RUNX3 Regulates Intercellular Adhesion Molecule 3 (ICAM-3) Expression during Macrophage Differentiation and Monocyte Extravasation
Figure 2
Identification and characterization of RUNX and C/EBP-binding elements within the ICAM-3 gene proximal regulatory region.
A. EMSA was performed on the indicated oligonucleotides spanning the −157/−14 region of the ICAM-3 promoter using nuclear extracts from THP-1, K-562 and Jurkat cells. The position of the major retarded species is indicated. B. EMSA was performed on the ICAM3.3 and ICAM3.5 oligonucleotides using nuclear extracts from the indicated COS-7 cells transfected with an empty expression vector (pCDNA3) or with either RUNX1 or RUNX3 together with CBF-β expression vector. The position of the RUNX1- and RUNX3-containing complex is shown. C. EMSA was performed on the ICAM3.5 and ICAM3.3 oligonucleotides using nuclear extracts from Jurkat cells in the absence (−) or presence of unlabeled competitor oligonucleotides (ICAM3.5, ICAM3.5mutRUNX, ICAM3.3, ICAM3.3mutRUNX, AMLcons) or polyclonal antisera against CD209 (Control antibody, Cnt Ab) or RUNX1 proteins (R-3034). The position of RUNX1-containing complexes are shown. Unlabeled competitor oligonucleotides were added at a 100-fold molar excess. D. EMSA was performed on the ICAM3.4 oligonucleotide using nuclear extracts from THP-1 cells in the absence (−) or presence of unlabeled competitor oligonucleotides (ICAM3.4, ICAM3.4mutCEBP, C/EBPcons) or polyclonal antibody against CD209 (Control antibody, Cnt Ab) or C/EBPα proteins (α-C/EBPα). The position of C/EBPα-containing complexes are shown. Unlabeled competitor oligonucleotides were added at a 100-fold molar excess. In A–D, EMSA's were performed twice with similar result and a representative experiment is shown. E. ICAM-3 promoter-based oligonucleotides with mutated nucleotides in lowercase and their relative positions. F. In vivo occupancy of the ICAM-3 promoter by RUNX1. Chromatin immunoprecipitation on Jurkat cells was performed with an affinity-purified polyclonal antisera specific for RUNX1 or purified rabbit IgG. Immunoprecipitated chromatin was analyzed by PCR using a pair of ICAM-3 promoter-specific primers that amplify a 234-bp fragment flanking the RUNX-binding sites at −80 and −29. ChIP experiment was performed twice with similar results, and a representative experiment is shown.