The far upstream chicken lysozyme enhancer at -6.1 kilobase, by interacting with NF-M, mediates lipopolysaccharide-induced expression of the chicken lysozyme gene in chicken myelomonocytic cells

Abstract

Macrophages respond to lipopolysaccharide (LPS) with the activation of various genes, including the lysozyme gene. Here, we show that the level of lysozyme mRNA increases following treatment of chicken myelomonocytic HD11 cells with LPS. By transient and stable transfection of the chloramphenicol acetyltransferase (CAT) gene controlled by regulatory elements of the lysozyme gene, we identified a subfragment of the -6.1 kilobase (kb) lysozyme enhancer that mediates the LPS-induced lysozyme expression. This subfragment contains two elements (D and E), each of which matches the highly degenerate consensus sequence of binding sites for C/EBP-like transcription factors. Furthermore, we found protein complexes to interact with elements D and E whose binding activity to elements D and E is LPS-inducible in myelomonocytic HD11 cells. Immunomobility shift assays show that NF-M, a myeloid-specific C/EBP beta-related transcription factor is an essential component of these protein complexes. Mutations of the C/EBP binding sites within D and E cause a reduction of basal activity and abolish LPS responsiveness of the -6.1 kb lysozyme enhancer. These results show that the -6.1 kb lysozyme enhancer, in addition to its role in cell type-specific expression, can mediate, by interacting with NF-M, LPS-induced expression of the lysozyme gene in chicken myelomonocytic cells.