27 research outputs found
Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1
No full textSeveral 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
Exzellente Lehre an Hochschulen – Preisträgerinnen und Preisträger des Ars legendi-Fakultätenpreis Mathematik und Naturwissenschaften 2022
No full textDer Ars legendi-Fakultätenpreis für exzellente Hochschullehre in Mathematik und den Naturwissenschaften 2022 geht an Dagmar Hann und Daniela Meilinger (Biologie, Ludwig-Maximilians-Universität München), Nele Milsch (Chemie, Georg-August-Universität Göttingen), Martin Schlather (Mathematik, Uni Mannheim) und Julia Sammet (Physik, Goethe-Universität Frankfurt/Main)
The Master Biology Entry Programme gewinnt den Ars legendi-Fakultätenpreis Biologie
No full textDagmar Hann und Daniela Meilinger von der Fakultät für Biologie der Ludwig-Maximilians-Universität München erhalten den diesjährigen Ars legendi-Fakultätenpreis Mathematik und Naturwissenschaften in der Kategorie Biologie. Die Preisträgerinnen haben ein umfassendes modulares Einführungsprogramm für Einsteigerinnen und Einsteiger internationaler biowissenschaftlicher Masterstudiengängen entwickelt. Die Angebote des The Master Biology Entry Programme tragen dazu bei, die internationalen Studierenden, die in Hinblick auf Theorie und Praxis recht unterschiedliche Qualifikationen mitbringen, auf einen vergleichbaren Wissensstand zu bringen
Global DNA hypomethylation prevents consolidation of differentiation programs and allows reversion to the embryonic stem cell state.
No full textDNA 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
Wie beeinflussen Umwelterfahrungen unser Genom? Regulation auf molekularer Ebene durch epigenetische Prozesse
No full textDifferenzierte Zelllinien, Organismen in verschiedenen Metamorphose-Stadien, Bienenköniginnen und -Arbeiterinnen – bei all diesen Beispielen ist die Basenabfolge der DNA jeweils (größtenteils) identisch, die Phänotypen zeigen jedoch extreme Abweichungen untereinander. Im letzten Beispiel ist der Effekt sogar von Umwelteinflüssen induziert. Versucht man diese Phänomene zu erklären, stößt man schnell an die Grenzen klassischer molekulargenetischer Konzepte. Denn die Organisation des Genoms, die Regulation der Transkription, die variable Anpassung auf Umweltreize bis hin zur Vererbung solcher Anpassungen werden epigenetisch vermittelt. Aufgrund der rasanten Entwicklung dieses Forschungsgebiets ist es meist schwer die Übersicht über “die Epigenetik” zu wahren. Dieser Beitrag stellt die wichtigsten fachlichen Grundlagen der molekularen Mechanismen zusammen, skizziert einen Unterrichtsvorschlag im Flipped-Classroom-Format und stellt eine Vielzahl von Arbeitsmaterial zur Verfügung
In Vivo Control of CpG and Non-CpG DNA Methylation by DNA Methyltransferases
No full textThe enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs
Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways
No full textDNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation
No full textDNMT1 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
Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells
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