DOI: 10.5061/dryad.547d7wmhr
title: Mature chromatin packing domains persist after RAD21 depletion in 3D

description: Understanding chromatin organization requires integrating measurements of
 genome connectivity and physical structure. It is well established that
 cohesin is essential for topologically associated domains (TAD) and loop
 connectivity features in Hi-C, but the corresponding change in physical
 structure has not been studied using electron microscopy. Pairing
 chromatin scanning transmission electron tomography with multi-omics
 analysis, and single molecule localization microscopy, we study the role
 of cohesin in regulating the conformationally defined chromatin nanoscopic
 packing domains. Our results indicate that packing domains are not
 physical manifestation of TADs. Using electron microscopy, we found that
 only 20% of packing domains are lost upon RAD21 depletion. The effect of
 RAD21 depletion is restricted to small, poorly packed (nascent) packing
 domains. Additionally, we present evidence that cohesin mediated
 loop-extrusion generates nascent domains that undergo maturation through
 nucleosome posttranslational modifications. Our results demonstrate that
 3D genomic structure, comprised of packing domains, is generated through
 cohesin activity and nucleosome modifications.
description: # Mature chromatin packing domains persist after RAD21 depletion in 3D
 [https://doi.org/10.5061/dryad.547d7wmhr](https://doi.org/10.5061/dryad.547d7wmhr) ## Description of the data and file structure Included zip files contain the respective code and data for generation of the files within the manuscript. Where relevant, script specific markdowns have been included. For the generation of the data from multi-color single molecule emission data, the files to upload need to be defined in the provided python script. For figures that rely on data from ENCODE, please download the respective files indicated in the supplementary table and set the file directories to load these data tables in the respective code. ChialoopsRad21.nb (Zenodo) was used to generate loop strengths in Figure 7. Please download the respective .bedpe files for this analysis and update the locations of files for import within the source code. Rad21 analysis.md (Zenodo) contains source code for specific multi-omics analysis included within the manuscript related to Mint-ChIP-Seq, Chia-PET, and Micro-C data not otherwise posted. **Usage Notes** All of the .tif files can be accessed with freely available ImageJ or FIJI software packages. The data files ending in .xslx can be opened with Microsoft Excel or other open access text editing tools (text editor) for viewing. ### Figure 1) Image files of chromatin electron microscopy tomograms (images) from cells are included, with: **Fig1a.tif** representing the chromatin electron microscopy tomogram of the cell. **Ctrldomain013d.tif** is a 3D rendition of a packing domain. **Ctrlloop649582.tif** is an observed chromatin loop with the accompanying **.avi** file representing the video projection of the observed loop. **domain1_properties.xslx** is the data showing the values of chromatin density extending from the domain interior to the periphery as a function of distance. The values included are radius (in nanometers), the mass (in a.u.), the standard deviation of the mass (in a.u.), the binarized chromatin volume concentration (CVC), the standard deviation of the binarized CVC, the gray-scale chromatin volume concentration (CVC), and the standard deviation of the gray-scale CVC. ### Figure 4) Image files of chromatin electron microscopy tomograms (images) from RAD21 depleted cells are included, with: **Fig4a.tif** representing the chromatin electron microscopy tomogram of the cell. **RAD21domain023D.tif** is a 3D rendition of a packing domain. **RAD21loop514629.tif** is an observed chromatin loop with the accompanying **.avi** file representing the video projection of the observed loop in these cells. ### Figure 5) This contains image files from two color single molecule localization microscopy (super-resolution imaging) of cells analyzing the distance between RAD21 to the nearest observed domain. **Composite.png** is a representative two color image. **Donut.pdf** represents the annular area around a domain with the captured RAD21 proteins within the area. **Storm association data.xlsx** represents the the quantified data from each cell to calculate the percentage of association within a domain. **Rad21_association-cell1_2_60nm.ipynb** is the script required to generate the observed associations from the super resolution images. The file directories under **SMLM CSV files** represent the replicates (**Control Replicate 1**, **Control Replicate 2**) with the acquired images numbered as files (Cell1-7 and Cell1-5) in each replicate numbered. The **edu** folder contains the emission events from the stained DNA. The **rad21** folder contains the emission events from RAD21. The reconstructed files are the .csv files and the resulting images are the .tif files. All of the .tif files can be accessed with freely available ImageJ or FIJI software packages. The ThunderStorm plug-in in the ImageJ environment can be used to import