Publications by authors named "Fabio Spada"

34 Publications

Active turnover of genomic methylcytosine in pluripotent cells.

Nat Chem Biol 2020 12 10;16(12):1411-1419. Epub 2020 Aug 10.

Department of Chemistry, Ludwig Maximilians University Munich and Center for Integrated Protein Science Munich (CIPSM), Munich, Germany.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.
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http://dx.doi.org/10.1038/s41589-020-0621-yDOI Listing
December 2020

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

Nat Protoc 2019 01;14(1):283-312

Center for Integrated Protein Science Munich (CiPSM), Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.
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http://dx.doi.org/10.1038/s41596-018-0094-6DOI Listing
January 2019

5-Formylcytosine to cytosine conversion by C-C bond cleavage in vivo.

Nat Chem Biol 2018 Jan 27;14(1):72-78. Epub 2017 Nov 27.

Center for Integrated Protein Science Munich CiPSM at the Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.
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http://dx.doi.org/10.1038/nchembio.2531DOI Listing
January 2018

Non-canonical Bases in the Genome: The Regulatory Information Layer in DNA.

Angew Chem Int Ed Engl 2018 04 8;57(16):4296-4312. Epub 2018 Mar 8.

Center for Integrated Protein Science, Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377, Munich, Germany.

Multicellular organisms developed the concept of specialized cells that perform specific functions. Examples are neurons and fibroblast to name just two out of more than 200. These cellular differences are established based on the same sequence information stored in the cell nucleus of all cells of an organism. The sequence information needs consequently different interpretations by the different cell types. During cellular development this interpretation of the genetic code has to be tightly regulated in space and time. Interpretation of the sequence information involves the controlled activation and silencing of specific genes so that certain proteins are made in one cell type but not in others. This involves an additional regulatory information layer beyond the pure base sequence. One aspect of this regulatory information layer relies on functional groups that are attached to the C(5) position of the canonical base dC. Currently four regulatory, non-canonical bases with a methyl (CH )-, a hydroxymethyl (CH OH)-, a formyl (CHO)- and a carboxyl (COOH)- group are known. While 5-methyl-cytidine is long recognised to be a regulatory base in the genome, the other three bases and the enzymes responsible for generating them, were just recently discovered.
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http://dx.doi.org/10.1002/anie.201708228DOI Listing
April 2018

5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells.

J Am Chem Soc 2017 08 21;139(30):10359-10364. Epub 2017 Jul 21.

Center for Integrated Protein Science at the Department of Chemistry, LMU Munich , Butenandtstrasse 5-13, Munich 81377, Germany.

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates.
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http://dx.doi.org/10.1021/jacs.7b04131DOI Listing
August 2017

Quantitative LC-MS Provides No Evidence for m dA or m dC in the Genome of Mouse Embryonic Stem Cells and Tissues.

Angew Chem Int Ed Engl 2017 09 30;56(37):11268-11271. Epub 2017 Mar 30.

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU München, Butenandtstr. 5-13, 81377, München, Germany.

Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N -methyldeoxyadenosine (m dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N -methyldeoxycytidine (m dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC-MS method. We further report quantitative data for m dC, hmdC, fdC, and cadC, but we were unable to detect either m dC or m dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.
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http://dx.doi.org/10.1002/anie.201700424DOI Listing
September 2017

Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release.

Nanoscale 2016 Apr;8(15):8101-10

Department of Chemistry, Nanosystems Initiative Munich (NIM), Center for Nano Science (CeNS), and Center for Integrated Protein Science Munich (CIPSM), University of Munich (LMU), Butenandtstr. 5-13, 81377 Munich, Germany.

Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.
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http://dx.doi.org/10.1039/c5nr08163gDOI Listing
April 2016

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

Cell-penetrating and neurotargeting dendritic siRNA nanostructures.

Angew Chem Int Ed Engl 2015 Feb 17;54(6):1946-9. Epub 2014 Dec 17.

Center for Integrative Protein Science, Department of Chemistry, Ludwig-Maximilians University München, Butenandtstrasse 5-13, 81377 Munich (Germany) http://www.carellgroup.de.

