Project Description
To cope with adverse environmental conditions such as elevated temperature, toxic chemicals, heavy metals, but also developmental and pathophysiological conditions cells mount a universally conserved homeostatic transcriptional program, the so-called heat shock response (HSR). In the center of the HSR is in all eukaryotic cells the conserved heat shock transcription factor HSF1. According to the current model Hsf1 is monomeric in unstressed metazoan cells and trimerizes and binds to DNA upon stressful impacts. We recently demonstrated that purified Hsf1 senses changes in temperature directly and trimerizes in a temperature and time of exposure-dependent way. We could further show that activation of Hsf1 is modulated by the molecular chaperone Hsp90. The aims of this project is to understand the regulation of Hsf1 monomer-dimer-trimer transition and DNA binding, to unravel the regulation of conformational dynamics of Hsf1 by chaperones and cochaperones during activation and attenuation of the HSR, and to elucidate the effect of SUMOylation on the conformational dynamics of Hsf1. This project will involve the use of hydrogen exchange mass spectrometry, fluorescence spectroscopy and other biochemical and biophysical methods with purified proteins, as well as, in vivo work in mammalian cells.
References::
Hentze N, Le Breton L, Wiesner J, Kempf G & Mayer MP (2016) Molecular mechanism of thermosensory function of human heat shock transcription factor Hsf1. Elife 5: e11576
Methods that will be used:
hydrogen exchange mass spectrometry, fluorescence spectroscopy, cross-linking mass spectrometry, other biochemical assays, flow cytometry, fluorescence microscopy, cell biological methods.
Profile of candidate’s qualification:
MSc in Biology or Biochemistry or equivalent Experience in protein purification Experience mammalian cell culture and/or microscopy would be an advantage
Keywords:
Heat shock response, Hsf1, Molecular chaperones, Hsp90, Hsp70, mass spectrometry, fluorescence spectroscopy
Histone variants are essential for many important cellular processes, for instance in DNA damage repair by phosphorylating H2A.X at sites of DNA breaks, or for chromosome segregation by incorporating the histone variant CENP-A to centromeric chromatin regions. The precise regulation and incorporation of many histone variants into chromatin is, however, still not fully understood. This projects will focus on the essential centromere-specific histone H3 variant CENP-A and its highly regulated loading process to centromeric regions during mitosis. Specifically, we would like to understand how post-translational modifications and nucleic acids stabilize the complex and introduce conformational changes to enabling a loading-competent complex to form. We will use a wide range of techniques, including fluorescence microscopy and spectroscopy, amide hydrogen (1H/2H)-exchange in combination with high-resolution mass spectrometry.
References:
Bade D, Paulau AL, Wendler A, Erhardt S.The E3 Ligase CUL3/RDX Controls Centromere Maintenance by Ubiquitylating and Stabilizing CENP-A in a CAL1-Dependent Manner. Developmental Cell 2014 Mar 10;28(5):508-19
Rošić S, Köhler F, Erhardt S. Repetitive centromeric satellite RNA is essential for kinetochore formation and cell division. J. Cell Biol. 2014 Nov 10;207(3):335-49.
This is a collaborative project of the Mayer and Erhardt group within the ZMBH
Profile of candidate’s qualification:
We are looking for a highly motivated and interactive PhD student with a strong background in protein biochemistry and molecular biology. The ideal candidate has a strong interest in chromatin biology and enjoys being part of an international and collaborative team.
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