Runx1 is a transcription factor essential for definitive hematopoiesis. In all vertebrates, the Runxl gene is transcribed from two promoters: a proximal promoter (P2), and a distal promoter (P1). We previously found that runx1 expression in a specific hematopoietic cell population in zebrafish embryos depends on cohesin. Here we show that zebrafish runxl is directly bound by cohesin and CCCTC binding factor (CTCF) at the P1 and P2 promoters, and within the intron between P1 and P2. Cohesin initiates expression of runx1 in the posterior lateral mesoderm and influences promoter use, while CTCF represses its expression in the newly emerging cells of the tail bud. The intronic binding sites for cohesin and CTCF coincide with histone modifications that confer enhancer-like properties, and two of the cohesin/CTCF sites behaved as insulators in an in vivo assay. The identified cohesin and CTCF binding sites are likely to be cis-regulatory elements (CREs) for runx1 since they also recruit RNA polymerase II (RNAPII). CTCF depletion excluded RNAPII from two intronic CREs but not the promoters of runx1. We propose that cohesin and CTCF have distinct functions in the regulation of runx1 during zebrafish embryogenesis, and that these regulatory functions are likely to involve runx1 intronic CREs. Cohesin (but not CTCF) depletion enhanced RUNX1 expression in a human leukemia cell line, suggesting conservation of RUNX1 regulation through evolution. (C) 2013 Elsevier B.V. All rights reserved.