Dynamics of Nucleosomes

Basics

Nucleosomes are the basic units of eukaryotic chromatin structure. They consist of approx. 150 bp DNA wrapped around a protein core formed by eight histone proteins.

In this highly stable complex the histone subunits hinder binding of other proteins to nucleosomal DNA. Thereby they play an important role in the regulation of all processes that require access to DNA, e.g. transcription and replication. The stability of nucleosomes suggests that elaborate mechanisms must have evolved to guide nucleosome dis- and reassembly and further structural changes. A detailed analysis of nucleosome dynamics is essential for a profound understanding of these processes. We analyse the dynamics of nucleosomes by measuring distances between different points within the nulceosome with Fluorescence Resonance Energy Transfer, a method to measure intramolecular distances. We use nucleosomes that are labeled with fluorescent dyes at specific positions. As a dynamic system nucleosomes adopt different conformations. To avoid averaging over different conformations and to unravel heterogeneities we also analyze these processes on a single-molecule level.

Histone Tail Dynamics

The nucleosome as the basic repeating unit of chromatin regulates DNA accessibility and has significant influence on genetic function. Two copies of each histone protein H2A, H2B, H3, H4 build up the protein octamer where short fragments of approximately 150 bp of DNA are wrapped around. This complex of DNA and histone octamer is called nucleosome. The N-terminal tails of the histone proteins are not part of the core structure and protrude from the nucleosome. It is known that they play an important role for inter- and intranucleosomal interactions. This project focuses mainly on the role of histone tails. One goal is to analyse the contribution of the tails to the overall stability of the nucleosome. To answer this question I will use truncated versions of H3 and H4 in which parts of the N-terminal tail regions are removed. With the help of this constructs I will experimentally control previously obtained results of MD simulations. Furthermore I will design site-specific mutated recombinant H2A proteins (R81A and R88A) with a single amino acid exchange at two important postitions. Until now it is not clear if these amino acids are crucial for the reconstitution of the nucleosome or if they only contribute to the stability of the complexe. To analyse the above mentioned issues I will perform an in vitro assay which is already established in the work group. This assay comprises fluorescently labeled nucleosome which are labeled on different positions within the histone core and along the DNA. The most important techniques for the analysis are flurorescence correlation sprectroscopy (FCS) and single pair Förster resonance energy transfer (spFRET). FCS measures the average diffusion coefficients of a fluorescently labeled sample by analyzing fluctuations in fluorescence emission. The diffusion coefficient provides informations about the molecule size and shape and can thereby be used to analyse potential interactions. The second technique spFRET is based on the non radiative energy transfer of two fluorophores which depends on the distance between them. This enables us to distinguish between particular subpopulations of a heterogenous sample. Both techniques will be used to investigate differences in the stability and dynamic of different compositions of in vitro reconstituted nucleosomes. The outcome of this study will increase and strengthen the current knowledge of nucleosome dynamics and thereby lead to a further understanding of the basic regulation of DNA accessibility and the associated regulation of gene function.

 

References:

  1. J. Erler, R. Zhang, L. Petridis, X. Cheng, J.C. Smith, J. Langowski: The role of histone tails in the nucleosome: A computational study. (2014) Biophys. J. 107(12), 2911-22. doi:10.1016/j.bpj.2014.10.065
  2. R. Zhang, J. Erler, J. Langowski: Molecular dynamics simulations of histone H3 and N4-terminal tail conformation in the presence and absence of nucleosome core. (2014) TASK Quarterly 18(3), 53-60
  3. K. Lehmann, R. Zhang, N. Schwarz,A. Gansen, N. Mücke,, J.Langowski and K. Tóth: Effects of charge-modifying mutations in histone H2A α3-domain on nucleosome stability. (2017) Scientific Reports 7, 13303

Modelling of Nucleosome Dynamics

The goal of the project is a deeper understanding of DNA compaction and thus gene expression and gene transcription based on an all atom model. In the nucleosome core particle, the basic unit of chromatin, DNA is wrapped around a protein core, build by an octamer of histone proteins H3, H4, H2A and H2B (see figure). For access to genomic DNA by proteins involved in the control and expression of the genome the nucleosome has to undergo structural remodeling including unwrapping of nucleosomal DNA segments from the nucleosome core. The positively charged tails of the histones seem to play an important role in this process. A former study showed that a truncation of the tails, similar to post-transcriptional modifications, such as acetylation, methylation or phosphorylation, results in a destabilization of the histone core, probably due to the absence of histone-histone and histone-DNA polar contacts (Ref. 1). The latter are known to influence nucleosome dynamics and gene transcription. A further project studying DNA unwrapping using coarse grained simulations showed a long-lived DNA detachment from the nucleosome. Part of the long-lived DNA detachment is a binding of the H3 tail to the protein core blocking the DNA from rebinding to that. A removal of the H3 tails causes the long-lived detachments to disappear. The results suggest that the H3 tail may stabilize the nucleosome in the open state during the initial stages of the nucleosome remodeling process (Ref. 2). 

The questions we aim to answer are: 

  • How do histone tails bind to the DNA surface?
  • Is there an influence of DNA or the nucleosome core on the secondary structure of histone tails?
  • What role do histone tails play in inter or intra nucleosomal interactions?

In addition to single MD simulations we are using replica exchange molecular dynamics (REMD) simulations to accelerate the generation of conformations and to overcome high energy barriers in the conformational space. The REMD method involves simulating multiple replicas of the system at different temperatures and randomly exchanging the complete state of two replicas which is comparable to a heating and cooling of the system.

The goal of a further study is to understand the effect of post-transcriptional modifications, such as acetylation or methylation on the nucleosome and tail dynamics. 

Results will be compared to results of spFRET measurements (see experimental part) where the DNA surface and histone tails are labeled to study the distance and thus a possible binding of the histone tails to the DNA surface.


References:

  1. M. Biswas, K. Voltz, J.C. Smith, J. Langowski:
    Role of histone tails in nucleosome stability. 
    PLoS Comp Biol 7(12), e1002279 (2011) DOI: 10.1371/journal.pcbi.1002279
  2. K. Voltz, J. Trylska, J.C. Smith, J. Langowski:
    Unwrapping of Nucleosomal DNA Ends: A Multiscale Molecular Dynamics Study.
    Biophys. J. 102 (4), 849-858; DOI:10.1016/j.bpj.2011.11.4028 (2012)
  3. R. Zhang, J. Erler, J. Langowski:
    Histone Acetylation Regulates Chromatin Accessibility: Role of H4K16 in Inter-nucleosome Interaction.
    (2017) Biophysical Journal112(3):450-459

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