The Gene-Silencing Protein MORC-1 Topologically Entraps DNA and Forms Multimeric Assemblies to Cause DNA Compaction

Abstract

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.

Keywords: DNA compaction; DNA-binding protein; GHKL ATPases; MORC; genome organization.

Figure 1.. MORC-1 is a DNA binding protein.

(A) Protein ([MORC-1] = 100 nM, 200 nM, 400 nM, 800 nM) was incubated with 32P labeled 250 bp DNA probe derived from λ-DNA and run on a 3% agarose gel, then dried before exposure to a phophor screen. (B) 200 nM MORC-1 was added to 0.5 nM 32P labeled 250 bp λ-DNA that was premixed with cold 250 bp λ-DNA, 250 bp scrambled DNA, or 50 bp λ-DNA probe (1 nM, 10 nM, 20 nM, 50 nM) and processed as described above. See also Figure S1.

Figure 2.. MORC-1 compacts DNA using a loop trapping mechanism.

(A) A schematic of the flow stretched DNA assay. (B) A representative kymograph demonstrating compaction of a quantum dot labelled λ-DNA over time. Scale bars represent 10 seconds and 5 μm. (C) Effect of ATP on MORC-1 compaction. Data was calculated from 48 different experiments, with a total of 1199 different trajectories. n=85, 48, 98, 122, 66, 84, 57, 97 (without ATP), and n=66, 94, 79, 52, 61, 76, 47, 67 (with 2 mM ATP). Error bars: SE. (D) AMP-PNP further stimulates DNA compaction. Compaction rates were from two independent experiments for each condition. Error bars represent the SE. n=45, 46, and 56 for experiments with no nucleotide, 2 mM ATP, and 2 mM AMP-PNP, respectively. (E) Left: schematic of DNA motion capture assay depicting the location of the five EcoRI binding sites. Middle: Plotted trajectories of location of EcoRI-E111Q conjugated quantum dots over time. Below: representative kymograph. [MORC-1] = 40 nM, [ATP] =1 mM. See also Figures S2 and S3, Video S1.

Figure 3.. MORC-1 forms bodies that grow in size with concentration.

(A) A representative kymograph showing Cy3 labeled MORC-1 cluster formation. (B) At varying MORC-1 concentrations, foci intensities were quantified at a fixed time point (140 seconds after flowing in sample) and presented as box plots. The number of clusters was n=21, 34, 55, and 34 for [MORC-1] = 2, 5, 10, and 40 nM, respectively. The red line inside each box corresponds to the median value while the bottom and top edges of each box correspond to the 25th and 75th percentiles, respectively. A Black dotted line connecting nearby median values was added for visual guidance and showed displayed a strong linear correlation (Pearson correlation coefficient: r = 0.99996). See also Figure S4, and Videos S2 and S3.

Figure 4.. MORC-1 is localized to bodies in vivo and forms condensates in vitro.

(A) MORC-1 are found in nuclear bodies of varying sizes in vivo. MORC-1 mitotic bodies (n=96) are on average (mean) 229 nm; MORC-1 pachytene bodies (n=53) are on average (mean) 170 nm. Scale bar represents 5 μm. (B) MORC-1 undergoes liquid-liquid phase separation. Unlabeled protein was buffer exchanged into 75 mM NaCl and mixed with labeled protein to achieve a final population of 1–2% labeling. Single color droplets were pre-formed by adding a final concentration of 10% PEG 3350. To image dual color droplets, pre-formed droplets were mixed together and incubated at room temperature before imaging. To image dual color MORC-1 on DNA, Cy5-MORC-1 was added to Cy3-MORC-1 preincubated with DNA. White represents areas of signal overlap. Scale bars represent 5 μm. See also Figure S5.

Figure 5.. MORC-1 foci topologically entrap DNA.

Representative kymographs of Cy3-MORC-1 foci movement on quantum dot labeled DNA (top panel, [MORC-1] = 5 nM, [ATP] = 2 mM) and on bare DNA (bottom panel, [MORC-1] = 10 nM, [ATP] = 2 mM). Schematics below the kymographs illustrate the experiment. MORC-1 is applied to the flow cell and allowed to form foci, then subjected to a 500 mM NaCl and 2 mM MgCl₂ wash in the presence of 2 mM ATP. Scale bars represent 20 seconds and 5 μm. SYTOX Orange stained DNA is shown to the right of the kymographs to indicate approximate location of foci on the DNA. Note that the quantum dot is not visible any of the displayed spectral channels. In the top panel, the top focus (magenta asterisk) contains approximately 2 MORC-1s and the bottom focus (lavender asterisk) contains approximately 7 MORC-1s. In the bottom panel, the time gap between the two events as marked by the dashed line is 7.2 seconds. The top focus (orange asterisk) has approximately 7 MORC-1s and the bottom (peach asterisk) has approximately 13 MORC-1s. See also Figure S6, and Videos S4, S5, and S6.

Figure 6.. MORC-1 selectively retains circular DNA after high salt washes.

(A) MORC-1 was incubated with DNA of varying topologies and precipitated with magnetic resin, then washed twice before proteinase K elution. Retained DNA is visualized by resolving samples on an 1% 1x TAE agarose gel and post-stained with ethidium bromide. (B) 400 nM MORC-1 was incubated with 50 ng open circular DNA that was made by Nt.BsmAI digestion and precipitated with magnetic resin, then washed twice with a high salt buffer and once with Restriction Enzyme (RE) buffer before digesting with restriction enzymes to alter the topology. The sample was then washed using low or high salt, then eluted using proteinase K. Retained DNA is visualized by resolving samples on an 1% 1x TAE agarose gel and post-stained with ethidium bromide. Note that PstI digestion was not complete and there were trace amounts of open circular plasmid remaining. However, MORC-1 washed with high salt only retains the open circular form and not the linearized material. See also Figure S6.

Figure 7.. MORC-1 compacts chromatin.

(A) Distribution of substrate lengths under flow. Mode of DNA substrates (n=301) = 10.03 μm; Mode of chromatin substrates (n=415) = 2.84 μm. Substrates were flowed into the flow cell and subsequently visualized with 3 μM SYTOX Orange. (B) Kymograph depicting MORC-1 mediated compaction of a chromatinized template. Scale bars represent 7 μm and 3 seconds, respectively. [MORC-1] = 75 nM. (C) MORC-1 compacts chromatinized substrates of varying lengths. [MORC-1] = 75 nM was flowed into the flow cell and compaction was allowed to proceed. Upper panel; scatter plot of individual trajectories plotted by the initial and final lengths. Bottom panel; projection of the scatter plot, binning the trajectories by final DNA length.