Biophysical Society Thematic Meeting| Les Houches 2019

Multiscale Modeling of Chromatin: Bridging Experiment with Theory

Tuesday Speaker Abstracts

POSITIONING THE LINKER HISTONE ON A CHROMATOSOME: WHAT ROLE DOES THE DNA PLAY? Madhura De 1 ; Rebecca C Wade 2 ; Katalin Tóth 1 ; 1 German Cancer Research Center, Biophysics of Macromolecules, Heidelberg, Baden- Württemberg, Germany 2 ZMBH, DKFZ-ZMBH Alliance and Interdisciplinary Center for Scientific Computing (IWR), Heidelberg University, Heidelberg, Baden-Württemberg, Germany The chromatosome comprises of the nucleosome with additional stretches of linker-DNA associated with the linker histone protein (LH). To study the effect of linker-DNA length and sequence on LH positioning, single-molecule FRET (sm-FRET) spectroscopy and sm-FRET with Alternating Laser Excitation (ALEX) on fluorescently labelled reconstituted chromatosomes were performed. sm-FRET measurements on chromatosomes labelled on the two linker-DNA arms confirmed that the LH ( Xenopus laevis H1.0b) compacted the chromatosome at low salt. This observation was further supported by EMSA. At a given salt, the distance between the dyes placed on the DNA, 20bp away from the two entry/exit sites were found to be similar in both symmetric chromatosomes with equally long 40bp linker-DNAs, or asymmetric chromatosomes with one 40bp, and the other, 24bp linker-DNA. To study the sequence effects of linker-DNA, we used a symmetric 226bp Widom 601 DNA with 11bp GC-rich region on one entry/exit site, and 11bp AT-rich region on the other entry/exit site. Chromatosomes were reconstituted, having the acceptor dye on one or the other linker-DNA, and the LH labelled with the donor dye at either the C-terminal domain (CTD) or the globular domain (GD). The distances between the CTD or the GD and either linker-DNA arms were equal in unmodified, symmetric chromatosomes. However, for symmetric chromatosomes with 11bp AT-rich region, the LH position was found to be askew: the CTD was closer to the GC-rich linker-DNA and the GD was closer to the modified AT-rich arm. On swapping the GC and the AT-rich regions, keeping the rest of the DNA unchanged, the GD was again found to be closer to the AT-rich arm. This suggests that the LH positioning on a single chromatosome is variable, and is affected, among other factors, by DNA sequence. This DNA-directed LH positioning may have implications in higher-order structures.


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