the .csv files to reconstruct images. Each imaging experiment is subdivided into folders labeled by the cell and the experimental condition (edu or RAD21). Composite.tif files represent the overlay of the two image sequences (edu and RAD21) into a single image file that can be opened with ImageJ or FIJI. Files that are labeled as ***-protocol.txt refer to the reconstruction parameter files for ThunderSTORM for the generation of the super resolution image contained for analysis. Files labeled as nucleus.tif in each folder are the respective output image of edu or RAD21 for that nucleus in the respective folder. ### Figure 8) Image files of chromatin electron microscopy tomograms (images) from cells with and without treatment by a histone deacetylase inhibitor are included, with: **Ctrl.tif** representing the chromatin electron microscopy tomogram of the control cell. **HDAC.tif** representing the chromatin electron microscopy tomogram of the cell.\ **control_hdac3_domain.tif** is an image slice from the rendition of a packing domain in control cells. **HDAC_Domain7_200nm.tif** is a 3D rendition of a packing domain in histone deacetylase treated cells. Accompanying it is a single image slice from the 3-D rendition titled **hdac3_inhibited domain.tif**\ **HDAC_loop_200nm.tif** is an observed chromatin loop in the histone deacetylase treated cells. ### HDAC3data.zip) This contains the relevant excel spreadsheets to calculate the packing domain properties upon inhibition with the histone deacetylsae inhibitor. hdac3controldomains.xlsx represents the values for domains in the control cells whereas hdac3inhibiteddomains.xlsx represents the treated domain properties. Domain properties include the average intensity within the domain (intensity), the scaling of chromatin packing values (D), the radius of the domain (Rmax), the packing efficiency (packing eff), and the radius of the elementary chain (Rmin). These data files can be opened with Microsoft excel or other open access text editing tools (text editor) for viewing. For analysis, the accompanying hdac3data.nb file is a Mathematica script containing the code to open these files and calculate the distribution of domain radius compared to packing efficiency as a reflection of the domain life cycle. This code can be run with Mathematica v13 or alternative opened with a text editor and transformed into comparable scripts in python or R. ### RAD21-main.zip) The domain1_properties.xslx file is the data showing the values of chromatin density extending from the domain interior to the periphery as a function of distance as in Figure 1. The values included are radius (in nanometers), the mass (in a.u.), the standard deviation of the mass (in a.u.), the binarized chromatin volume concentration (CVC), the standard deviation of the binarized CVC, the gray-scale chromatin volume concentration (CVC), and the standard deviation of the gray-scale CVC. The control packing efficiency.xlsx represents the values for domains in the control cells whereas RAD21 packing efficiency.xlsx represents the RAD21 depleted domain properties. Domain properties include the average intensity within the chromatin volume concentration (binarized CVC), domain (grayscale_density), the scaling of chromatin packing values (D), the radius of the domain (domain radius), the packing efficiency. These data files can be opened with Microsoft excel or other open access text editing tools (text editor) for viewing. For analysis, the accompanying rad21 analysis.nb file is a Mathematica script containing the code to open these files and calculate the distribution of domain radius compared to packing efficiency as a reflection of the domain life cycle for these excel files. This code can be run with Mathematica v13 or alternative opened with a text editor and transformed into comparable scripts in python or R. The RAD21 depletion PWS data.xlsx file represents the average chromatin scaling within nuclei of RAD21 depleted cells (HCT116-RAD21-AID2 + 4 hr Auxin) compared to the accompanying control cells (HCT116 RAD-AID2 - Auxin). These were obtained using live cell PWS microscopy and the accompanying PWS RAD21 Data.nb file is a Mathematica script to plot the values within this spreadsheet. These can be transformed into comparable scripts in python or R. The Rad21domainSizeInKbps.xlsx is the calculated DNA content within packing domains in control cells (Control) in compared to packing domains in RAD21 depleted cells (RAD21). This can be opened with Microsoft Excel or an open access text editor for viewing. It can be opened with RAD21Analysis.nb to generate the respective plots using Mathematica v13. Included within RAD21Analysis.nb is a subset of script to analyze atac-seq data available through ENCODE. The accession values for the ENCODE files are ENCFF107INQ.bed (control cells) and NCFF460JUY.bed (RAD21 depleted cells). ## Access information Other publicly accessible locations of the data: * ENCODE Consortium with accession numbers for respective data listed within the supplemental materials of this manuscript Data was derived from the following sources: * ENCODE Consortium with the files listed in the supplemental materials of the manuscript