DOI: 10.5061/dryad.r2280gbkf
title: Data from: Coactivator condensation drives cardiovascular cell lineage specification

description: During development, cells make switch-like decisions to activate new gene
 programs specifying cell lineage. The mechanisms underlying these decisive
 choices remain unclear. Here, we show that the cardiovascular
 transcriptional coactivator, Myocardin (MYOCD), activates cell identity
 genes by concentration-dependent and switch-like formation of
 transcriptional condensates. MYOCD forms such condensates and activates
 cell identity genes at critical concentration thresholds achieved during
 smooth muscle cell and cardiomyocyte differentiation. The C-terminal
 disordered region of MYOCD is necessary and sufficient for condensate
 formation. Disrupting this region’s ability to form condensates disrupts
 gene activation and smooth muscle cell reprogramming. Rescuing condensate
 formation by replacing this region with disordered regions from
 functionally unrelated proteins rescues gene activation and smooth muscle
 cell reprogramming. Our findings demonstrate that MYOCD condensate
 formation is required for gene activation during cardiovascular
 differentiation. We propose that the formation of transcriptional
 condensates at critical concentrations of cell type-specific regulators
 provides a molecular switch underlying the activation of key cell identity
 genes during development.
description: The following datasets pertain to the manuscript entitled
 "Coactivator condensation drives cardiovascular cell lineage
 specification" by Gan Peiheng et al. (2023-12-28). These data are
 primarily micrographs acquired by spinning disk confocal
 microscopy.
description: # Data from: Coactivator condensation drives cardiovascular cell lineage
 specification Dataset DOI: 10.5061/dryad.r2280gbkf Description of the data
 and file structure ## GENERAL INFORMATION **Dataset overview** This
 dataset has been generated primarily by confocal fluorescence microscopy.
 All raw image files can be found here.  **Corresponding author
 information** Name: Benjamin Sabari ORCID:
 [https://orcid.org/0000-0002-6399-575X](https://orcid.org/0000-0002-6399-575X) Affiliation: Green Center for Reproductive Biology, University of Texas Southwestern, Dallas, Texas, United States email: [benjamin.sabari@utsouthwestern.edu](mailto:benjamin.sabari@utsouthwestern.edu) **Related publication** Peiheng Gan et al. ,Coactivator condensation drives cardiovascular cell lineage specification.Sci. Adv.10,eadk7160(2024).DOI:10.1126/sciadv.adk7160 Files and variables File: 2024_Ganetal_SupplementalFigures.tar.gz Description: This zipped file contains folders pertaining to the supplemental figures. These are raw micrographs. File names include date, cell type, and the different fluorescently labelled components of the image that was analyzed. The zipped folder contains subfolders corresponding to each figure and is labeled accordingly: Figure S1 A-B-E-F-G-H-&gt; Fluorescence confocal imaging with immunofluorescence of COS7 cells ectopically expressing MYOCD-mEGFP (fluorescence), V5-MEF2C(immunofluorescence), Flag-NFYA(immunofluorescence), or Myc-MYOCD(immunofluorescence). Figure S1 C-D-&gt; Fluorescence confocal imaging in live COS7 cells that are ectopically expressing MYCOD mEGFP. These data are from fluorescence recovery after photobleaching experiments. Figure S2 C-&gt; Sanger sequencing plot of mEGFP insertion to the C-terminus of endogenous MYOCD in hiPSCs. Figure S2 E-G-&gt; Fluorescence confocal imaging of purified recombinant GFP used as concentration standards for estimating nuclear MYOCD concentration in individual cells. Figure S5 C-D and F-G-&gt; Fluorescence confocal microscopy of in vitro droplet formation in purified recombinant MYOCD TAD domain fused to mEGFP, the MYOCD TAD domain in which all aromatic residues are substituted with alanine (FWY to A), or mEGFP alone. Figure S6 B-C-&gt; Fluorescence confocal imaging with immunofluorescence of COS7 cells ectopically expressing MYOCD with the TAD replaced with FUS IDR or TAF15 IDR fused to mEGFP (fluorescence) and Flag-SRF (immunofluorescence). Figure S7A-&gt; Fluorescence confocal imaging of