Fig. 4: dCENP-A binds directly to Spt6 and is affected by mutating phosphoresidues.
a Drosophila Spt6 domain organization based on Pfam73: acidic (red), Helix-turn-Helix (green), YqgF/RNaseH-like domain (purple), Helix-hairpin-Helix (yellow), S1 RNA-binding domain (magenta), tandem SH2 (orange). Corresponding histone and RNAPII-binding domains based on S. cerevisiae39,63. b Alignment of the histone-binding domain of S. cerevisiae Spt6 (residues 239–314; blue) based on McDonald et al.39 with the bacterially expressed fragment of Drosophila Spt6 (199–338; red). Alignments were performed on Uniprot using the Clustal Omega program74. Asterisk indicates fully conserved residues, colon indicates strongly similar residues, and period indicates weakly similar residues. c Pull-down experiments of purified recombinant 6his-smt3-Spt6 (199–338) or 6his-smt3 as a negative control with recombinant H3/H4 or dCENP-A-ΔNT (101–225) are shown on a Coomassie-stained SDS-PAGE. N = 3 independent experiments. d Western blot (α FLAG, α Spt6) showing co-IPs of Spt6 with WT dCENP-AFLAG and dCENP-AFLAG bearing phosphorylation-abolishing (S to A) or phosphomimetic (S to D) mutations at phosphorylation sites S20, S75 and S77. N = 5 independent experiments. WT wildtype, FT flowthrough; IP immunoprecipitate. dC-A dCENP-A. e Western blot (α FLAG, α Spt6, α H3) showing co-IPs of endogenous Spt6 (two bands) with dCENP-AFLAG (top) or dCENP-AFLAG (S20/75/77A) (bottom) and H3 exposed to low (150 mM) and high (750 mM) salt wash conditions. N = 5 independent experiments. Source data are provided as a Source Data file.
