Journal: bioRxiv

Article Title: In vivo epigenetic editing of sema6a promoter reverses impaired transcallosal connectivity caused by C11orf46/ARL14EP neurodevelopmental risk gene

doi: 10.1101/491779

Figure Lengend Snippet: a Flow diagrams for immunoaffinity purification of C11orf46 followed by mass spectrometric analysis. b Eight clones of inducible HEK293 cell lines expressing full length 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 (C11orf46Δ) (clone 7 and 8) in the presence of doxycycline (Dox+). SETDB1 was consistently co-precipitated with full-length C11orf46, but not with C11orf46Δ. c Silver staining of proteins coprecipitated with affinity-purified wildtype C11orf46 (clone 10) or C11orf46Δ (clone 8). SETDB1, MCAF1 and KAP1 were detected in co-precipitate from full-length C11orf46, but not of C11orf46Δ. d Top scoring proteins co-precipitated with C11orf46 in cell lines overexpressed with C11orf46 (clone 10), C11orf46Δ (clone 8), and control cells are shown with % of amino acid sequence coverage, see Supplementary Table 1 for details. e Protein abundance (in femtomoles) in affinity-purified C11orf46 complexes. Notice prominence of SETDB1-MCAF1-KAP1 repressor proteins (black). Bar graphs indicate mean±S.E.M. n = 3 independent mass spectrometry quantification. f Schematic diagrams indicating position of C11orf46 arginine to histidine substitution at codon 236 (R236H) in highly 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, C11orf46Δ, or C11orf46 R236H showed that absence of CRD domain and R236H mutation is associated with massively weakened or non-detectable C11orf46-SETDB1 binding and partial loss of binding to other components of the complex. Notice that R236H mutation does not fully disrupt C11orf46-KAP1 binding. i R236H mutation and structural deletion of CRD at the C-terminal 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 (48hours after Dox treatment). l, m Silver staining of proteins co-precipitated with affinity-purified SETDB1. Both endogenous C11orf46 and SETDB1 binding partners such as MCAF1 and KAP1 were detected and verified by mass spectrometry. Top scoring proteins co-precipitated with SETDB1 in cell lines overexpressed with SETDB1 (clone 1) and control cells are shown with % of amino acid sequence coverage. For detail of SETDB1-binding proteins that were identified in these experiments, Supplementary Table 2 . n (left) C11orf46-SETDB1 co-immunoprecipitation in postmortem 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.

Article Snippet: For immunoblots, the following antibodies were used: rabbit monoclonal antibody against the C-terminus of human SETDB1 (Cell Signaling technology, Cat #2196S), KAP1 (cell signaling technology, Cat#4124S); rabbit polyclonal antibody against SETDB1 (Santa Cruz, Cat #sc-66884X), MCAF1 (Novus biological, Cat #NB100-438), Histone H3 C-terminus (Millipore, Cat #07-690).

Techniques: Immunoaffinity Purification, Clone Assay, Expressing, Silver Staining, Affinity Purification, Sequencing, Mass Spectrometry, Immunoprecipitation, Mutagenesis, Binding Assay