Additional file 1 of Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells

Additional file 1: Supplementary Figure S1. Related to Figure 1. Validation and characterizing of our DOX-Inducible system for manipulating FOS expression in Muscle Progenitor Cells Ex Vivo. (A) Representative images of cultured muscle progenitor cells in GM supplemented with increasing amounts of Doxycycline (0, 0.005 μg/ml, 0.01 μg/ml, 0.05 μg/ml, 0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 2.5 μg/ml, 5 μg/ml) for 48 hours in culture. Scale bars represent 50 microns. (B) Quantification of the total number of Hoechst+ cells after 48 hours in GM supplemented with the indicated concentration of DOX (n=cells from 3mice). (C) Experimental Flowchart for detecting FOS protein (sc-7292) using ProteinSimple WES platform and analysis using the COMPASS software. (D) Virtual bands showing gradual increase of FOS protein with increasing amounts of DOX. Raw area of signal is shown for loading control (GAPDH) and FOS. FOS signal normalized to GAPDH is displayed, highlighting that 1 ug/ml of DOX was the lowest concentration that gave the maximal induction of FOS protein. (E) 20X images of two-week cultured muscle progenitor cells grown in GM for 48 hours or differentiated in DM for 72 hours and stained for PAX7 and MyoG. Nuclei stained with DAPI. Scale bar represents 50 microns. (F) Corrected total cell fluorescence (CTCF) for PAX7 (left) and MYOG (right) quantified in (E). n= 98-4184 cells (PAX7) and n= 2169-46118 cells (MYOG). (G) Corrected total cell fluorescence (CTCF) for PAX7 (left) and MYOG (right) in two-week cultured pSLIK-Fos and pSLIK-Gfp muscle progenitor cells. n=2295-2816 (PAX7) and n=1432-3779 (MYOG). Mean comparisons using One-way ANOVA with post-hoc Tukey test (B) and Mann Whitney U-test (F, G). Supplementary Figure S2. Related to Figure 3. FOS-dependent suppression of myogenic genes in muscle progenitor cells is reversible upon DOX removal. pSLIK-Fos cells were treated with 1 ug/ml DOX for 72-hours and then chased with media devoid of DOX for 3 and 5 days followed by RT-qPCR. Mean relative mRNA expression (+/-SD) and normalized (to Gapdh) for Fos, MyoD, MyoG, Tcap, and Ttn. Data shows that upon removal of DOX, mRNA expression for MyoD, MyoG, Tcap, and Ttn are de-repressed concomitant with FOS returning to basal levels. Mean comparisons were performed with a one-way ANOVA with post hoc Tukey test. N= 5-6 replicate cultures from cells pooled from 4 mice. Supplementary Figure S3. Related to Figure 3. Chromatin Immunoprecipitation (ChIP)-qPCR analysis for H3K27Me3 occupancy at the promoter region of Bmp6, an intergenic region, MyoD, and Ttn in pSLIK-Fos and pSLIK-Gfp cultured muscle progenitor cells. (A) Experimental design: Primary satellite cells were infected with virus expressing pSLIK-Fos or pSLIK-Gfp constructs, selected with 100 μg/mL of hygromycin for 6 days, and expanded for approximately 3 weeks to generate enough cells for ChIP-qPCR. Once at the desired cell density, pSLIK-Fos and pSLIK-Gfp cells were cultured in GM-supplemented with 1μg/mL doxycycline (DOX) for 48 hours. Chromatin was IP’d using an H3K27Me3- or immunoglobulin G (IgG)-specific antibodies followed by qPCR. Plot shows mean percent of input (+/- SD) for each amplicon targeting the first 500bps upstream of the TSS for Bmp6 and an intergenic region (left) and MyoD and Ttn (right). Location of primers relative to gene TSS is shown in schematic above each plot. Statistical comparisons were performed with two-way ANOVA with post hoc Holm-Sidak test. N= 8-9 replicate IPs were performed on cells pooled from 4 mice. Supplementary Figure S4. Related to Figure 4. Quality Metrics of Hi-C Datasets. (A) Pairwise comparison of all Hi-C heatmaps for each replicate of pSLIK-Gfp and pSLIK-Fos datasets showing all the chromosomes and the inter-chromosomal interactions. (B) Scaling plot showing the interaction frequency as a function of genomic distance, demonstrating that pSLIK-Fos and pSLIK-Gfp muscle progenitor cells have similar rates of decay of interaction frequency. (C) Representative higher resolution images of Hi-C datasets of pSLIK-Gfp and pSLIK-Fos muscle progenitor cells. Supplementary Figure S5. Related to Figure 5. Compartment Analysis of Hi-C Data. (A-B) Heatmap for the pSLIK-Fos and pSLIK-Gfp cells showing the compartment strength, quantified by plotting interaction frequencies in 250kb bins arranged by their values along the first eigenvector (PC1/EV1) to obtain compartmentalization saddle plots. The average interaction frequencies of the observed / expected interactions between pairs of loci (250kb bins) were calculated and arranged by their compartment signal (1st eigenvector value) for pSLIK-Fos and pSLIK-Gfp muscle progenitor cells. In these plots the upper left quadrant represents B-B interactions, and the lower right corner represents A-A interactions. (C) Comparison of the intra- and inter-compartmental interaction frequencies indicates that FOS induction (i.e., pSLIK-Fos cells) results in significantly higher interactions within the A-type and B-type compartments (p < 2.2 x 10-16, Wilcoxon rank-sum test). In contrast, the inter-compartmental (between the A-type and B-type compartments) interactions were significantly decreased (p < 2.2 x 10-16, Wilcoxon rank-sum test) in pSLIK-Fos cells when compared to pSLIK-Gfp cells. (D) Gene ontology terms of differentially expressed transcripts whose coding regions have switched from open (A-type) to closed (B-type) compartmentalization. (E). Gene ontology terms of differentially expressed transcripts whose coding regions have switched from closed (B-type) to open (A-type) compartmentalization. Supplementary Figure S6. Related to Figure 6. Analysis of TAD boundaries. (A) A representative Hi-C heatmap at 40kb resolution for a 10 megabase genomic region (chr1:186,000,000-196,000,000) with insulation plots depicted on the bottom showing that most TADs are stable in pSLIK-Fos cells relative to pSLIK-Gfp cells. (B) Violin plot demonstrating similar TAD boundary strengths between pSLIK-Gfp and pSLIK-Fos datasets (p-value: Wilcoxon rank-sum test). Supplementary Figure S7. Related to Figure 6. Analysis of looping events. (A) Hi-C heatmap of pSLIK-Gfp and pSLIK-Fos datasets showing a reduction of a loop formation at the Myl1 gene locus, which is associated with a decrease in Myl1 gene expression (pSLIK-Fos vs. pSLIK-Gfp log2FC = -1.7). The z-scores of the looping interaction are depicted on the figure. (B) Overlap in MyoD-specific looping events in [38] that overlaps with FOS-specific looping events identified in this study.

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