We report the development of dendritic siRNA nanostructures that are able to penetrate even difficult to transfect cells such as neurons with the help of a special receptor ligand. The nanoparticles elicit strong siRNA responses, despite the dendritic structure. An siRNA dendrimer directed against the crucial rabies virus (RABV) nucleoprotein (N protein) and phosphoprotein (P protein) allowed the suppression of the virus titer in neurons below the detection limit. The cell-penetrating siRNA dendrimers, which were assembled using click chemistry, open up new avenues toward finding novel molecules able to cure this deadly disease.
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http://dx.doi.org/10.1002/anie.201409803DOI Listing
February 2015

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Nat Chem Biol 2014 Jul 18;10(7):574-81. Epub 2014 May 18.

Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.
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http://dx.doi.org/10.1038/nchembio.1532DOI Listing
July 2014

Poly(ADP-ribose) polymerase 1 (PARP1) associates with E3 ubiquitin-protein ligase UHRF1 and modulates UHRF1 biological functions.

J Biol Chem 2014 Jun 29;289(23):16223-38. Epub 2014 Apr 29.

From the Poly(ADP-ribosyl)ation and Genome Integrity Group, Equipe Labellisée Ligue Nationale Contre le Cancer, Laboratoire d'Excellence Medalis, Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg, UMR7242, Centre Nationale de la Recherche Scientifique/Université de Strasbourg, Boulevard Sebastien Brant, BP10413, 67412 Illkirch, France,

Poly(ADP-ribose) polymerase 1 (PARP1, also known as ARTD1) is an abundant nuclear enzyme that plays important roles in DNA repair, gene transcription, and differentiation through the modulation of chromatin structure and function. In this work we identify a physical and functional poly(ADP-ribose)-mediated interaction of PARP1 with the E3 ubiquitin ligase UHRF1 (also known as NP95, ICBP90) that influences two UHRF1-regulated cellular processes. On the one hand, we uncovered a cooperative interplay between PARP1 and UHRF1 in the accumulation of the heterochromatin repressive mark H4K20me3. The absence of PARP1 led to reduced accumulation of H4K20me3 onto pericentric heterochromatin that coincided with abnormally enhanced transcription. The loss of H4K20me3 was rescued by the additional depletion of UHRF1. In contrast, although PARP1 also seemed to facilitate the association of UHRF1 with DNMT1, its absence did not impair the loading of DNMT1 onto heterochromatin or the methylation of pericentric regions, possibly owing to a compensating interaction of DNMT1 with PCNA. On the other hand, we showed that PARP1 controls the UHRF1-mediated ubiquitination of DNMT1 to timely regulate its abundance during S and G2 phase. Together, this report identifies PARP1 as a novel modulator of two UHRF1-regulated heterochromatin-associated events: the accumulation of H4K20me3 and the clearance of DNMT1.
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http://dx.doi.org/10.1074/jbc.M113.527424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047392PMC
June 2014

Intrinsic and extrinsic connections of Tet3 dioxygenase with CXXC zinc finger modules.

PLoS One 2013 14;8(5):e62755. Epub 2013 May 14.

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

Tet proteins are emerging as major epigenetic modulators of cell fate and plasticity. However, little is known about how Tet proteins are targeted to selected genomic loci in distinct biological contexts. Previously, a CXXC-type zinc finger domain in Tet1 was shown to bind CpG-rich DNA sequences. Interestingly, in human and mouse the Tet2 and Tet3 genes are adjacent to Cxxc4 and Cxxc10-1, respectively. The CXXC domains encoded by these loci, together with those in Tet1 and Cxxc5, identify a distinct homology group within the CXXC domain family. Here we provide evidence for alternative mouse Tet3 transcripts including the Cxxc10-1 sequence (Tet3(CXXC)) and for an interaction between Tet3 and Cxxc4. In vitro Cxxc4 and the isolated CXXC domains of Tet1 and Tet3(CXXC) bind DNA substrates with similar preference towards the modification state of cytosine at a single CpG site. In vivo Tet1 and Tet3 isoforms with and without CXXC domain hydroxylate genomic 5-methylcytosine with similar activity. Relative transcript levels suggest that distinct ratios of Tet3(CXXC) isoforms and Tet3-Cxxc4 complex may be present in adult tissues. Our data suggest that variable association with CXXC modules may contribute to context specific functions of Tet proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062755PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653909PMC
December 2013

Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.

PLoS One 2012 27;7(12):e52629. Epub 2012 Dec 27.

Department of Biology II, Ludwig Maximilians University Munich, Planegg-Martinsried, Germany.

