Fig. 3
C11orf46 binds to the SETDB1 complex. a Flow diagrams for immunoaffinity purification of C11orf46 followed by mass spectrometric analysis. b Eight clones of inducible HEK293 cell lines expressing FLAG-tagged C11orf46 (clone 1–5 and 10) and a truncated form C11orf46 lacking amino acids 209–237 at the C-terminal region which corresponds to cysteine rich domain (CRD) of C11orf46 (C11orf46∆) (clone 7 and 8) in the presence of doxycycline (Dox+). SETDB1 was co-precipitated with C11orf46, but not with C11orf46∆. c Silver staining of proteins co-precipitated with C11orf46 (clone 10) or C11orf46∆ (clone 8). SETDB1, MCAF1 and KAP1 were detected in co-precipitate from C11orf46, but not of C11orf46∆. d Top scoring proteins co-precipitated with C11orf46 in cell lines overexpressed with C11orf46, C11orf46∆, and control cells are shown with % of amino acid sequence coverage. e Protein abundance in affinity purified C11orf46 complexes. Notice prominence of SETDB1-MCAF1-KAP1 repressor proteins (black). Bar graphs indicate mean ± S.E.M. n = 3 independent quantification. f Schematic diagrams indicating position of C11orf46 arginine to histidine substitution at codon 236 (R236H) in conserved cysteine rich domain (CRD; amino acids 209–237) between human and mouse. g, h Co-immunoprecipitation experiments using protein lysates from HEK293 cells overexpressing FLAG-tagged C11orf46, C11orf46∆, or C11orf46R236H showed that absence of CRD domain and R236H mutation is associated with weakened or non-detectable C11orf46-SETDB1 binding and partial loss of binding to other components. R236H mutation does not fully disrupt C11orf46-KAP1 binding. i R236H mutation and CRD deletion of C11orf46 do not affect binding to histone H3. j Flow diagrams for immunoaffinity purification of SETDB1 followed by mass spectrometric analysis. k Inducible SETDB1 expression system in HEK293 cell lines (48 h after Dox treatment). l, m Silver staining of proteins co-precipitated with SETDB1. Both endogenous C11orf46 and SETDB1 binding partners were detected by mass spectrometry. Top scoring proteins co-precipitated with SETDB1 in cell lines overexpressed with SETDB1 and control cells are shown. n (left) C11orf46-SETDB1 co-immunoprecipitation in human cerebral cortex. (right) SETB1 co-immunoprecipitates include C11orf46, HP1γ, and MCAF1 in HeLa nuclear extract. o Schematic summary of above data representing working model of C11orf46-SETDB1 complex in relation to histone H3
