Fig. 1: SNAI2 is highly expressed in RMS and is regulated by a MYOD bound super enhancer.

a Violin plot showing expression (log₂FPKM) of SNAI2 across RMS and normal tissue. FPKM, Fragments Per Kilo base of transcript per Million mapped reads. b Representative western blot (n = 3 biologically independent experiments) of SNAI2 expression in different RMS cell lines. c Representative SNAI2 immunohistochemical staining in RMS primary tumors (14 positive of 19 FN-RMS and 3 positive of 4 FP-RMS) compared to normal muscle and Isotype control antibody. Scale Bar = 100 μM. d Topological interactions (Hi-C data from IMR90)¹⁷ of TAD containing the SNAI2 locus. e H3K27ac ChIP-seq data at SNAI2 TAD showing recurrent super enhancers (SEs) in a panel of FP-RMS (red), FN-RMS (blue) primary tumor samples and cell lines, myoblasts, myotubes, and skeletal muscle cells (yellow). Solid horizontal blocks show ___location of predicted super enhancers. f SNAI2 promoter and enhancer bound by MYOD and loaded with active histone mark H3K27ac in FN-RMS cell lines. * Previously reported MYOD peak near SNAI2²¹. RRPM, Reference-adjusted Reads Per Million Mapped Reads. g Topological interactions in SMS-CTR (HiChIP of H3K27ac) of SEs in the SNAI2 locus. h siRNA targeting MYOD1 was used to knock down MYOD expression in SMS-CTR cells. MYOD and SNAI2 expression was detected by western blot (top) and qRT-PCR (bottom). Data was normalized to cells treated with scramble siRNA (n = 3 biologically independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test, exact p values are reported in the figure). i Targeted disruption of MYOD binding sites in enhancers surrounding the SNAI2 gene in SMS-CTR cells, using sgRNAs to deliver dCas9-KRAB suppressor. Location of enhancers E1–E5 are shown above. Schematic of experimental workflow shown in bottom left. Bar chart of qRT-PCR measurements for SNAI2 expression after dCas9-KRAB perturbation with various guides is shown in the bottom right; p values shown were calculated among biological triplicates using t-test with Welch’s correction. Error bars represent the SD among the 3 biologically independent replicates.