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bioRxiv · 10.1101/2023.01.04.522433

Dimensional Reduction for Single Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67

Benning, N. A.·Kaestel-Hansen, J.·Rashid, F.·Park, S.·Urteaga, R. M.·Liao, T.-W.·Hao, J.·Berger, J. M.·Hatzakis, N. S.·Ha, T.
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Abstract

Macromolecules organize themselves into discrete membrane-less compartments. Mounting evidence has suggested that nucleosomes as well as DNA itself can undergo clustering or condensation to regulate genomic activity. Current in vitro condensation studies provide insight into the physical properties of condensates, such as surface tension and diffusion. However, such studies lack the resolution needed for complex kinetic studies of multicomponent condensation. Here, we use a supported lipid bilayer platform in tandem with total internal reflection microscopy to observe the 2-dimensional movement of DNA and nucleosomes at the single-molecule resolution. This dimensional reduction from 3-dimensional studies allows us to observe the initial condensation events and dissolution of these early condensates in the presence of physiological condensing agents. Using polyamines, we observed that the initial condensation happens on a timescale of minutes while dissolution occurs within seconds upon charge inversion. Polyamine valency, DNA length and GC content affect threshold polyamine concentration for condensation. Protein-based nucleosome condensing agents, HP1 and Ki-67, have much lower threshold concentration for condensation than charge-based condensing agents, with Ki-67 being the most effective as low as 100 pM for nucleosome condensation. In addition, we did not observe condensate dissolution even at the highest concentrations of HP1 and Ki-67 tested. We also introduce a two-color imaging scheme where nucleosomes of high density labeled in one color is used to demarcate condensate boundaries and identical nucleosomes of another color at low density can be tracked relative to the boundaries after Ki-67 mediated condensation. Our platform should enable the ultimate resolution of single molecules in condensation dynamics studies of chromatin components under defined physicochemical conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/522433v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ed0cc9org.highwire.dtl.DTLVardef@1e26b5dorg.highwire.dtl.DTLVardef@1f6e73corg.highwire.dtl.DTLVardef@c7227b_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Benning, N. A., Kaestel-Hansen, J., Rashid, F., Park, S., Urteaga, R. M., Liao, T.-W., Hao, J., Berger, J. M., Hatzakis, N. S., Ha, T.. 2023-01-04. Dimensional Reduction for Single Molecule Imaging of DNA and Nucleosome Condensation by Polyamines, HP1α and Ki-67. https://doi.org/10.1101/2023.01.04.522433

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