DNA methylation patterns change dynamically during mammalian development and lineage specification, yet scarce information is available about how DNA methylation affects gene expression profiles upon differentiation. Here we determine genome-wide transcription profiles during undirected differentiation of severely hypomethylated (Dnmt1⁻/⁻) embryonic stem cells (ESCs) as well as ESCs completely devoid of DNA methylation (Dnmt1⁻/⁻;Dnmt3a⁻/⁻;Dnmt3b⁻/⁻ or TKO) and assay their potential to transit in and out of the ESC state. We find that the expression of only few genes mainly associated with germ line function and the X chromosome is affected in undifferentiated TKO ESCs. Upon initial differentiation as embryoid bodies (EBs) wild type, Dnmt1⁻/⁻ and TKO cells downregulate pluripotency associated genes and upregulate lineage specific genes, but their transcription profiles progressively diverge upon prolonged EB culture. While Oct4 protein levels are completely and homogeneously suppressed, transcription of Oct4 and Nanog is not completely silenced even at late stages in both Dnmt1⁻/⁻ and TKO EBs. Despite late wild type and Dnmt1⁻/⁻ EBs showing a much higher degree of concordant expression, after EB dissociation and replating under pluripotency promoting conditions both Dnmt1⁻/⁻ and TKO cells, but not wild type cells rapidly revert to expression profiles typical of undifferentiated ESCs. Thus, while DNA methylation seems not to be critical for initial activation of differentiation programs, it is crucial for permanent restriction of developmental fate during differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052629PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531338PMC
June 2013

Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers.

Nucleic Acids Res 2012 Jul 2;40(12):5368-77. Epub 2012 Mar 2.

Department of Biology, Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Germany.

Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2'-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes.
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http://dx.doi.org/10.1093/nar/gks199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384321PMC
July 2012

Characterization of PvuRts1I endonuclease as a tool to investigate genomic 5-hydroxymethylcytosine.

Nucleic Acids Res 2011 Jul 4;39(12):5149-56. Epub 2011 Mar 4.

Ludwig Maximilians University Munich, Department of Biology and Center for Integrated Protein Science Munich, 82152 Planegg-Martinsried, Germany.

In mammalian genomes a sixth base, 5-hydroxymethylcytosine ((hm)C), is generated by enzymatic oxidation of 5-methylcytosine ((m)C). This discovery has raised fundamental questions about the functional relevance of (hm)C in mammalian genomes. Due to their very similar chemical structure, discrimination of the rare (hm)C against the far more abundant (m)C is technically challenging and to date no methods for direct sequencing of (hm)C have been reported. Here, we report on a purified recombinant endonuclease, PvuRts1I, which selectively cleaves (hm)C-containing sequences. We determined the consensus cleavage site of PvuRts1I as (hm)CN(11-12)/N(9-10)G and show first data on its potential to interrogate (hm)C patterns in mammalian genomes.
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http://dx.doi.org/10.1093/nar/gkr118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130283PMC
July 2011

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

PLoS One 2011 Feb 2;6(2):e16627. Epub 2011 Feb 2.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Planegg, Germany.

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016627PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032784PMC
February 2011

Usp7 and Uhrf1 control ubiquitination and stability of the maintenance DNA methyltransferase Dnmt1.

J Cell Biochem 2011 Feb;112(2):439-44

Department of Biology II, Ludwig Maximilians University Munich, Planegg-Martinsried, Germany.

In mammals Dnmt1 is the DNA methyltransferase chiefly responsible for maintaining genomic methylation patterns through DNA replication cycles, but how its maintenance activity is controlled is still not well understood. Interestingly, Uhrf1, a crucial cofactor for maintenance of DNA methylation by Dnmt1, is endowed with E3 ubiquitin ligase activity. Here, we show that both Dnmt1 and Uhrf1 coprecipitate with ubiquitin specific peptidase 7 (Usp7), a de-ubiquitinating enzyme. Overexpression of Uhrf1 and Usp7 resulted in opposite changes in the ubiquitination status and stability of Dnmt1. Our findings suggest that, by balancing Dnmt1 ubiquitination, Usp7 and Uhrf1 fine tune Dnmt1 stability.
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http://dx.doi.org/10.1002/jcb.22998DOI Listing
February 2011

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA.

Nucleic Acids Res 2010 Oct 4;38(19):e181. Epub 2010 Aug 4.

Department of Biology, Center for Integrated Protein Science Munich, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.
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http://dx.doi.org/10.1093/nar/gkq684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965258PMC
October 2010

The multi-domain protein Np95 connects DNA methylation and histone modification.

