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Unterschleißheim, Germany

Lang R.,Intervet International GmbH | Vogt L.,Cytos Biotechnology AG | Zurcher A.,Molecular Partners | Winter G.,Ludwig Maximilians University of Munich
American Laboratory | Year: 2010

A study was conducted to demonstrate an improved asymmetric flow field-flow fractionation (AF4) for the investigation of virus-like particles (VLP). It was possible to obtain the molecular weight distributions of the fractionated species by coupling the technique with a multiangle laser light-scattering (MALLS) detector. AF4 analysis was performed in a hollow channel consisting of two plates separated by a spacer foil. The upper channel plate was impermeable, while the other plate was permeable and made of a porous frit material. High-resolution separation was achieved within a significantly thin parabolic channel flow against which a perpendicular cross-flow was applied. The flow channel allowed the parabolic flow profile to be created due to the laminar flow of the liquid. A stressed VLP sample was analyzed by AF4 using in the study using a standard separation channel or small separation channel.

Qin W.,Ludwig Maximilians University of Munich | Qin W.,University of Insubria | Wolf P.,Ludwig Maximilians University of Munich | Wolf P.,University of Insubria | And 27 more authors.
Cell Research | Year: 2015

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. © 2015 IBCB, SIBS, CAS.

Upadhyaya S.,Pirbright Institute | Ayelet G.,National Veterinary Institute | Paul G.,Intervet International GmbH | King D.P.,Pirbright Institute | And 2 more authors.
Vaccine | Year: 2014

Foot-and-mouth disease viruses (FMDV) from serotype A exhibit high antigenic diversity. Within the Middle East, a strain called A-Iran-05 emerged in 2003, and subsequently replaced the A-Iran-96 and A-Iran-99 strains that were previously circulating in the region. Viruses from this strain did not serologically match with the established A/Iran/96 vaccine, although most early samples matched with the older A22/Iraq vaccine. However, many viruses from this strain collected after 2006 had poor serological match with the A22/Iraq vaccine necessitating the development of a new vaccine strain (A/TUR/2006). More recently, viruses from the region now exhibit lower cross-reactivity with the A/TUR/2006 antisera highlighting the inadequacy of the serotype A vaccines used in the region. In order to understand the genetic basis of these antigenic phenotypes, we have determined the full capsid sequence for 57 Middle Eastern viruses isolated between 1996 and 2011 and analysed these data in context of antigenic relationship (r1) values that were generated using antisera to A22/Iraq and A/TUR/2006. Comparisons of capsid sequences identified substitutions in neutralising antigenic sites (1, 2 and 4), which either individually or together underpin these observed antigenic phenotypes. © 2013 The Authors.

Pichler G.,Ludwig Maximilians University of Munich | Wolf P.,Ludwig Maximilians University of Munich | Schmidt C.S.,Ludwig Maximilians University of Munich | Meilinger D.,Ludwig Maximilians University of Munich | And 6 more authors.
Journal of Cellular Biochemistry | Year: 2011

Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1 -/- embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells. Copyright © 2011 Wiley-Liss, Inc.

Frauer C.,Ludwig Maximilians University of Munich | Rottach A.,Ludwig Maximilians University of Munich | Meilinger D.,Ludwig Maximilians University of Munich | Bultmann S.,Ludwig Maximilians University of Munich | And 9 more authors.
PLoS ONE | Year: 2011

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 dnmt-/- ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites. © 2011 Frauer et al.

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