Structure function-analysis of DUX by 2C-like cell conversion.
a. (Top) Schematic of constructs used for flow cytometry: mCherry-tagged FL, ΔC12345+14aa, C1+14aa, C2+14aa, C3+14aa, C4+14aa, and C5+14aa. (Bottom) Flow cytometry for MERVL::GFP reporter given mCherry expression in Dux-/- mESCs with 18hr overexpression of indicated constructs. *p-value < 0.05, student’s t-test. (n=3 biological replicates). Constructs with a single black line have the linker GGGGS2 and constructs with two black lines have linkers with GGGGS2 and GAGAS2, respectively.
b. (Top) Schematic of constructs used for flow cytometry: mCherry-tagged FL, C3+14aa, C3Δ14aa, C5+14aa, C5Δ14aa. (Bottom) Flow cytometry for MERVL::GFP reporter given mCherry expression in Dux-/- mESCs following 18hr expression of indicated constructs. *p-value < 0.05, student’s t-test. (n=3 biological replicates)
c. (Top) DUX domain chimera construct design for C1-C3 fusions with (top) schematic of constructs and (bottom) specific chimera cut-offs for C1-C3 a,b,d (pink to blue) and C3-C1 a,b,c (blue to pink) constructs. (Bottom) Schematic of DUX domain chimera constructs used for flow cytometry: mCherry-tagged full-length DUX (FL), C1C3a, C1C3b, C1C3c, C3C1a, C3C1b, C3C1c. Symbols adjacent to alignments: red circle denotes transcriptionally inactive repeats and the green circle denotes transcriptionally active repeats. (Right) Flow cytometry for MERVL::GFP reporter given mCherry expression in Dux-/- mESCs folllowing 18hr expression of indicated constructs. *p-value < 0.05, student’s t-test. (n=3 biological replicates)
d. Flow cytometry for MERVL::GFP reporter given mCherry expression in Dux-/- mESCs following 18hr expression of point mutation constructs: (Top) FL-DUX, C3+14aa, C3 D438E+14aa, C3 D438G+14aa, and C3F443L+14aa. (Bottom) FL-DUX, C1+14aa, C3+14aa, C1DPLELF+14aa (substation of C3 amino acids into C1+14aa), and C3GPLELL+14aa (substitution of C2 and C4 6aa sequence into C3+14aa). *p-value < 0.05, student’s t-test. (n=3 biological replicates)
e. Principle component analysis (PCA) of RNA-seq analysis for 18hr overexpression of DUX domain chimera constructs, full-length DUX, and mCherry alone (n=2). Colored squares beside sample legends denote the inability to activate MERVL::GFP reporter (red) or ability to activate MERVL::GFP reporter (green).
f. Heat map of RNA-seq at DUX target genes (n=456) from 18hr expression of DUX domain chimera constructs, full-length DUX, and mCherry alone (n=2). Colored rectangles above samples denote inability to activate MERVL::GFP reporter (red) or ability to activate MERVL::GFP reporter (green).