The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.
Emperle et al. (2019) Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns. Nucleic Acids Res. 47(21):11355-11367
Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3' side of the CpG site, where they resemble DNMT3B, while 5' flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.
Lungu et al. (2017) Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites. Nature Communications 8:649
Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live-cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing defined epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.Tools for imaging epigenetic modifications can shed light on the regulation of epigenetic processes. Here, the authors present a fluorescence complementation approach for detection of DNA and histone methylation at endogenous genomic sites allowing following of dynamic changes of these marks by live-cell microscopy.
Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL–AF9;NrasG12D-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.
Publikationsliste von Dr. Rathert
- 28. T. L. Bauer u. a., „Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development“, Cancers, Bd. 13, Nr. 15, Art. Nr. 15, 2021, doi: 10.3390/cancers13153801.
- 27. J. Diesch u. a., „Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis“, Nature Communications, Bd. 12, Nr. 1, Art. Nr. 1, 2021, doi: 10.1038/s41467-021-26258-z.
- 26. S. Pinter u. a., „A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation“, Nucleic Acids Res, Bd. 49, Nr. 8, Art. Nr. 8, 2021, doi: 10.1093/nar/gkab180.
- 25. P. Rathert, „Structure, Activity and Function of the NSD3 Protein Lysine Methyltransferase“, Life, Bd. 11, Nr. 8, Art. Nr. 8, 2021, doi: 10.3390/life11080726.
- 24. J. Broche, G. Kungulovski, P. Bashtrykov, P. Rathert, und A. Jeltsch, „Genome-wide investigation of the dynamic changes of epigenome modifications after global DNA methylation editing“, Nucleic Acids Res, Dez. 2020, doi: 10.1093/nar/gkaa1169.
- 23. A. Bröhm u. a., „Somatic Cancer Mutations in the SUV420H1 Protein Lysine Methyltransferase Modulate Its Catalytic Activity“, Journal of Molecular Biology, Bd. 431, Nr. 17, Art. Nr. 17, 2019, doi: 10.1016/j.jmb.2019.06.021.
- 22. M. Emperle u. a., „Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns“, Nucleic Acids Res, Bd. 47, Nr. 21, Art. Nr. 21, 2019, doi: 10.1093/nar/gkz911.
- 21. W. S. Lieb u. a., „The GEF‐H1/PKD3 signaling pathway promotes the maintenance of triple negative breast cancer stem cells“, International Journal of Cancer, S. ijc.32798, Nov. 2019, doi: 10.1002/ijc.32798.
- 20. S. Sdelci u. a., „MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation“, Nature Genetics, Bd. 51, Nr. 6, Art. Nr. 6, 2019, doi: 10.1038/s41588-019-0413-z.
- 19. P. R. Carolin Kroll, „Stable Expression of Epigenome Editors via Viral Delivery and Genomic Integration“, Methods in Molecular Biology, Nr. 1767, Art. Nr. 1767, 2018.
- 18. A. Rajavelu u. a., „Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2“, Nucleic Acids Research, Bd. 46, Nr. 17, Art. Nr. 17, 2018, doi: doi:10.1093/nar/gky715.
- 17. C. Lungu, S. Pinter, J. Broche, P. Rathert, und A. Jeltsch, „Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites“, Nature Communications, Bd. 8, 2017, doi: 10.1038/s41467-017-00457-z.
- 16. S. Sdelci u. a., „Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk“, Nature chemical biology, Bd. 12, Nr. 7, Art. Nr. 7, 2016, doi: 10.1038/nchembio.2080.
- 15. P. Rathert u. a., „Transcriptional plasticity promotes primary and acquired resistance to BET inhibition“, Nature, Bd. 525, Nr. 7570, Art. Nr. 7570, Sep. 2015, doi: 10.1038/nature14898.
- 14. S. Kudithipudi, C. Lungu, P. Rathert, N. Happel, und A. Jeltsch, „Substrate specificity analysis and novel substrates of the protein lysine methyltransferase NSD1“, Chemistry and Biology, Bd. 21, Nr. 2, Art. Nr. 2, 2014, doi: 10.1016/j.chembiol.2013.10.016.
