HUMAN GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ENHANCER FUNCTION IS ASSOCIATED WITH COOPERATIVE INTERACTIONS BETWEEN AP-1 AND NFATPC/

The promoter of the human granulocyte-macrophage colony-stimulating fa ctor gene is regulated by an inducible upstream enhancer. The enhancer encompasses three previously defined binding sites for the transcript ion factor NFAT (GM170, GM330, and GM550) and a novel NFAT site define d here as the GM420 element. While there was considerable redundancy w ithin the enhancer, the GM330, GM420, and GM550 motifs each functioned efficiently in isolation as enhancer elements and bound NFATp and AP- 1 in a highly cooperative fashion. These three NFAT sites closely rese mbled the distal interleukin-2 NFAT site, and methylation interference assays further defined GGA(N)(9)TCA as a minimum consensus sequence f or this family of NFAT sites. By contrast, the GM170 site, which also had conserved GGA and TCA motifs but in which these motifs were separa ted by 15 bases, supported strong independent but no cooperative bindi ng of AP-1 and NFATp, and this site functioned poorly as an enhancer e lement. While both the GM330 and GM420 elements were closely associate d with the inducible DNase I-hypersensitive site within the enhancer, the GM420 element was the only NFAT site located within a 160-bp HincI I-BalI fragment defined by deletion analysis as the essential core of the enhancer. The GM420 element was unusual, however, in containing a high-affinity NFATp/c-binding sequence (TGGAAAGA) immediately upstream of the sequence TGACATCA which more closely resembled a cyclic AMP re sponse-like element than an AP-1 site. We suggest that the cooperative binding of NFATp/c and AP-1 requires a particular spacing of sites an d that their cooperativity and induction via independent pathways ensu re very tight regulation of the granulocyte-macrophage colony-stimulat ing factor enhancer.