Fig. 7. CHD3 KD activates TWIST2-repressed myogenic genes in FN-RMS cells.

(A) Left: EdU (magenta) immunocytochemistry of RD cells infected with a shScramble control or shRNAs to knockdown CHD3 (shCHD3#1 or shCHD3#2) for 3 days followed by EdU incorporation for 4 hours. Nuclei were stained with DAPI (blue). Phase contrast images are shown below. Right: Quantification of the percentage of EdU⁺ nuclei. Scale bar, 50 μm. n = 3 biologically independent samples. (B) qRT-PCR analysis of indicated genes in RD cells infected with shScramble, shCHD3#1, or shCHD3#2 for 3 days (relative to shScramble, normalized to 18S rRNA). n = 3 biologically independent samples. (C) Model describing the mechanism of TWIST2 function in FN-RMS cells. In cycling cells, TWIST2 interacts with CHD3 at loci in myogenic genes to restrict H3K27ac deposition and turn off gene expression. TWIST2 also interacts with SMARCA4, and at growth gene loci, these proteins together enable H3K27ac deposition and activate gene expression. This is reversed in TWIST2 KD FN-RMS cells, where myogenic genes are activated fand growth genes are repressed, leading to cell cycle exit and terminal myogenic differentiation. All statistical comparisons between groups were evaluated by unpaired Student’s t test, ****P < 0.0001, ***P < 0.001, and **P < 0.01. Data are presented as the mean ± SEM.