purified recombinant GFP used as concentration standards for estimating nuclear MYOCD concentration in individual cells. Figure S7D-E and S8 D-E-&gt; Fluorescence confocal imaging of 10T1/2 cells that were subjected to differentiation-promoting conditions. Cells are ectopically expressing WT MYOCD, MYOCD without the TAD domain, or MYOCD in which all aromatic residues within the TAD have been substituted to alanine (FWY to A) and have been immunofluorescently stained to mark Acta2 expression levels. Figure S8 C-&gt; Western blot for GFP or B-tubulin in 10T1/2 cells ectopically expressing mEGFP alone, WT MYCOD, MYOCD without the TAD, MYOCD-FWY to A, MYOCD fused to FUS IDR, MYOCD fused to TAF15 IDR, MYOCD fused to NPHS1 IDR, MYOCD fused to DDX4 IDR, or MYOCD fused to CDT1 IDR. File: Data_Repository.xlsx Description: The file contains sheets with specific data tables pertaining to each figure. Each sheet is labelled with the respective figure to which the data corresponds. Each sheet also contains a brief description of the data in each sheet, a description of the column headers, and other contextual details when necessary. Variables Variables are described by columns in the Excel sheet.  File: 2024_Ganetal_MainFigures.tar.gz Description: This zipped file contains folders pertaining to the main figures. These are raw micrographs. File names include date, cell type, and the different fluorescently labelled components of the image that was analyzed. The zipped folder contains subfolders corresponding to each figure and is labeled accordingly: Figure 1 A-F -&gt; Fluorescence confocal imaging with immunofluorescence of COS7 cells ectopically expressing MYOCD-mEGFP (fluorescence), Flag-SRF(immunofluorescence), Flag-NRF1(immunofluorescence), or Myc-MYOCD(immunofluorescence). Figure 1 G_S1I-L-&gt;Fluorescence confocal imaging with immunofluorescence of COS7 cells ectopically expressing MYOCD-mEGFP (fluorescence) and immunofluorescence for endogenous H3K27ac, RPB1, HP1a, or NPM1. Figure 2 B-D-&gt; Fluorescence confocal imaging of hiPSC differentiated into cardiomyocytes or smooth muscle cells. MYOCD is endogenously tagged with mEGFP at the C-terminus. This folder includes 1) live cell imaging and 2) fixed cell imaging in which immunofluorescence for Acta2 is performed. Figure 2D-F, 4 A-C, 5A-H, K-L-&gt; Fluorescence confocal imaging of COS7 cells ectopically expressing MYOCD-mEGFP. Figure 3-&gt;Fluorescence confocal imaging of hiPSC-derived cardiomyocytes or smooth muscle cells in which MYOCD is endogenously tagged with mEGFP at the C-terminus. These data represent RNA FISH for either Acta2, Ebf4, Myl4, Tbx20, or Amhr2. Figure 4 D-F and S4 F-H-&gt;Fluorescence confocal imaging of COS7 cells ectopically expressing MYOCD-mEGFP and the plasmid-based fluorescent protein reporter. Figure 5 D-E and I-J and M-N-&gt;Fluorescence confocal imaging of COS7 cells ectopically expressing MYOCD-mEGFP, MYOCD without TAD-mEGFP, MYOCD with FWY to A TAD, MYOCD in which the TAD is replaced with FUS or TAF15. Figure 6 A-J-&gt;Fluorescence confocal imaging of 10T1/2 cells that were subjected to differentation conditions. Cells are ectopically expressing MYOCD-mEGFP, MYOCD without TAD-mEGFP, MYOCD with FWY to A TAD, and MYOCD in which the TAD is replaced with FUS or TAF15. Figure 7 A-C and S9A-C-&gt; Fluorescence confocal imaging in COS7 cells in which cells are immunofluorescently stained for endogenous p300 or RPB1 and are ectopically expressing either: WT MYCOD, MYOCD in which the TAD is replaced with FUS IDR or CDT1 IDR. Figure 7 D-E and S9 D-E-&gt;Fluorescence confocal imaging in U2OS 263 cells in which cells are immunofluorescently stained for endogenous p300 or RPB1 and are ectopically expressing either: CFP-LacI- no fusion, CFP-LacI-MYCOD TAD, CFP-LacI-FUS IDR, CFP-LacI-CDT1 IDR. Access information Other publicly accessible locations of the data: RNA-seq and ChIP-seq data from hiPSC presented here are available for download at GEO under the accession number GSE235928.  RNA-seq and ChIP-seq data from 10T1/2 cells presented here are available for download at GEO under accession number GSE250206. Data was derived from the following sources: All data is original to this study.