Compromised 2-start zigzag chromatin folding in immature mouse retina cells driven by irregularly spaced nucleosomes with short DNA linkers

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 re..., 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/carsonpurn..., , # 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 orig...,