TY  - JOUR
A1  - Groeneweg, Femke L.
A1  - van Royen, Martin E.
A1  - Fenz, Susanne
A1  - Keizer, Veer I. P.
A1  - Geverts, Bart
A1  - Prins, Jurrien
A1  - de Kloet, E. Ron
A1  - Houtsmuller, Adriaan B.
A1  - Schmidt, Thomas S.
A1  - Schaaf, Marcel J. M.
T1  - Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP
JF  - PLOS ONE
N2  - Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (<= 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.
KW  - NF-KAPPA-B
KW  - image correlation spectroscopy
KW  - human mineralocorticoid receptor
KW  - nuclear-pore complexes
KW  - in-vivo
KW  - living cells
KW  - mobility
KW  - transcription
KW  - protein
KW  - reveals
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117085
VL  - 9
IS  - 3
ER  - 
TY  - JOUR
A1  - Wolf, Annette
A1  - Akrap, Nina
A1  - Marg, Berenice
A1  - Galliardt, Helena
A1  - Heiligentag, Martyna
A1  - Humpert, Fabian
A1  - Sauer, Markus
A1  - Kaltschmidt, Barbara
A1  - Kaltschmidt, Christian
A1  - Seidel, Thorsten
T1  - Elements of Transcriptional Machinery Are Compatible among Plants and Mammals
JF  - PLoS ONE
N2  - In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-\(\kappa\) B in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-\(\kappa\) B such as its inhibitor I\(\kappa\)B isoform \(\alpha\) that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-\(\kappa\)B in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-\(\kappa\)B signaling pathways. The system successfully enabled the controlled manipulation of NF-\(\kappa\)B activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e. g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway.
KW  - complexes
KW  - in vivo
KW  - DNA-binding
KW  - nuclear proe
KW  - gene expression
KW  - NF-KAPPA-B
KW  - RNA-binding protein
KW  - alpha
KW  - inflammation
KW  - homodimers
Y1  - 2013
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131203
VL  - 8
IS  - 1
ER  -