Figure 7. Phf5a loss leads to Paf1C destabilization and inhibits myogenic differentiation.

(a) Western blot analysis of myoblast self-renewal and myotube differentiation markers (myosin heavy chain and Pax7, respectively) following shControl or shPhf5a knockdown (see Supplementary Figure 7). (b) Schematic of Tet-inducible Rosa26^rtTACol1a1^TREshRNA animals for the derivation of primary myoblasts. Addition of doxycycline drives expression of shPhf5a from the Col1a1 locus. LSL: LoxP-stop-LoxP cassette. (c and d) Myosin heavy chain (MHC) immunofluorescence on primary myotubes purified from Rosa26^rtTACol1a1^TREshRNA animals (c) and CRISPR-Cas9-mediated Phf5a silencing on C2C12 cells (d), respectively, depicting suppression of myoblast differentiation. Scale bars, 100 µm. (e) Western blot analysis of Phf5a on primary myotubes purified from Rosa26^rtTACol1a1^TREshRNA animals. Addition of Doxycyclin induces shRNA hairpin expression and the silencing of Phf5a (see Supplementary Figure 7). (f) Venn diagram of Leo1-bound genes in myoblasts and myotubes following ChIP-sequencing. (g) Genome browser tracks showing peaks of Leo1 ChIP-sequencing for representative genes in myoblasts and myotubes. Histone-1 cluster genes, Myog, Myo1c, Myom3 and Ubc are shown as examples. (h) Venn diagram of Leo1 bound genes in myotube differentiation in the presence or absence of Phf5a following ChIP-sequencing. (i) Genome browser tracks showing peaks of Leo1 ChIP-sequncing for representative genes in shControl and shPhf5a conditions, respectively. Myog, and several olfactory, taste and smell receptors and G-protein coupled receptors, ion channels and neurotransmitter receptors are shown as examples. (j) Western blot analysis of Paf1C subunit composition using immunoprecipitations in C2C12 cells differentiated for 72h following knockdown with shControl or shPhf5a, respectively. A significant loss among Paf1C subunit interactions is observed upon Phf5a silencing (see Supplementary Figure 7).