Sun M et al. (2023), Monitoring the compaction of single DNA molecul...

XB-ART-59570

Proc Natl Acad Sci U S A 2023 Mar 21;12012:e2221309120. doi: 10.1073/pnas.2221309120.

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Monitoring the compaction of single DNA molecules in Xenopus egg extract in real time.

Sun M , Amiri H , Tong AB , Shintomi K , Hirano T , Bustamante C , Heald R .

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DNA compaction is required for the condensation and resolution of chromosomes during mitosis, but the relative contribution of individual chromatin factors to this process is poorly understood. We developed a physiological, cell-free system using high-speed Xenopus egg extracts and optical tweezers to investigate real-time mitotic chromatin fiber formation and force-induced disassembly on single DNA molecules. Compared to interphase extract, which compacted DNA by ~60%, metaphase extract reduced DNA length by over 90%, reflecting differences in whole-chromosome morphology under these two conditions. Depletion of the core histone chaperone ASF1, which inhibits nucleosome assembly, decreased the final degree of metaphase fiber compaction by 29%, while depletion of linker histone H1 had a greater effect, reducing total compaction by 40%. Compared to controls, both depletions reduced the rate of compaction, led to more short periods of decompaction, and increased the speed of force-induced fiber disassembly. In contrast, depletion of condensin from metaphase extract strongly inhibited fiber assembly, resulting in transient compaction events that were rapidly reversed under high force. Altogether, these findings support a speculative model in which condensin plays the predominant role in mitotic DNA compaction, while core and linker histones act to reduce slippage during loop extrusion and modulate the degree of DNA compaction.

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Species referenced: Xenopus laevis
Genes referenced: asf1a
GO keywords: mitotic cell cycle [+]

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	Fig.1. Assay using Xenopus egg extracts and optical tweezers to monitor compaction of a single DNA. (A) Xenopus sperm nuclei were added to high-speed extract (HSE) arrested in either interphase or metaphase. Sperm nuclei decondensed in interphase HSE to form rounded clusters of sperm chromosomes, whereas single mitotic chromosomes were visible in metaphase extracts. (B) Optical tweezer setup in which a linear DNA tether is coupled to two beads; one bead is attached to a micropipette and the other positioned in an optical trap. (C) Example trace showing DNA contour in base pairs and force exerted on the trapped bead over time. Alternating between low (1.5 pN) and high (15 pN) force after introduction of HSE results in rapid cycles of compaction and decompaction.
	Fig.2. Cell cycle–dependent DNA compaction requires ATP. (A) DNA extension in base pairs over time following release from high force in interphase-arrested HSE with added ATP (blue), metaphase-arrested HSE with added ATP (red), and metaphase-arrested HSE without added ATP and treated with apyrase to degrade ATP (cyan). The thick line in each plot is the median trace and the bands indicate the upper and lower quartiles. Data are displayed at 5 Hz. Full-length (6,256 bp) and zero-length DNA contours are indicated with gray dashed lines. (B) Degree of compaction in each of the conditions in panel A. Individual traces are shown in SI Appendix, Fig. S3.
	Fig.3. Core and linker histones contribute to single DNA compaction. (A) Compaction time course profiles upon no depletion (red), ASF1 depletion (purple), or linker histone H1.8 depletion (brown). The thick line in each plot is the median trace and the bands indicate the upper and lower quartiles. Data are displayed at 5 Hz. (B) Relative effects of ASF1 and H1.8 depletion on final compaction level. (C) Compaction dynamics in control and histone-depleted reactions. Curves show median trace of actively compacting fibers (SI Appendix). Fibers under depletion conditions condense more slowly after 1 s, indicating slippage. (D) Compaction velocities (mean ± SEM) as a function of relative compaction state. Rates of overall compaction are higher in control reactions, but do not vary significantly with the relative level of compaction in control or following core or linker histone depletion. SI Appendix, Fig. S3 (E) A subset of traces is shown to illustrate that more frequent upward movements are observed upon depletion of ASF1 or H1.8. See SI Appendix, Fig. S4B for quantification. Individual traces under each condition are shown in SI Appendix, Fig. S3.
	Fig.4. Core and linker histones contribute to the compacted state of the DNA fiber. (A) Decompaction time course profiles at high force in HSE with no depletion (red), ASF1 depletion (purple), or linker H1.8 depletion (brown). The thick line in each plot is the median trace and the bands indicate the upper and lower quartiles. Data are displayed at 5 Hz. (B) Complementary cumulative distribution function (CCDF) fitting to decompaction times show the extent to which ASF1 and H1.8 depletions reduce decompaction time. Individual traces for each condition are shown in SI Appendix, Fig. S3.
	Fig.5. Effects of condensin depletion on compaction and decompaction dynamics. Time course profiles for compaction at low force (A) and decompaction at high force (B) in HSE with no depletion (red) and condensin depletion (green). The thick line in each plot is the median trace and the bands indicate the upper and lower quartiles. Data are displayed at 5 Hz. (C) Model in which condensin-extruded DNA loops are stabilized by formation of nucleosomes as well as nonnucleosomal H1.8-DNA interactions that inhibit condensin slippage.
	Fig. S1. Optical tweezers DNA contour length analysis. (A) Force-extension curve of the ~6.2 Kb DNA tether in gray, and an Extensible Worm Like Chain (XWLC) fit in black. (B) The contour length calculated based on the XWLC parameters remains flat if the DNA is simply pulled or relaxed. (C) Example trace showing raw data of DNA extension length over time in the top panel converted to contour length in base pairs in the middle panel. The lower panel shows force exerted on the trapped bead.
	Fig. S2. Western blots of ASF1, histone H1.8, and condensin depletions.
	Fig. S3. Individual experiment traces for all conditions tested in this study. (A) Compaction traces showing DNA contour length over time following release from high force. (B) Decompaction traces for the same conditions following return to high force. Individual traces are in gray. The median trace and the quartile bands are colored. The dashed line at 6256 bp corresponds to the expected contour length of full-length naked DNA.
	Fig S4. Frequency of short decompaction phases and quantification of step sizes. (A) Quantification of compaction versus slippage. The ratio between the distance moved downwards (compaction) versus total movement (upwards + downwards) was calculated for each compaction trace. With no depletion, 95% of movement is compaction, compared to 72% and 67% for ASF1 and H1.8 depletions, respectively. The black line at 0.5 indicates the expected ratio with no net compaction, as is the case for most condensin depletion traces. Each point represents a single trace with the mean and standard deviation indicated by solid lines. (B) Step size distribution of decompaction traces. The decompaction step sizes show a similar distribution across the different conditions tested. The median step size is ~200 bp and the mean is ~250bp

References [+] :

Arimura, Structural features of nucleosomes in interphase and metaphase chromosomes. 2021, Pubmed, Xenbase