Single-molecule FRET; Chromatin; Epigenetics; Dynamics; Pioneer transcription factor
Mivelaz Maxime, Cao Anne-Marinette, Kubik Slawomir, Zencir Sevil, Hovius Ruud, Boichenko Iuliia, Stachowicz Anna Maria, Kurat Christoph F., Shore David, Fierz Beat (2020), Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor, in Molecular Cell
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Boichenko Iuliia, Fierz Beat (2019), Chemical and biophysical methods to explore dynamic mechanisms of chromatin silencing, in Current Opinion in Chemical Biology
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Fierz Beat, Poirier Michael G. (2019), Biophysics of Chromatin Dynamics, in Annual Review of Biophysics
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Kilic Sinan, Felekyan Suren, Doroshenko Olga, Boichenko Iuliia, Dimura Mykola, Vardanyan Hayk, Bryan Louise C., Arya Gaurav, Seidel Claus A. M., Fierz Beat (2018), Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α, in Nature Communications
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Kilic Sinan, Boichenko Iuliia, Lechner Carolin C., Fierz Beat (2018), A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage, in Chemical Science
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The structure and internal motions of chromatin are a determining factor in all chromatin transactions, including gene activation, transcription, DNA replication or DNA repair. Thus a deep understanding of the molecular organization of chromatin is of great importance. Chromatin structure and function is regulated by the incorporation of histone variants, modifications of the DNA and, in particular, post-translational modifications (PTMs) of the histones, in combination with protein effectors. Studies using X-ray and electron microscopy approaches revealed that chromatin fibers are organized in stacks of compact tetranucleosome units. However, as a whole, chromatin structure and dynamics are still enigmatic as it is a conformationally and chemically heterogeneous and highly dynamic complex. Hence, new approaches are required.A key process governed by local chromatin organization and conformational dynamics is gene regulation through transcription factors. Repressed genes reside in a compact chromatin compartment, heterochromatin, with restricted biochemical accessibility. A first step to activate such genes, e.g. during development, requires an initial, or pioneer transcription factor (pTF) to access target sites in compact chromatin. Such pTFs have the ability to invade compact chromatin, remodel its structure and initiate the assembly of further factors to subsequently enact a change in the chromatin state (i.e. the presence of histone variants, PTMs, the positioning of nucleosomes). We hypothesize that the ability of pTFs to bind target sites on nucleosomes, combined with the transient exposure of such sites due to conformational fluctuations in compact chromatin, enables their pioneering activity. When bound, pTFs then enact dynamic opening of the structure allowing gene transcription to take place. How pTFs can bypass chromatin inhibition and then remodel the structure is not well understood and a major current biological question.Here, we will elucidate dynamic chromatin structure and the molecular basis of pTF function using a novel combination of single-molecule and chemical biology approaches. In recent years, we have developed a suite of chemical and biophysical methods to mechanistically probe chromatin regulatory processes in highly defined systems with single-molecule resolution. We are thus uniquely positioned to investigate fundamental chromatin processes at unprecedented spatiotemporal resolution, and answer the following questions:1. What is the dynamic organization of chromatin fibers? Here we will implement a single-molecule fluorescence resonance energy transfer (smFRET) approach to directly determine local chromatin structure and dynamics as a function of nucleosome positioning and fiber architecture.2. Which interactions govern local dynamics in chromatin arrays? Key contacts between nucleosomes are modulated by histone variants and PTMs. We will use our smFRET approach to reveal how chromatin structure is dynamically remodeled by these modifications, enabling TF access to internal sites.3. How do pioneer TFs access target sites in compact chromatin? Using the yeast pTF Rap1 as a model, we will gain mechanistic insight into pTF action in compact chromatin fiber of defined architecture.Taken together, these studies will provide, for the first time, direct insight into the conformational heterogeneity, dynamics and energetics of chromatin fibers, and how they are accessed by pTFs. We are convinced that our methods will pave the way to a molecular understanding of chromatin regulation.