Publikationen / Publications 2012

C. Bönisch, K. Schneider, S. Pünzeler, S.M. Wiedemann, C. Bielmeier, M. Bocola, H.C. Eberl, W. Kuegel, J. Neumann, E. Kremmer, H. Leonhardt, M. Mann, J. Michaelis, L. Schermelleh and S.B. Hake
H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive
characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
Nucleic Acids Research (2012) 1-14
doi: 10.1093/nar/gks267

B. Treutlein, A. Muschielok, J. Andrecka, A. Jawhari, C. Buchen, D. Kostrewa, F. Hög, P. Cramer and J. Michaelis
Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex
The open promoter complex (OC) is a central intermediate during transcription initiation that contains a DNA bubble. Here, we employ singlemolecule Fo¨ rster resonance energy transfer experiments and Nano-Positioning System analysis to determine the three-dimensional architecture of a minimal OC consisting of promoter DNA, including a TATA box and an 11-nucleotide mismatched region around the transcription start site, TATA box-binding protein (TBP), RNA polymerase (Pol) II, and general transcription factor (TF)IIB and TFIIF. In this minimal OC, TATA-DNA and TBP reside above the Pol II cleft between clamp and protrusion domains. Downstream DNA is dynamically loaded into and unloaded
from the Pol II cleft at a timescale of seconds. The TFIIB core domain is displaced from the Pol II wall, where it is located in the closed promoter complex. These results reveal large overall structural changes during the initiation-elongation transition, which are apparently accommodated by the intrinsic flexibility of TFIIB.
Molecular Cell 46 (2012) 136-146
doi: 10.1016/j.molcel.2012.02.008

W. Kügel, A. Muschielok and J. Michaelis
Bayesian-Inference-Based Flourescence Correlation Spectroscopy and Single-Moledule Burst Analysis Reveal the Influence of Dye Selection on DNA Hairpin Dynamics
Fluorescence correlation spectroscopy (FCS) is a powerful tool to gain information about dynamics of biomolecules. However, the key problem is to extract the rates hidden in the FCS data by fitting the data to a meaningful model. A number of different fitting approaches have been described in recent years but the extraction of relevant information to date has still been limited by numerous experimental problems and the fact that the set of starting parameter values chosen could often predefine the result. We establish a new way to globally analyze FCS data based on Bayesian inference to overcome these issues. Moreover, the influence of other remaining experimental error sources, for example, photophysics, is excluded by additional means. Using this approach in combination with the results from single-molecule burst analysis, we investigate the kinetics of DNA hairpins labeled with a variety of different fluorescent probes as a function of the salt concentration. We find that the rates of hairpin opening and closing as well as the equilibrium constant of the transition depend on the characteristics of the dye molecules used to label the hairpin. Thus, great caution has to be used when utilizing dye molecules as reporters for the kinetics of dynamic macromolecular structures.
ChemPhysChem 13 (2012) 1013-1022
DOI: 10.1002/cphc.201100720