- 13. I. Bock, A. Dhayalan, S. Kudithipudi, O. Brandt, P. Rathert, und A. Jeltsch, „Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays“, Epigenetics, Bd. 6, Nr. 2, Art. Nr. 2, 2011, doi: 10.4161/epi.6.2.13837.
- 12. A. Dhayalan, S. Kudithipudi, P. Rathert, und A. Jeltsch, „Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase“, Chemistry and Biology, Bd. 18, Nr. 1, Art. Nr. 1, 2011, doi: 10.1016/j.chembiol.2010.11.014.
- 11. A. Dhayalan u. a., „The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation“, Journal of Biological Chemistry, Bd. 285, Nr. 34, Art. Nr. 34, 2010, doi: 10.1074/jbc.M109.089433.
- 10. Y. Zhang u. a., „Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail“, Nucleic Acids Research, Bd. 38, Nr. 13, Art. Nr. 13, 2010, doi: 10.1093/nar/gkq147.
- 9. A. Dhayalan, E. Dimitrova, P. Rathert, und A. Jeltsch, „A continuous protein methyltransferase (G9a) assay for enzyme activity measurement and inhibitor screening“, Journal of Biomolecular Screening, Bd. 14, Nr. 9, Art. Nr. 9, 2009, doi: 10.1177/1087057109345528.
- 8. A. Jeltsch und P. Rathert, „Putting the pieces together: histone H2B ubiquitylation directly stimulates histone H3K79 methylation“, ChemBioChem, Bd. 9, Nr. 14, Art. Nr. 14, 2008, doi: 10.1002/cbic.200800414.
- 7. P. Rathert u. a., „Protein lysine methyltransferase G9a acts on non-histone targets“, Nature Chemical Biology, Bd. 4, Nr. 6, Art. Nr. 6, 2008, doi: 10.1038/nchembio.88.
- 6. P. Rathert, X. Zhang, C. Freund, X. Cheng, und A. Jeltsch, „Analysis of the substrate specificity of the Dim-5 histone lysine methyltransferase using peptide arrays“, Chemistry and Biology, Bd. 15, Nr. 1, Art. Nr. 1, 2008, doi: 10.1016/j.chembiol.2007.11.013.
- 5. P. Rathert, A. Dhayalan, H. Ma, und A. Jeltsch, „Specificity of protein lysine methyltransferases and methods for detection of lysine methylation of non-histone proteins“, Molecular BioSystems, Bd. 4, Nr. 12, Art. Nr. 12, 2008, doi: 10.1039/b811673c.
- 4. R. Goyal, P. Rathert, H. Laser, H. Gowher, und A. Jeltsch, „Phosphorylation of serine-515 activates the Mammalian maintenance methyltransferase Dnmt1“, Epigenetics, Bd. 2, Nr. 3, Art. Nr. 3, 2007, doi: 10.4161/epi.2.3.4768.
- 3. A. Jeltsch, R. Jurkowska, T. Jurkowski, K. Liebert, P. Rathert, und M. Schlickenrieder, „Application of DNA methyltransferases in targeted DNA methylation“, Applied Microbiology and Biotechnology, Bd. 75, Nr. 6, Art. Nr. 6, 2007, doi: 10.1007/s00253-007-0966-0.
- 2. P. Rathert u. a., „Reversible inactivation of the CG specific SssI DNA (cytosine-C5)-methyltransferase with a photocleavable protecting group“, ChemBioChem, Bd. 8, Nr. 2, Art. Nr. 2, 2007, doi: 10.1002/cbic.200600358.
- 1. P. Rathert, X. Cheng, und A. Jeltsch, „Continuous enzymatic assay for histone lysine methyltransferases“, BioTechniques, Bd. 43, Nr. 5, Art. Nr. 5, 2007, doi: 10.2144/000112623.