Andrea Rottach

Ludwig-Maximilians-Universität München

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Heinrich Leonhardt

Ludwig-Maximilians-Universität München

Bianca Bertulat

Heidelberg University

Juan Sandoval

Saint Aloysius Gonzaga National University

M. Cristina Cardoso

Technical University of Darmstadt

Fabio Spada

Guy's and St Thomas' NHS Foundation Trust

Sebastian Bultmann

Ludwig-Maximilians-Universität München

Weihua Qin

North University of China

Manuela Milden

Technical University of Darmstadt

Stefan Hasenöder

Helmholtz Zentrum München

Mengxi Wang

Shaanxi University of Science and Technology

Cooperative Institutions

Ludwig-Maximilians-Universität München

Center for Integrated Protein Science Munich

Max Delbrück Center

Technical University of Darmstadt

University of Zurich

Institut d'Investigació Biomédica de Bellvitge

Helmholtz Zentrum München

Heidelberg University

Leibniz-Forschungsinstitut für Molekulare Pharmakologie

LMU Klinikum

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Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1

PLoS ONE (2011)

Carina Frauer Andrea Rottach Daniela Meilinger Sebastian Bultmann Karin Fellinger

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.

DNMT1

CpG site

DNA methyltransferase

10.1371/journal.pone.0016627

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Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

PLoS ONE (2011)

Katharina Laurence Jost Andrea Rottach Manuela Milden Bianca Bertulat Annette Becker

Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro.

MeCP2

Polyclonal antibodies

Immunofluorescence

Rett Syndrome

10.1371/journal.pone.0026499

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Citations (21)