Transcriptional activation of Il36A by C/EBPβ via a half-CRE*C/EBP element in murine macrophages is independent of its CpG methylation level

Abstract

Interleukin-36(Il-36) is a member of the novel Il-1-like proin-flammatory cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types. We have recently shown that CCAAT enhancer binding protein{beta} (C/EBP{beta}) binds specifically to an essential half cAMP response element (half-CRE)*C/EBP motif in the Il36A promoter to induce Il36A expression upon LPS stimulation. C/EBPs are transcription factors belonging to the basic leucine zipper (bZIP) family of transcriptional regulators. C/EBP proteins can form homo- and heterodimers and regulate gene expression by binding to C/EBP specific recognition sequences and composite sites that can contain 5'-cytosine-phosphate-guanine-3'dinucleotides (CpG). CpG methylation of such elements has been shown to influence transcription factor binding and gene expression. Here we show that the half-CREoC/EBP element in the Il36A promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. By using electrophoretic mobility gel shift and fluorescence polarization assays we demonstrate that C/EBP{beta} binding to the half-CRE*C/EBP element in the Il36A promoter following LPS stimulation is insensitive to CpG methylation. Transfection assays also show that methylation of the CpG in the half-CRE*C/EBP element does not alter LPS-induced Il36A promoter activity. A direct comparison of Il36A mRNA copy numbers as well as the pro-Il-36 protein level in RAW264.7 and primary macrophages revealed similar amounts in both cell types. Taken together, our data suggest that C/EBP{beta} binding to the half-CRE*C/EBP element and C/EBP{beta} mediated gene activation occurs independently of the CpG methylation status of the target DNA sequence and underline the potential of C/EBP{beta} to recognize methylated as well as unmethylated binding sites.