Interaction of RUNX1 and TAL1. Affinity purification of bio-tagged TAL1 reveals TAL1 interaction with RUNX1. (A) Experimental flow scheme. Bio-tagged TAL1 and bound interaction partners were affinity purified and identified by SILAC-based MS analysis. RUNX1 and the RUNX1 heterodimerization partner core binding factor β (CBFbeta) were identified as part of the TAL1 interactome. (B) MS-spectrum of the “heavy” SILAC-labeled triply charged peptide ASLNHSTAFNPQPSQMQDTR10 (m/z = 795.37) derived from RUNX1; the “light” version of that peptide (ASLNHSTAFNPQPSQMQDTR; m/z = 792.03) showed at least a 10-fold decreased intensity. (C) Interaction of endogenous RUNX1 with TAL1. TAL1 was immunoprecipitated from K562 cell lysates with an anti-TAL1 antibody (IP). Coprecipitated RUNX1 was visualized with an anti-RUNX1 antibody (CoIP). (D) Coimmunoprecipitation of transfected HA-tagged RUNX1 with Flag-tagged TAL1 in HEK-293 cells. Flag-tagged TAL1 was coexpressed with hemagglutinin (HA)-tagged RUNX1. Flag-TAL1 was immunoprecipitated using anti-FLAG-beads (IP), and coprecipitation of RUNX1 was visualized using an anti-HA-tag antibody (CoIP) (left). The corresponding input controls are shown (right). (E) GST pulldown with recombinant GST-TAL1 and in vitro translated ³⁵S-labeled full-length RUNX1. (F) Mapping of the interaction domain of TAL1 with RUNX1 using different recombinant GST-TAL1 deletion constructs and ³⁵S-labeled full-length RUNX1. (G) Mapping of the interaction domain of RUNX1 with TAL1 using different recombinant GST-RUNX1 deletion constructs and ³⁵S-labeled full-length TAL1. (H) ChIP-ReChIP shows coocupancy of TAL1 with RUNX1 at the KLF1 promoter. ChIP-ReChIP was performed with the sequential use of the given antibodies. Semiquantitative ChIP-PCR was performed with a primer pair close to the KLF1 transcription start site with 40 PCR cycles. (I) Schematic representation of TAL1 and RUNX1. The regions involved in the interaction are marked. AD, activation domain; bHLH, basic helix loop helix; RHD, runt domain.