Constitutive Expression of Murine Decay-Accelerating Factor 1 Is Controlled by the Transcription Factor Sp1

Abstract

The complement regulatory protein decay-accelerating factor (DAF or CD55) protects host tissue from complement-mediated injury by inhibiting the classical and alternative complement pathways. Besides its role in complement regulation, DAF has also been shown to be a key player in T cell immunity. Modulation of DAF expression could therefore represent a critical regulatory mechanism in both innate and adaptive immune responses. To identify and characterize key transcriptional regulatory elements controlling mouse Daf1 expression, a 2.5-kb fragment corresponding to the 5′ flanking region of the mouse Daf1 gene was cloned. Sequence analysis showed that the mouse Daf1 promoter lacks conventional TATA and CCAAT boxes and displays a high guanine and cytosine content. RACE was used to identify one major and two minor transcription start sites 47, 20, and 17 bp upstream of the translational codon. Positive and negative regulatory regions were identified by transiently transfecting sequential 5′deletion constructs of the 5′flanking region into NIH/3T3, M12.4, and RAW264.7 cells. Mutational analyses of the promoter region combined with Sp1-specific ELISA showed that the transcription factor Sp1 is required for basal transcription and LPS-induced expression of the Daf1 gene. These findings provide new information on the regulation of the mouse Daf1 promoter and will facilitate further studies on the expression of Daf1 during immune responses.

A functional SNP in the regulatory region of the decay-accelerating factor gene associates with extraocular muscle pareses in myasthenia gravis

“…LPS-induced upregulation of the regulatory activity and the expression of Daf1 were shown in both murine lymphocyte and macrophage cell lines but not in fibroblasts, suggesting tissue specificity of LPS-induced DAF upregulation. 28 The LPS-induced DAF upregulation in these mouse cells were predominantly dependent on the two Sp1 sites most proximal to the transcription start site, 28 excluding the herein described putative Sp1 site lost with the SNP. Although our studies in human control lymphoblasts showed the expected LPS treatment-induced DAF expression, the lymphoblast containing the c.-198C4G SNP showed a reduction in gDAF mRNA and protein levels after LPS stimulation.…”

“…Transfection of the Mut DAF 5 0 -flanking region (containing a mutation mimicking the SNP) cloned into a luciferase reporter vector shows the SNP to be functional and capable of driving luciferase expression in three different cell lines under basal conditions. Similarly, absolute DAF mRNA expression in EBVtransformed lymphoblasts from two individuals with 28 Two of three Sp1 sites in the Daf1 promoter ( þ 1 to À619) were critical for the basal Daf1 promoter activity in several cell lines, whereas the remaining non-critical Sp1 site that equates with the Sp1 site lost in the human c.-198C4G SNP did not influence basal transcription. 28 Interestingly, deletion of the latter Sp1 site in mice also resulted in the activation of Daf1-driven luciferase activity in mouse B cells.…”

“…Similarly, absolute DAF mRNA expression in EBVtransformed lymphoblasts from two individuals with 28 Two of three Sp1 sites in the Daf1 promoter ( þ 1 to À619) were critical for the basal Daf1 promoter activity in several cell lines, whereas the remaining non-critical Sp1 site that equates with the Sp1 site lost in the human c.-198C4G SNP did not influence basal transcription. 28 Interestingly, deletion of the latter Sp1 site in mice also resulted in the activation of Daf1-driven luciferase activity in mouse B cells. 28 Several studies have shown that bacterial LPS, a major component of Gram-negative bacterial cell walls, upregulates DAF expression (Cauvi et al 28 ).…”

“…28 Interestingly, deletion of the latter Sp1 site in mice also resulted in the activation of Daf1-driven luciferase activity in mouse B cells. 28 Several studies have shown that bacterial LPS, a major component of Gram-negative bacterial cell walls, upregulates DAF expression (Cauvi et al 28 ). LPS-induced upregulation of the regulatory activity and the expression of Daf1 were shown in both murine lymphocyte and macrophage cell lines but not in fibroblasts, suggesting tissue specificity of LPS-induced DAF upregulation.…”

“…25 The 'minimal DAF promoter' or the 5 0 -flanking region has several regulatory regions that show functional variation in different cell lines, suggesting tissue specificity in transcriptional regulation. [26][27][28] Furthermore, there is increasing evidence of single nucleotide polymorphisms (SNPs) in regulatory elements influencing variation in gene expression and susceptibility to complex diseases. 29 On the basis of these observations, we hypothesize that DAF expression may be lower than normal or not properly upregulated during 'inflammatory stress,' resulting in inadequate complement protection in the EOMs of a subgroup of individuals with MG. We screened the DAF gene sequence of subjects with the severe EOM phenotype focusing initially on exons encoding the regions important in the regulation of the classical complement pathway, and second on the DAF gene regulatory sequence, which would influence gene expression.…”