DOI: 10.5061/dryad.bcc2fqzpz
title: Compromised 2-start zigzag chromatin folding in immature mouse retina cells driven by irregularly spaced nucleosomes with short DNA linkers

description: The formation of condensed heterochromatin is critical for establishing
 cell-specific transcriptional programs. To reveal structural transitions
 underlying heterochromatin formation in maturing mouse rod photoreceptors,
 we apply cryo-EM tomography, AI-assisted denoising, and molecular
 modeling. We find that chromatin isolated from immature retina cells
 contains many closely apposed nucleosomes with extremely short or absent
 nucleosome linkers, which are inconsistent with the typical two-start
 zigzag chromatin folding. In mature retina cells, the fraction of
 short-linker nucleosomes is much lower, supporting stronger chromatin
 compaction. By Cryo-EM-assisted nucleosome interaction capture, we observe
 that chromatin in immature retina is enriched with i±1 interactions while
 chromatin in mature retina contains predominantly i±2 interactions typical
 of the two-start zigzag. By mesoscale modeling and computational
 simulation, we clarify that the unusually short linkers typical of
 immature retina are sufficient to inhibit the two-start zigzag and
 chromatin compaction by the interference of very short linkers with linker
 DNA stems. We propose that this short linker composition renders
 nucleosome arrays more open in immature retina and that, as the linker DNA
 length increases in mature retina, chromatin becomes globally condensed
 via tight zigzag folding. This mechanism may be broadly utilized to
 introduce higher chromatin folding entropy for epigenomic plasticity.
description: Retina chromatin samples with a concentration of about 0.2 mg/ml
 DNA were mixed with a suspension of 10 nm fiduciary gold particles (Sigma
 Aldrich cat # 741957) treated with bovine serum albumin to prevent
 clustering. 3 μl chromatin samples were applied to Quantifoil R2/2 200
 mesh copper grids (EMS Q250-CR2). Vitrification by plunging into liquid
 ethane was conducted using the FEI Vitrobot Mk IV Grid Plunging System at
 100% humidity, 4 °C, and setting the blotting strength at 5, and blotting
 time at 3.5 sec.  Cryo-EM imaging was conducted on a Titan Krios G3i 300
 kV electron microscope, equipped with a K3 direct electron detector
 (Gatan, CA) at the Penn State Hershey cryo-EM core as described  (61).
 Tilt series were aligned using fiducials, CTF corrected, and SIRT
 reconstructed using the IMOD software suite (https://bio3d.colorado.edu/imod/). Regression denoising was accomplished in Dragonfly (ORS) using data synthesized by cryo-TomoSim (CTS, https://github.com/carsonpurnell/cryotomosim_CTS). Regression denoised images were exported as .tiff files and converted to MRC using the <em>mrc2tif</em> program within IMOD. The regression-denoised images were segmented into smaller subtomograms by IMOD/3dmod and inverted using “newstack” to generate subtomograms with positive intensity corresponding to high density. Data was visualized either with IMOD or Chimera.  For stereological measurements, the SIRT-reconstructed tomograms were segmented into smaller subtomograms by IMOD/3dmod,  inverted using “newstack”, and filtered by nonlinear anisotropic diffusion using IMOD command: “nad_eed_3d -n 30 -f -k 50”  to reduce noise and enhance chromatin edges. The filtered subtomograms (both SIRT-reconstructed and regression-denoised) were exported into UCSF Chimera (RBVI, Univ. San Francisco, CA) for interactive visualization and analysis of nucleosome structures. In Chimera, the filtered volumes were fitted with nucleosome core X-ray crystal structure (pdb 2CV5 (63)) semi-automatically to correspondent electron densities in the volume using the ‘fitmap’ command and the nucleosomes were overlaid with centroids, center-to-center axes, and nucleosome planes using the structure analysis ‘Axes/Planes/Centroids’ tool. Each nucleosome in an array was numbered and the following measurements were recorded: center-to-center distance D to the next nucleosome (n+1) in the array; center-to-center distance N to the nearest nucleosome (n<sub>x</sub>) in the 3D space; angle α between the two axes connecting each nucleosome with the previous one (n-1) and the