Runx1 is a transcription factor essential for definitive hematopoiesis. In all 
vertebrates, the Runx1 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 runx1 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.