we successfully triggered a direct, transient switch of pancreatic alpha to insulin-producing cells opening a future research on promising therapeutic avenue for diabetes management.
Open Access
H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1
(2017) Jurkowska, Renata Z.; Qin, Su; Kungulovski, Goran; Tempel, Wolfgang; Liu, Yanli; Bashtrykov, Pavel; Stiefelmaier, Judith; Jurkowski, Tomasz P.; Kudithipudi, Srikanth; Weirich, Sara; Tamas, Raluca; Wu, Hong; Dombrovski, Ludmila; Loppnau, Peter; Reinhardt, Richard; Min, Jinrong; Jeltsch, Albert
SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.
Open Access
The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes
(2018) Emperle, Max; Dukatz, Michael; Kunert, Stefan; Holzer, Katharina; Rajavelu, Arumugam; Jurkowska, Renata Z.; Jeltsch, Albert
The DNA methyltransferase DNMT3A R882H mutation is observed in 25% of all AML patients. DNMT3A is active as tetramer and the R882H mutation is located in one of the subunit/subunit interfaces. Previous work has reported that formation of mixed wildtype/R882H complexes leads to a strong loss of catalytic activity observed in in vitro DNA methylation assays (Russler-Germain et al., 2014, Cancer Cell 25:442–454). To investigate this effect further, we have prepared mixed wildtype/R882H DNMT3A complexes by incubation of individually purified subunits of the DNMT3A catalytic domain and full-length DNMT3A2. In addition, we have used a double affinity tag approach and specifically purified mixed catalytic domain complexes formed after co-expression of R882H and wildtype subunits in E. coli cells. Afterwards, we determined the catalytic activity of the mixed complexes and compared it to that of purified complexes only consisting of one subunit type. In both settings, the expected catalytic activities of mixed R882H/wildtype complexes were observed demonstrating an absence of a dominant negative effect of the R882H mutation in purified DNMT3A enzymes. This result suggests that heterocomplex formation of DNMT3A and R882H is unlikely to cause dominant negative effects in human cells as well. The limitations of this conclusion and its implications are discussed.