Human Granulocyte-Macrophage Colony-Stimulating Factor Enhancer Function Is Associated with Cooperative Interactions between AP-1 and NFATp/c

Abstract

The promoter of the human granulocyte-macrophage colony-stimulating factor gene is regulated by an inducible upstream enhancer. The enhancer encompasses three previously defined binding sites for the transcription factor NFAT (GM170, GM330, and GM550) and a novel NFAT site defined here as the GM420 element. While there was considerable redundancy within 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 resembled the distal interleukin-2 NFAT site, and methylation interference assays further defined GGA(N) 9 TCA as a minimum consensus sequence for this family of NFAT sites. By contrast, the GM170 site, which also had conserved GGA and TCA motifs but in which these motifs were separated by 15 bases, supported strong independent but no cooperative binding of AP-1 and NFATp, and this site functioned poorly as an enhancer element. While both the GM330 and GM420 elements were closely associated with the inducible DNase I-hypersensitive site within the enhancer, the GM420 element was the only NFAT site located within a 160-bp HincII-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 response-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 and that their cooperativity and induction via independent pathways ensure very tight regulation of the granulocyte-macrophage colony-stimulating factor enhancer.

A Distal Enhancer in the Interferon-γ (IFN-γ) Locus Revealed by Genome Sequence Comparison

“…Potentially, the method is capable of identifying gene regulatory elements that are located in distal intergenic regions or even in intronic regions of neighboring genes as shown for the CD4 gene and the imprinted Igfr2 gene (47,48). The correspondence of CNS regions, DNase I hypersensitive sites, and distal enhancer elements has been reported for several cytokine genes, including IL-4, IL-13, and GM-CSF (6,28,49,50), revealing a common theme in regulation of cytokine genes. Once the general conserved regions have been identified, further fine sequence comparisons can then be utilized to identify conserved binding sites for relevant transcription factors, especially if the sequences of multiple mammalian species are compared (Cartwheel program, cartwheel.caltech.edu/).…”

Partners in transcription: NFAT and AP-1

“…Upstream of the GM-CSF gene lies an enhancer element that controls GM-CSF expression and contains four NFAT sites, three of which support cooperative binding with AP-1 (Cockerill et al, 1995). The cell-type speci®city of GM-CSF expression di ers from that of IL-3: GM-CSF is expressed by lymphocytes as well as myeloid cells, while IL-3 is exclusively expressed by lymphoid cells.…”

Negative Regulation of the NFAT1 Factor by CD45: Implication in HIV-1 Long Terminal Repeat Activation

“…Since the transcription factor AP-1 has been known to be an important cooperating factor for NFAT (40,79), EMSA experiments were performed with a probe consisting of an AP-1-binding site (Fig. 4B).…”