Analysis of Il36a induction by C/EBP\u03b2 via a half-CRE\u2022C/EBP element in murine macrophages in dependence of its CpG methylation level

Nina Janze,Ralph Goethe,Andreas Nerlich

doi:10.1038/s41435-021-00153-5

Abstract

Interleukin-36α is a novel member of the IL-1 cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types after induction. The transcription factor (TF) C/EBPβ binds specifically to an essential half-CRE•C/EBP motif in the Il36a promoter to induce Il36a expression upon LPS stimulation. C/EBPs regulate gene expression by binding to recognition sequences that can contain 5′-cytosine-phosphate-guanine-3′ dinucleotides (CpG), whose methylation can influence TF binding and gene expression. Herein we show that the half-CRE•C/EBP element in the Il36a promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. We demonstrate that C/EBPβ binding to the half-CRE•C/EBP element in the Il36a promoter following LPS stimulation is insensitive to CpG methylation and that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36a promoter activity which correlated with similar Il36a mRNA copy numbers and pro-IL-36α protein amount in both cell types. Taken together, our data indicate that C/EBPβ binding to the half-CRE•C/EBP element and subsequent gene activation occurs independently of the CpG methylation status of the half-CRE•C/EBP motif and underlines the potential of C/EBPs to recognize methylated as well as unmethylated motifs.

Highlights

The interleukin (IL)-36 cytokines constitute a subfamily of the IL-1 cytokine family and consist of three agonistic cytokines (IL-36α, IL36β IL-36γ), and the IL-36 receptor antagonist (IL-36RA) that play important roles in host immunity [1]
Similar to the receptor for IL-1, the IL-36 receptor recruits the transmembrane ILl-1 receptor accessory protein upon ligand binding that subsequently activates intracellular signaling via mitogen-activated protein kinases and nuclear factor-κB (NF-κB) activated transcription leading to inflammatory responses [10,11,12]
We have previously shown that LPS-induced Il36a mRNA was assessed with different approaches

Summary

Introduction

The interleukin (IL)-36 cytokines constitute a subfamily of the IL-1 cytokine family and consist of three agonistic cytokines (IL-36α, IL36β IL-36γ), and the IL-36 receptor antagonist (IL-36RA) that play important roles in host immunity [1]. DNA methylation of gene promoters was considered to silence transcription either directly by inhibiting binding of certain transcription factors (TF) to their binding sites [14,15,16,17] or indirectly by binding of TFs containing a methyl-CpG (mCpG)-binding domain (MBD), that in turn recruits histone deacetylases which subsequently promote local chromatin condensation [18]. Examples include transcriptional activators that contain a C2H4 zinc-finger domain like Kaiso and Krueppel-like factor 4 [19, 20], proteins with a helix-turn-helix DNA-binding domain like Zhx1/2 and Pax6 [21, 22], helix-loop-helix domaincontaining TFs like c-MYC and ARNT2 [23, 24], and TFs belonging to the family of bZIP proteins like C/EBPs [25, 26]

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