Cockerill P N, Bert A G, Jenkins F, Ryan G R, Shannon M F, Vadas M A
Division of Human Immunology, Hanson Centre for Cancer Research, Institute for Medical and Veterinary Science, Adelaide, Australia.
Mol Cell Biol. 1995 Apr;15(4):2071-9. doi: 10.1128/MCB.15.4.2071.
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)9TCA 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.
人类粒细胞-巨噬细胞集落刺激因子基因的启动子受一个可诱导的上游增强子调控。该增强子包含转录因子NFAT的三个先前确定的结合位点（GM170、GM330和GM550）以及一个在此定义为GM420元件的新NFAT位点。虽然增强子内存在相当程度的冗余，但GM330、GM420和GM550基序各自作为增强子元件单独发挥作用效率很高，并以高度协同的方式结合NFATp和AP-1。这三个NFAT位点与远端白细胞介素-2的NFAT位点非常相似，甲基化干扰试验进一步确定GGA(N)9TCA为此类NFAT位点家族的最小共有序列。相比之下，GM170位点也有保守的GGA和TCA基序，但这些基序被15个碱基隔开，它支持AP-1和NFATp的强独立结合但不支持协同结合，并且该位点作为增强子元件功能较差。虽然GM330和GM420元件都与增强子内可诱导的DNase I超敏位点紧密相关，但GM420元件是唯一位于通过缺失分析定义为增强子核心的160 bp HincII-BalI片段内的NFAT位点。然而，GM420元件不同寻常之处在于，在TGACATCA序列的紧邻上游含有一个高亲和力的NFATp/c结合序列（TGGAAAGA），该序列与环磷酸腺苷反应样元件的相似性高于与AP-1位点的相似性。我们认为，NFATp/c和AP-1的协同结合需要特定的位点间距，并且它们通过独立途径的协同性和诱导作用确保了对粒细胞-巨噬细胞集落刺激因子增强子的非常严格的调控。
