Fig. 5: Downstream BMP and RARA transcription factors cooperate to determine cell fate, following exposure to RA.
A Heatmap showing ChIP-seq peaks for RARA, SMAD4, and SMAD9 genome-wide in CHP-134 cells following treatment with DMSO control or 2 µM ATRA for 1 day. The color scale represents binding peak intensity, scaled between 0 and 5 (see Methods). Each genomic region (y-axis) was centered on the binding peak, displaying 1 kb upstream and downstream, sorted by average peak intensity across all transcription factors. Two biologically independent replicates are shown. B Venn diagram showing the number and proportion of overlapped binding regions between RARA, SMAD4, and SMAD9 in DMSO-treated CHP-134 cells. C GSEA plot showing the top 4 enriched pathways (defined in BioPlanet 2019) of SMAD9 bound genes that gain RARA peaks following 2 µM ATRA treatment for 1 day in CHP-134 cells (see Methods). Running Enrichment Scores (colored lines). The vertical-colored lines indicate the members of each gene set, ranked by baseline SMAD9 binding intensity (y-axis, bottom panel). P-values were calculated by a one-sided empirical permutation test. D Volcano plot showing the differentially expressed genes (red or blue points) after 1, 3, or 6 days of 2 µM ATRA exposure in CHP-134 cells. Differential expression cutoffs: log2 fold change > 1 or < -1 and FDR < 0.05. E Bar plot showing the percentage of up or down-regulated genes that are bound by SMAD4 and RARA at 1, 3, and 6 days (x-axis) following 2 µM ATRA exposure in CHP-134 cells. P-values indicate the enrichment of RARA and SMAD4 bound genes among the differentially expressed genes, calculated using a two-sided Fisher’s exact test. P-values from left to right are 3.78 × 10−8, 5.98 × 10−8, 7.53 × 10−16, and 1.60 × 10−8. F Bar plot showing the number of up or down (blue) regulated genes (y-axis), bound by both RARA and SMAD4, following at 1-, 3-, and 6-days exposure 2 µM ATRA. Genes are grouped by their functional annotation (see Methods) in processes related to apoptosis (apop), cell cycle (cycle), and differentiation (diff). G Bar blot showing the expression (y-axis) of selected BMP signaling pathway genes (x-axis) in CHP-134 cells treated with ATRA for 1, 3, or 6 days (colors). Data represents the mean ± SD of two independent replicates. H Top-ranked consensus motif enrichments in the RARA binding regions of 1-day ATRA-treated CHP-134 cells, which are all basic helix-loop-helix (bHLH) factors. I Western blots (upper panel) of c-PARP expression in wild-type (−) and ID2-overexpressed (+) CHP-134 cells following 3 days of ATRA treatment at concentrations indicated. A representative result of three independent experiments is shown. GAPDH was used as a loading control. J Viability of wild-type (WT) and ID2-overexpressing CHP-134 cells after ATRA treatment for 6 days. Data represents the mean ± SD of three independent replicates. P = 1.43 × 10−5 at the arrow indicated point (two-sided t-test). K A conceptual model for how cells with high or low BMP signaling are directed into different fates upon RA exposure. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are provided as a Source Data file.