Nucleic Acids Res 2010 Apr 21;38(6):1796-804. Epub 2009 Dec 21.

Ludwig Maximilians University Munich, Department of Biology II and Center for Integrated Protein Science Munich, Grosshaderner Str. 2, 82152 Planegg-Martinsried, Germany.

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ss. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.
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http://dx.doi.org/10.1093/nar/gkp1152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847221PMC
April 2010

Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells.

EMBO Rep 2009 Nov 2;10(11):1259-64. Epub 2009 Oct 2.

Department of Biology II, Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians University Munich, Grosshaderner Street 2, 82152 Planegg-Martinsried, Germany.

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi-methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.
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http://dx.doi.org/10.1038/embor.2009.201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2756565PMC
November 2009

DNA methylation-mediated epigenetic control.

J Cell Biochem 2009 Sep;108(1):43-51

Department of Biology II and Munich Center for Integrated Protein Science CiPSM, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

During differentiation and development cells undergo dramatic morphological and functional changes without any change in the DNA sequence. The underlying changes of gene expression patterns are established and maintained by epigenetic processes. Early mechanistic insights came from the observation that gene activity and repression states correlate with the DNA methylation level of their promoter region. DNA methylation is a postreplicative modification that occurs exclusively at the C5 position of cytosine residues (5mC) and predominantly in the context of CpG dinucleotides in vertebrate cells. Here, three major DNA methyltransferases (Dnmt1, 3a, and 3b) establish specific DNA methylation patterns during differentiation and maintain them over many cell division cycles. CpG methylation is recognized by at least three protein families that in turn recruit histone modifying and chromatin remodeling enzymes and thus translate DNA methylation into repressive chromatin structures. By now a multitude of histone modifications have been linked in various ways with DNA methylation. We will discuss some of the basic connections and the emerging complexity of these regulatory networks.
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http://dx.doi.org/10.1002/jcb.22253DOI Listing
September 2009

Visualization and measurement of DNA methyltransferase activity in living cells.

Curr Protoc Cell Biol 2008 Jun;Chapter 22:Unit 22.12

Ludwig Maximilians University Munich, Department of Biology II, Martinsried, Germany.

In this unit, a live-cell assay to measure DNA (cytosine-5) methyltransferase (MTase) activity at the single-cell level is described. This method takes advantage of the irreversible binding of enzymatically active MTases to genomic DNA substituted with the mechanism-based inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). The procedure comprises incorporation of this nucleoside analog into DNA during replication and quantification of the time-dependent MTase immobilization by fluorescence recovery after photobleaching (FRAP). This trapping assay monitors kinetic properties and activity-dependent immobilization of MTases in their native environment and enables direct comparison of mutations and inhibitors that affect MTase regulation and catalytic activity in single living cells. In addition, a simplified protocol to obtain qualitative information on the activity of either endogenously or exogenously expressed MTases is provided.
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http://dx.doi.org/10.1002/0471143030.cb2212s39DOI Listing
June 2008

The PHD domain of Np95 (mUHRF1) is involved in large-scale reorganization of pericentromeric heterochromatin.

Mol Biol Cell 2008 Aug 28;19(8):3554-63. Epub 2008 May 28.

Department of Structural and Functional Biology, University of Insubria, 21052 Busto Arsizio (VA), Italy.

Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone-binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes, like HDAC and DNMT1, required for the replication and formation of PH.
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http://dx.doi.org/10.1091/mbc.e07-10-1059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2488286PMC
August 2008

A mutagenesis strategy combining systematic alanine scanning with larger mutations to study protein interactions.

Anal Biochem 2008 Feb 18;373(1):176-8. Epub 2007 Oct 18.

Department of Biology and Munich Center for Integrated Protein Science, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

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http://dx.doi.org/10.1016/j.ab.2007.10.016DOI Listing
February 2008

Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation.

Nucleic Acids Res 2007 18;35(13):4301-12. Epub 2007 Jun 18.

Ludwig Maximilians University Munich, Department of Biology II, 82152 Martinsried, Germany.

