51 research outputs found
Proteindynamik im Zellkern: Auswirkungen auf die Genexpression
No full textMithilfe der Mikroskopietechnik "Fluorescence
Recovery After Photobleaching" (FRAP) wurden die
kinetischen Eigenschaften der Proteine der Polycomb
Gruppe in lebenden Drosophila Embryonen und
verschiedenen Geweben (Imaginalscheibe der Flügel und
Speicheldrüse) charakterisiert. Dieses sind meines
Wissens die ersten kinetischen Bleichexperimente, die
an kompletten Embryonen und lebenden Geweben
durchgeführt wurden.
Die Gene der Polycomb Gruppe (PcG) sind essentielle
Gene in höheren Eukaryoten, die für die
Aufrechterhaltung der räumlich spezifischen
Unterdrückung für die Entwicklung wichtiger
Regulatoren, wie der homeotischen Gene, nötig sind.
Deren Abwesenheit sowie deren Überexpression führt zu
Transformationen der axialen Segmente des Körpers.
Obwohl PcG Proteinkomplexe bereits ex vivo isoliert wurden, ist bisher nur
wenig über deren Stabilität und den exakten Mechanismus
der Repression in vivo
bekannt.
Ich habe den Diffusionskoeffizieten von PcG Proteinen
und deren Dissotiationskonstante und Aufenthaltszeit in
Komplexen in verschiedenen Entwicklungsstadien
bestimmen können. In polytenen Zellkernen suggerieren
die Ratenkonstanten eine Heterogenität der Komplexe.
Zum Vergleich mit den experimentellen Daten wurden
Computersimulationen mit neuartigen Modellen
durchgeführt, die die räumlich inhomogene Verteilung
der Proteine berücksichtigen.
Daraus ergaben sich Schätzungen sowohl für die vorwärts
gerichtete als auch die rückwärtige Ratenkonstante der
Komplexbildung. Der Austausch in den Komplexen
geschieht in einer Zeitspanne von einer bis zu zehn
Minuten, also etwa eine Größenordnung schneller als die
Zellzykluszeit. Dies spricht gegen Modelle der
Repression, in denen die Zugänglichkeit von
Transkriptionsaktivatoren zum Chromatin permanent
eingeschränkt wird und zeigt, dass die
Langzeitunterdrückung hauptsächlich durch das chemische
Gleichgewicht in Verbindung mit dem
Massenwirkungsgesetz erreicht wird.
Mithilfe der Computersimulation von Diffusion und
räumlich lokalisierter Bindungsstellen unter
Berücksichtigung der Randbedingungen konnte ich die
gemessenen biophysikalischen Prozesse, die dem FRAP
Experiment zugrunde liegen, genau beschreiben und
daraus einen optimierten experimentellen Ablauf und
eine quantitative Interpretation der Ergebnisse
ableiten.Fluorescence recovery after photobleaching (FRAP)
microscopy was used to determine the kinetic properties
of Polycomb group proteins in whole living Drosophila
organisms (embryos) and tissues (wing imaginal discs
and salivary glands). These are the first
photobleaching experiments performed in whole embryos
and tissues. Polycomb group (PcG) genes are essential
genes in higher eukaryotes responsible for the
maintenance of the spatially distinct repression of
developmentally important regulators such as the
homeotic genes. Their absence, as well as
overexpression, causes transformations in the axial
organization of the body. Although protein complexes
have been isolated in vitro, little is known about
their stability or exact mechanism of repression
in vivo.
I determined the translational diffusion coefficients
of PcG proteins, dissociation constants and residence
times for complexes in vivo
at different developmental stages. In polytene nuclei
the rate constants suggest heterogeneity of the
complexes. Computer simulations with new models for
spatially distributed protein complexes were performed
in systems showing both diffusion and binding
equilibria and the results compared with the
experimental data. I was able to determine forward and
reverse rate constants for complex formation. Complexes
exchanged within a period of one to ten minutes, more
than an order of magnitude faster than the cell cycle
time, ruling out models of repression in which access
of transcription activators to the chromatin is limited
and demonstrating that longterm repression primarily
reflects mass-action chemical equilibria.
