Abstract
By means of Silicon Nano Tweezers (SNTs) the effects on the mechanical properties of λ-phage DNA during interaction with calf thymus nucleosome to form an artificial chromatin analog were measured. At a concentration of 100 nM, a nucleosome solution induced a strong stiffening effect on DNA (1.1 N m−1). This can be compared to the effects of the histone proteins, H1, H2A, H3 where no changes in the mechanical properties of DNA were observed and the complex of the H3/H4 proteins where a smaller increase in the stiffness is observed (0.2 N m−1). Para-sulphonato-calix[4]arene, SC4, known for epigenetic activity by interacting specifically with the lysine groups of histone proteins, was studied for its effect on an artificial chromatin. Using a microfluidic SNT device, SC4 was titrated against the artificial chromatin, at a concentration of 1 mM in SC4 a considerable increase in stiffness, 15 N m−1, was observed. Simultaneously optical microscopy showed a physical change in the DNA structure between the tips of the SNT device. Electronic and Atomic Force microscopy confirmed this structural re-arrangement. Negative control experiments confirmed that these mechanical and physical effects were induced neither by the acidity of SC4 nor through nonspecific interactions of SC4 on DNA.
Highlights
With the recent and better knowledge on how gene regulation occurs and after intense debate in the academic community[1], epigenetics has been commonly defined as “the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence”[2]
Epigenetics paradoxically possesses a reversible nature[5] that can be illustrated through its two main covalent modifications either directly on DNA with the methylation of cytosine nucleic bases or indirectly with various post translational modifications on proteins interacting with DNA such as histone proteins or other proteins called writers, erasers or readers[6]
While the ATPase dependent method produces a periodic nucleosome array with any DNA sequence of indefinite length, the ATPase-independent reconstitution of nucleosomes is random along the DNA position[41]. We selected this latter method as it presents the advantage of producing a pure chromatin unencumbered by histone chaperones or other biomolecules that could interfere with the subsequent epigenetic assessments
Summary
With the recent and better knowledge on how gene regulation occurs and after intense debate in the academic community[1], epigenetics has been commonly defined as “the study of changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence”[2]. For reversing or preventing a specific epigenetic modification in order to re-establish the tissue homeostasis, full understanding of how the DNA and its associated proteins interact with their environment while maintaining their normal cellular functions, is required. Such understanding requires suitable methods to study the properties of DNA during its rearrangement in the nucleus, (folding-unfolding) and how the mechanical properties of DNA can weigh on the nucleosome complexes of Chromatin[23]. This forms an octameric complex consisting of eight globular proteins, with the two amino-terminal domains of the H3 proteins pointing outward[3]
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