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Abstract
ABSTRACTChromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape.
Highlights
Histones are the basic structural components of chromatin
Chromatibody is a dynamic chromatin probe that can be modulated We explored the dynamic properties of chromatibody and bivalent chromatibody, in order to introduce avidity and engineer binding molecules with increased functional affinity (Olichon and Surrey, 2007; Hultberg et al, 2011; Cardoso et al, 2014)
Our experimental set-up did not allow us to estimate the t1/2 for GFP or H2B–GFP as these were, respectively, too fast and too low to be calculated. These data indicate that the chromatibody interaction with chromatin is highly dynamic, being far less tightly associated to chromatin than H2B– GFP, suggesting that they are labile probes for chromatin studies. These results show that chromatibody is a dynamic chromatin probe that can be modulated for its binding properties
Summary
Histones are the basic structural components of chromatin. Eukaryotic DNA is wrapped around histone octamers, containing two copies of each of the core histones (H2A, H2B, H3 and H4) (Luger et al, 1997). Small basic proteins that can be covalently modified at their N- or C-terminal tails, as well as on their globular domains (Bannister and Kouzarides, 2011). Histone post-translational modifications modulate their interaction with both DNA and effector proteins, influencing chromatin structure and function (Bhaumik et al, 2007; Rothbart and Strahl, 2014). Chromatin function and dynamics are involved in many cellular processes, including gene expression regulation, DNA repair or meiosis.
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