With the help of computer programs built to simulate
diffusion in boundary conditions and binding kinetics
of proteins to localized binding sites in the genome I
describe in detail the observed biophysical processes
underlying FRAP and offer guidance for a better setup,
optimization and interpretation of photobleaching
experiments
Loss of 5-hydroxymethylcytosine in cancer: Cause or consequence?
No full textAbstractDiscovery of the enzymatic activity that catalyses oxidation of 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) mediated by the MLL (KMT2A) fusion partner TET1 has sparked intense research to understand the role this new DNA modification has in cancer. An unambiguous picture has emerged where tumours are depleted of 5hmC compared to corresponding normal tissue, but it is not known whether lack of 5hmC is a cause or a consequence of tumourigenesis. Experimental data reveals a dual tumour-suppressive and oncogenic role for TET proteins. Tet2 mutations are drivers in haematological malignancies but Tet1 had an oncogenic role in MLL-rearranged leukaemia, where Tet1 is overexpressed. Overexpression of Tet2 in melanoma cells re-established the 5hmC landscape and suppressed cancer progression but inhibiting Tet1 in non-transformed cells did not initiate cellular transformation. In this review we summarise recent findings that have shaped the current understanding on the role 5hmC plays in cancer
Polycomb group protein complexes exchange rapidly in living Drosophila
No full textFluorescence recovery after photobleaching (FRAP) microscopy was used to determine the kinetic properties of Polycomb group (PcG) proteins in whole living Drosophila organisms (embryos) and tissues (wing imaginal discs and salivary glands). PcG genes are essential genes in higher eukaryotes responsible for the maintenance of the spatially distinct repression of developmentally important regulators such as the homeotic genes. Their absence, as well as overexpression, causes transformations in the axial organization of the body. Although protein complexes have been isolated in vitro, little is known about their stability or exact mechanism of repression in vivo. We determined the translational diffusion constants of PcG proteins, dissociation constants and residence times for complexes in vivo at different developmental stages. In polytene nuclei, the rate constants suggest heterogeneity of the complexes. Computer simulations with new models for spatially distributed protein complexes were performed in systems showing both diffusion and binding equilibria, and the results compared with our experimental data. We were able to determine forward and reverse rate constants for complex formation. Complexes exchanged within a period of 1-10 minutes, more than an order of magnitude faster than the cell cycle time, ruling out models of repression in which access of transcription activators to the chromatin is limited and demonstrating that long-term repression primarily reflects mass-action chemical equilibria
The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach
No full textGerman Research Council (DFG) as part of the
Collaborative Research Center "Physical modeling of
non-equilibrium processes in biological systems"
(SFB 1027) and the Cluster of Excellence on
Multimodal Computing and Interaction at Saarland
Universit
An shRNA kinase screen identifies regulators of UHRF1 stability and activity in mouse embryonic stem cells
No full textPropagation of DNA methylation through cell division relies on the recognition of methylated cytosines by UHRF1. In reprogramming of mouse embryonic stem cells to naive pluripotency (also known as ground state), despite high levels of Uhrf1 transcript, the protein is targeted for degradation by the proteasome, leading to DNA methylation loss. We have undertaken an shRNA screen to identify the signalling pathways that converge upon UHRF1 and control its degradation, using UHRF1-GFP fluorescence as readout. Many candidates we identified are key enzymes in regulation of glucose metabolism, nucleotide metabolism and Pi3K/AKT/mTOR pathway. Unexpectedly, while downregulation of all candidates we selected for validation rescued UHRF1 protein levels, we found that in some of the cases this was not sufficient to maintain DNA methylation. This has implications for development, ageing and diseased conditions. Our study demonstrates two separate processes that regulate UHRF1 protein abundance and activity
Possible transitions of the 9 different states of a CpG site.
No full textMethyl groups are a) removed after cell division, b) added due to maintenance (μm) or de novo methylation (μd) and c) are hydroxylated (η) by Tet enzymes.</p
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