TY - JOUR A1 - Schul, Daniela A1 - Schmitt, Alexandra A1 - Regneri, Janine A1 - Schartl, Manfred A1 - Wagner, Toni Ulrich T1 - Bursted BMP Triggered Receptor Kinase Activity Drives Smad1 Mediated Long-Term Target Gene Oscillation in c2c12 Cells JF - PLoS ONE N2 - Bone Morphogenetic Proteins (BMPs) are important growth factors that regulate many cellular processes. During embryogenesis they act as morphogens and play a critical role during organ development. They influence cell fates via concentration-gradients in the embryos where cells transduce this extracellular information into gene expression profiles and cell fate decisions. How receiving cells decode and quantify BMP2/4 signals is hardly understood. There is little data on the quantitative relationships between signal input, transducing molecules, their states and location, and ultimately their ability to integrate graded systemic inputs and generate qualitative responses. Understanding this signaling network on a quantitative level should be considered a prerequisite for efficient pathway modulation, as the BMP pathway is a prime target for therapeutic invention. Hence, we quantified the spatial distribution of the main signal transducer of the BMP2/4 pathway in response to different types and levels of stimuli in c2c12 cells. We found that the subcellular localization of Smad1 is independent of ligand concentration. In contrast, Smad1 phosphorylation levels relate proportionally to BMP2 ligand concentrations and they are entirely located in the nucleus. Interestingly, we found that BMP2 stimulates target gene expression in non-linear, wave-like forms. Amplitudes showed a clear concentration-dependency, for sustained and transient stimulation. We found that even burst-stimulation triggers gene-expression wave-like modulations that are detectable for at least 30 h. Finally, we show here that target gene expression oscillations depend on receptor kinase activity, as the kinase drives further expression pulses without receptor reactivation and the target gene expression breaks off after inhibitor treatment in c2c12 cells. KW - gene expression KW - BMP signaling KW - SMAD signaling KW - genetic oscillators KW - cell fusion KW - DNA-binding proteins KW - luciferase KW - kinase inhibitors Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130131 VL - 8 IS - 4 ER - TY - JOUR A1 - Salat, Daniela A1 - Winkler, Anja A1 - Urlaub, Henning A1 - Gessler, Manfred T1 - Hey bHLH Proteins Interact with a FBXO45 Containing SCF Ubiquitin Ligase Complex and Induce Its Translocation into the Nucleus JF - PLoS One N2 - The Hey protein family, comprising Hey1, Hey2 and HeyL in mammals, conveys Notch signals in many cell types. The helix-loop-helix (HLH) domain as well as the Orange domain, mediate homo- and heterodimerization of these transcription factors. Although distinct interaction partners have been identified so far, their physiological relevance for Hey functions is still largely unclear. Using a tandem affinity purification approach and mass spectrometry analysis we identified members of an ubiquitin E3-ligase complex consisting of FBXO45, PAM and SKP1 as novel Hey1 associated proteins. There is a direct interaction between Hey1 and FBXO45, whereas FBXO45 is needed to mediate indirect Hey1 binding to SKP1. Expression of Hey1 induces translocation of FBXO45 and PAM into the nucleus. Hey1 is a short-lived protein that is degraded by the proteasome, but there is no evidence for FBXO45-dependent ubiquitination of Hey1. On the contrary, Hey1 mediated nuclear translocation of FBXO45 and its associated ubiquitin ligase complex may extend its spectrum to additional nuclear targets triggering their ubiquitination. This suggests a novel mechanism of action for Hey bHLH factors. KW - ubiquitination KW - glycerol KW - transcription factors KW - DNA-binding proteins KW - immunoprecipitation KW - protein interactions KW - protein domains Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125769 VL - 10 IS - 6 ER - TY - JOUR A1 - Gaubatz, Stefan A1 - Esterlechner, Jasmina A1 - Reichert, Nina A1 - Iltzsche, Fabian A1 - Krause, Michael A1 - Finkernagel, Florian T1 - LIN9, a Subunit of the DREAM Complex, Regulates Mitotic Gene Expression and Proliferation of Embryonic Stem Cells JF - PLoS ONE N2 - The DREAM complex plays an important role in regulation of gene expression during the cell cycle. We have previously shown that the DREAM subunit LIN9 is required for early embryonic development and for the maintenance of the inner cell mass in vitro. In this study we examined the effect of knocking down LIN9 on ESCs. We demonstrate that depletion of LIN9 alters the cell cycle distribution of ESCs and results in an accumulation of cells in G2 and M and in an increase of polyploid cells. Genome-wide expression studies showed that the depletion of LIN9 results in downregulation of mitotic genes and in upregulation of differentiation-specific genes. ChIP-on chip experiments showed that mitotic genes are direct targets of LIN9 while lineage specific markers are regulated indirectly. Importantly, depletion of LIN9 does not alter the expression of pluripotency markers SOX2, OCT4 and Nanog and LIN9 depleted ESCs retain alkaline phosphatase activity. We conclude that LIN9 is essential for proliferation and genome stability of ESCs by activating genes with important functions in mitosis and cytokinesis. KW - cell cycle KW - cell division KW - cell differentation KW - DNA-binding proteins KW - gene expression KW - gene regulation KW - gene targeting KW - microarrays KW - pluripotency Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96922 ER -