next one (n+1) in a chain: angle β between the planes of consecutive nucleosomes n and n+1; and angle <em>para</em> between the plane of each nucleosome (n) and the plane of the nearest nucleosome (n<sub>x</sub>) in the 3D space. The chromatin mesoscale model combines coarse-grained representations of nucleosome cores, histone tails, linker DNA, and LHs within chromatin fiber arrays. To introduce nucleosome cores with 0 bp linker DNA, we connect two nucleosomes by a spring of 3 nm equilibrium length. Overlapping nucleosomes are allowed due to soft excluded volume terms for nucleosome-nucleosome interactions. We simulate 20 copies each for PN1 and PN56 by Monte Carlo sampling, recording for each an ensemble of 2000 configurations. For each ensemble, we calculate the packing ratio as the number of nucleosomes in 11 nm of fiber, the internucleosome interactions, and the tail interactions. For a single trajectory of PN1 and PN56, we create fan plots to represent, along each nucleosomal plane, the linker DNA cumulative and average positional distribution across a single trajectory. For single trajectories, we also assess quantitatively the degree of stem formation, as follows. We measure the distances between the average positions of each pair of beads on the two linker DNAs associated with the same core and assume that stem formation requires the distance between two DNA beads to be less than 2.5 nm.
description: # Compromised 2-start zigzag chromatin folding in immature mouse retina
 cells driven by irregularly spaced nucleosomes with short DNA linkers
 [https://doi.org/10.5061/dryad.bcc2fqzpz](https://doi.org/10.5061/dryad.bcc2fqzpz) ## Description of the data and file structure These files contain data related to the manuscript "Compromised 2-start zigzag chromatin folding in immature mouse retina cells driven by irregularly spaced nucleosomes with short DNA linkers" by Kable et al. 1. tilt_series_data - Contains all the original cryo-EM tilt series data generated by IMOD and used for analysis in the paper. 2. CAP_models - Each subfolder contains folders with both the filtered *.mrc volume and its corresponding *.py session file generated by CHIMERA. 3. mesoscale_models - Contains Matlab scripts needed to visualize chromatin configurations of each trajectory for PN1 and PN56 mesoscale models. ### Files and variables #### File: tilt\_series\_data.zip **Description:** Contains all the original cryo-EM tilt series data. To view the data, you will need to download and install IMOD: &lt;[https://bio3d.colorado.edu/imod/&gt;](https://bio3d.colorado.edu/imod/&gt;) To view a single tilt series, use the “3dmod”. Like this: 3dmod tilt_series_name.mrc #### File: CAP\_models.zip **Description:** Each subfolder contains folders with both the filtered .mrc volume and its corresponding .py file containing fitted-in nucleosome core PDB structures and Centroid/Axis/Plane models used for stereology. To open this data, you will need to download and install UCSF Chimera. &lt;[https://www.cgl.ucsf.edu/chimera/&gt;](https://www.cgl.ucsf.edu/chimera/&gt;) To view the data and the model, open Chimera and use it to open the .py file within one of the directories. It should load the corresponding .mrc and display the placement of each centroid, axis, and plane within it. ### File: mesoscale\_models.zip **Description:** These are the MATLAB scripts needed to visualize chromatin configurations of each trajectory. The main script to run is nonuniform_LH_video.m For visualization, you will have to: 1\) change the name of the dimension file on line 33 of nonuniform_LH_video.m This will be PN56_dim.in or PN1_dim.in, depending on which of the two systems you are visualizing 2\) change the name of the trajectory file on line 10 of load_MC.m This can be, for example, PN56_150mM.dat To run the visualization, you will need the dimension and trajectory files in the same folder that you have the MATLAB scripts. After you run the script, it will ask you in the command window to Enter first frame number: , you can type 2000, and then it will ask you to Enter last frame number: , you can type 2000 again. This will create a 3D plot of frame 2000. If you want to visualize many frames, you can type, for example, 1 as the first frame and 100 as the last frame.