Postreplicative maintenance of genomic methylation patterns was proposed to depend largely on the binding of DNA methyltransferase 1 (Dnmt1) to PCNA, a core component of the replication machinery. We investigated how the slow and discontinuous DNA methylation could be mechanistically linked with fast and processive DNA replication. Using photobleaching and quantitative live cell imaging we show that Dnmt1 binding to PCNA is highly dynamic. Activity measurements of a PCNA-binding-deficient mutant with an enzyme-trapping assay in living cells showed that this interaction accounts for a 2-fold increase in methylation efficiency. Expression of this mutant in mouse dnmt1-/- embryonic stem (ES) cells restored CpG island methylation. Thus association of Dnmt1 with the replication machinery enhances methylation efficiency, but is not strictly required for maintaining global methylation. The transient nature of this interaction accommodates the different kinetics of DNA replication and methylation while contributing to faithful propagation of epigenetic information.
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http://dx.doi.org/10.1093/nar/gkm432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1934996PMC
August 2007

DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells.

J Cell Biol 2007 Feb 20;176(5):565-71. Epub 2007 Feb 20.

Department of Biology II and 2Department of Chemistry, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen-binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference-mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.
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http://dx.doi.org/10.1083/jcb.200610062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064015PMC
February 2007

Conserved patterns of nuclear compartmentalization are not observed in the chordate Oikopleura.

Biol Cell 2007 May;99(5):273-87

Sars Centre for Marine Molecular Biology, Bergen High Technology Centre, University of Bergen, Thormøhlensgt 55, Bergen, Norway.

Background Information: Recent results from a limited number of eukaryotic model organisms suggest that major principles governing spatial organization of the genome in functionally distinct nuclear compartments are conserved through evolution.

Results: We examined the in situ spatial organization of major nuclear components and nuclear patterns of gene loci with strictly defined expression patterns in endocycling cells of the transparent urochordate Oikopleura dioica, a complex metazoan with a very compact genome. Endocycling cells with different functions and similar DNA content displayed distinct topologies of nuclear components. However, the generation of the diverse nuclear architectures did not involve specific local organization of active genes or their preferential amplification. Interestingly, endocycling cells lacked nuclear-envelope-associated heterochromatin and prominent splicing-factor domains, which in mammalian cells associate with transcriptionally silent and active loci respectively. In addition, no correlation was found between transcriptional activity of a locus and its association with chromatin domains rich in specific histone modifications.

Conclusions: Together, these findings and the absence of typical eukaryotic replication patterns reveal a surprisingly limited functional compartmentalization of O. dioica endocycling nuclei. This indicates that robust cell-type-specific gene expression does not necessarily require high levels of spatial genome organization.
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http://dx.doi.org/10.1042/BC20060124DOI Listing
May 2007

Regulation of DNA methyltransferase 1.

Adv Enzyme Regul 2006 20;46:224-34. Epub 2006 Jul 20.

Biocenter, Department of Biology II, Ludwig Maximilians University Munich, Germany.

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http://dx.doi.org/10.1016/j.advenzreg.2006.01.011DOI Listing
January 2007

Trapped in action: direct visualization of DNA methyltransferase activity in living cells.

Nat Methods 2005 Oct;2(10):751-6

Ludwig Maximilians University Munich, Department of Biology II, Planegg-Martinsried, Germany.

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2'-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.
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http://dx.doi.org/10.1038/nmeth794DOI Listing
October 2005

Restricted mobility of Dnmt1 in preimplantation embryos: implications for epigenetic reprogramming.

BMC Dev Biol 2005 Aug 24;5:18. Epub 2005 Aug 24.

Department of Biology II, Ludwig Maximilians University Munich, Grosshadernerstr. 2, 82152 Planegg-Martinsried, Germany.

Background: Mouse preimplantation development is characterized by both active and passive genomic demethylation. A short isoform of the prevalent maintenance DNA methyltransferase (Dnmt1S) is found in the cytoplasm of preimplantation embryos and transiently enters the nucleus only at the 8-cell stage.

Results: Using GFP fusions we show that both the long and short isoforms of Dnmt1 localize to the nucleus of somatic cells and the cytoplasm of preimplantation embryos and that these subcellular localization properties are independent of phosphorylation. Importantly, photobleaching techniques and salt extraction revealed that Dnmt1S has a very restricted mobility in the cytoplasm, while it is highly mobile in the nucleus of preimplantation embryos.

Conclusion: The restricted mobility of Dnmt1S limits its access to DNA and likely contributes to passive demethylation and epigenetic reprogramming during preimplantation development.
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http://dx.doi.org/10.1186/1471-213X-5-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1208874PMC
August 2005