Publications by authors named "Martha Smets"

8 Publications

  • Page 1 of 1

Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in -ITD AML with concurrent epigenetic mutations.

Oncotarget 2017 Dec 16;8(65):108738-108759. Epub 2017 Oct 16.

Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.

Effectively targeting leukemia-initiating cells (LIC) in -ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates -ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show -ITD occurs within the most primitive Lin/CD33/CD45/CD34CD38 LIC compartment. Crenolanib alone could not target -ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC and severely impaired engraftment in NSG mice. Strikingly, -mutated samples harboring mutations were completely resistant to crenolanib whereas neither nor mutations influenced response. Conversely, primary AML LIC harboring either or mutations did not show increased sensitivity to AZA. In summary, resistance of -ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of -ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.
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http://dx.doi.org/10.18632/oncotarget.21877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752477PMC
December 2017

Ubiquitome Analysis Reveals PCNA-Associated Factor 15 (PAF15) as a Specific Ubiquitination Target of UHRF1 in Embryonic Stem Cells.

J Mol Biol 2017 12 18;429(24):3814-3824. Epub 2017 Oct 18.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany. Electronic address:

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.
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http://dx.doi.org/10.1016/j.jmb.2017.10.014DOI Listing
December 2017

L1 retrotransposition is activated by Ten-eleven-translocation protein 1 and repressed by methyl-CpG binding proteins.

Nucleus 2017 09 19;8(5):548-562. Epub 2017 May 19.

a Department of Biology , Technical University Darmstadt , Darmstadt , Germany.

One of the major functions of DNA methylation is the repression of transposable elements, such as the long-interspersed nuclear element 1 (L1). The underlying mechanism(s), however, are unclear. Here, we addressed how retrotransposon activation and mobilization are regulated by methyl-cytosine modifying ten-eleven-translocation (Tet) proteins and how this is modulated by methyl-CpG binding domain (MBD) proteins. We show that Tet1 activates both, endogenous and engineered L1 retrotransposons. Furthermore, we found that Mecp2 and Mbd2 repress Tet1-mediated activation of L1 by preventing 5hmC formation at the L1 promoter. Finally, we demonstrate that the methyl-CpG binding domain, as well as the adjacent non-sequence specific DNA binding domain of Mecp2 are each sufficient to mediate repression of Tet1-induced L1 mobilization. Our study reveals a mechanism how L1 elements get activated in the absence of Mecp2 and suggests that Tet1 may contribute to Mecp2/Mbd2-deficiency phenotypes, such as the Rett syndrome. We propose that the balance between methylation "reader" and "eraser/writer" controls L1 retrotransposition.
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http://dx.doi.org/10.1080/19491034.2017.1330238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703239PMC
September 2017

DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation.

Hum Mol Genet 2017 04;26(8):1522-1534

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.
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http://dx.doi.org/10.1093/hmg/ddx057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393148PMC
April 2017

Comparative Analysis of Single-Cell RNA Sequencing Methods.

Mol Cell 2017 Feb;65(4):631-643.e4

Anthropology & Human Genomics, Department of Biology II, Ludwig-Maximilians University, Großhaderner Straße 2, 82152 Martinsried, Germany. Electronic address:

Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs). Power simulations at different sequencing depths showed that Drop-seq is more cost-efficient for transcriptome quantification of large numbers of cells, while MARS-seq, SCRB-seq, and Smart-seq2 are more efficient when analyzing fewer cells. Our quantitative comparison offers the basis for an informed choice among six prominent scRNA-seq methods, and it provides a framework for benchmarking further improvements of scRNA-seq protocols.
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http://dx.doi.org/10.1016/j.molcel.2017.01.023DOI Listing
February 2017

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

Cell Res 2015 Aug 12;25(8):911-29. Epub 2015 Jun 12.

1] Department of Biology II, Ludwig Maximilians University Munich, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany [2] Center for Integrated Protein Science Munich (CIPSM), Via Manara 7, 21052 Busto Arsizio (VA), Italy [3] Nanosystems Initiative Munich (NIM), Via Manara 7, 21052 Busto Arsizio (VA), Italy.

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.
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http://dx.doi.org/10.1038/cr.2015.72DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528052PMC
August 2015

A modular open platform for systematic functional studies under physiological conditions.

Nucleic Acids Res 2015 Sep 24;43(17):e112. Epub 2015 May 24.

Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Großhaderner Strasse 2, 82152 Planegg-Martinsried, Germany

Any profound comprehension of gene function requires detailed information about the subcellular localization, molecular interactions and spatio-temporal dynamics of gene products. We developed a multifunctional integrase (MIN) tag for rapid and versatile genome engineering that serves not only as a genetic entry site for the Bxb1 integrase but also as a novel epitope tag for standardized detection and precipitation. For the systematic study of epigenetic factors, including Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2, Tet3 and Uhrf1, we generated MIN-tagged embryonic stem cell lines and created a toolbox of prefabricated modules that can be integrated via Bxb1-mediated recombination. We used these functional modules to study protein interactions and their spatio-temporal dynamics as well as gene expression and specific mutations during cellular differentiation and in response to external stimuli. Our genome engineering strategy provides a versatile open platform for efficient generation of multiple isogenic cell lines to study gene function under physiological conditions.
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http://dx.doi.org/10.1093/nar/gkv550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787826PMC
September 2015

Long-chain fatty acid analogues suppress breast tumorigenesis and progression.

Cancer Res 2014 Dec 10;74(23):6991-7002. Epub 2014 Oct 10.

Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem, Israel.

Obesity and type 2 diabetes (T2D) are associated with increased breast cancer incidence and mortality, whereas carbohydrate-restricted ketogenic diets ameliorate T2D and suppress breast cancer. These observations suggest an inherent efficacy of nonesterified long-chain fatty acids (LCFA) in suppressing T2D and breast tumorigenesis. In this study, we investigated novel antidiabetic MEDICA analogues consisting of methyl-substituted LCFA that are neither β-oxidized nor esterified to generate lipids, prompting interest in their potential efficacy as antitumor agents in the context of breast cancer. In the MMTV-PyMT oncomouse model of breast cancer, in which we confirmed that tumor growth could be suppressed by a carbohydrate-restricted ketogenic diet, MEDICA treatment suppressed tumor growth, and lung metastasis, promoting a differentiated phenotype while suppressing mesenchymal markers. In human breast cancer cells, MEDICA treatment attenuated signaling through the STAT3 and c-Src transduction pathways. Mechanistic investigations suggested that MEDICA suppressed c-Src-transforming activity by elevating reactive oxygen species production, resulting in c-Src oxidation and oligomerization. Our findings suggest that MEDICA analogues may offer therapeutic potential in breast cancer and overcome the poor compliance of patients to dietary carbohydrate restriction.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0385DOI Listing
December 2014