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

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

3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets

Identifizierung und Charakterisierug von basic Helix-Loop-Helix (bHLH)- Transkriptionsfaktoren im Zusammenhang mit dem interstitiellen Stammzell-System des Süßwasserpolypen Hydra

Meine Arbeit befasst sich mit der Superfamilie der basic Helix-Loop-Helix (bHLH)-Transkriptionsfaktoren, im Zusammenhang mit Differenzierungs-mechanismen interstitieller Stammzellen (I-Zellen) im Süßwasserpolypen Hydra. Da bHLH-Dimere konservierte Funktionen bei der Auswahl von Vorläuferzellen und deren Differenzierung und Subspezifizierung zu einzelnen Zelltypen übernehmen, interessierte mich, welche bHLH-Faktoren an der Spezifizierung des I-Zell-Systems beteiligt sind, das in Hydra neben Gameten und Drüsenzellen alle neuronalen Zelltypen hervorbringt. Mein Fokus richtete sich dabei speziell auf die beiden neuronalen Differenzierungswege in Hydra (Nervenzell- und Nematocyten-differenzierungsweg) und die neuronalen bHLH-Familien des Achaete/Scute-Complexes (AS-C) und der Atonal Related Proteine (ARP). Vor dem Hintergrund, dass bislang nur ein AS-C-Homolog (Cnidarian Achaete/Scute-Homolog, Cnash) in Hydra beschrieben werden konnte (Grens et al., 1995) gelang es mir vier ARP- und zwei AS-C-Homolge zu isolieren, von denen das AS-C-Homolog hyLiAS (Hydra Like Achaete/Scute) weiter charakterisiert werden konnte (Stenotelen und Desmonemen). Zusätzlich gelang es LiAS eine Interaktion mit der cytoplasmatischen Hydra Cadherin-Domäne nachzuweisen, wodurch sich eine duale Funktion dieses neuen AS-C-Homologs mit Aufgaben außerhalb des Zellkerns andeutet. Meine Arbeit zeigt, dass bereits in den Anfängen der Nervensystem-Evolution vor etwa 650 Millionen Jahren ein complexes Netzwerk von bHLH-faktoren der AS-C- und ARP-Familie vorlag und unterstreicht den Wert der Cnidarier als Modellorganismen für die Evolution von Differenzierungsmechanismen und Signalwegen

Microwave Induced Electroporation of Adherent Mammalian Cells at 18 GHz

This paper discusses microwave-induced electroporation as a promising alternative to conventional transfection methods. Adherent C2C12 mouse cells are successfully transfected with a 5TAMRA redlabeled peptide by using a recently developed planar microwave electroporation tool. It allows to monitor the uptake kinetics with live-cell confocal microscopy and is suitable to culture, manipulate, and observe the adherent cells over several days. Viability tests with the Calcein blue AM proof the vitality of the treated cells after 72 h. The question of whether the observed effects are temperature or field induced is tackled. For this reason, comprehensive coupled full-wave electromagnetic-thermal simulations are aligned with temperature measurements. The temperature at the position of the cells does not exceed 34 °C for an input power of 24 dBm. The corresponding electric field strength is evaluated at the position of the cells. A value of 150 V/cm is not exceeded, which is at least a factor of 10 below the field strength of the conventional electroporation. Consequently, almost no cell mortality does occur during the treatment. Comparative thermal tests without a microwave field but with a successively increased temperature up to 42 °C show no uptake. In contrast, the successful uptake follows the pattern of the microwave field although the temperature distribution is homogeneous. We rate this as evidence that the uptake is induced by the high-frequency electromagnetic field rather than the temperature

3D-Image analysis platform monitoring relocation of pluripotency genes during reprogramming.

Nuclear organization of chromatin is an important level of genome regulation with positional changes of genes occurring during reprogramming. Inherent variability of biological specimens, wide variety of sample preparation and imaging conditions, though pose significant challenges to data analysis and comparison. Here, we describe the development of a computational image analysis toolbox overcoming biological variability hurdles by a novel single cell randomizing normalization. We performed a comparative analysis of the relationship between spatial positioning of pluripotency genes with their genomic activity and determined the degree of similarity between fibroblasts, induced pluripotent stem cells and embryonic stem cells. Our analysis revealed a preferred positioning of actively transcribed Sox2, Oct4 and Nanog away from the nuclear periphery, but not from pericentric heterochromatin. Moreover, in the silent state, we found no common nuclear localization for any of the genes. Our results suggest that the surrounding gene density hinders relocation from an internal nuclear position. Altogether, our data do not support the hypothesis that the nuclear periphery acts as a general transcriptional silencer, rather suggesting that internal nuclear localization is compatible with expression in pluripotent cells but not sufficient for expression in mouse embryonic fibroblasts. Thus, our computational approach enables comparative analysis of topological relationships in spite of stark morphological variability typical of biological data sets

Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure

The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function