Hirofumi Kai

Myeloid ELF-1-like factor up-regulates lysozyme transcription in epithelial cells

Lysozyme is an important component of innate immunity against common pathogens at mucosal surfaces. We previously cloned and characterized the bovine lysozyme 5A (lys5A) promoter with the purpose of determining cis- andtrans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ETS consensus sequence located approximately at −46 to −40 bp from the transcription start site. The identity of the ETS-related protein responsible for gene transactivation was unknown. In this study, we screened six ETS-related proteins by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, the myeloid Elf-1-like factor (MEF) was the most potent. Gel shift analysis of epithelial cell nuclear extracts using a lys5A probe including the ETS-binding site (−50/−31) yielded a single band with retarded mobility. This band was supershifted by an antibody directed against MEF. Supporting the possibility that MEF is responsible for functional transactivation of lysozyme in epithelial cells, we found that antisense MEF mRNA decreased lys5A promoter activity and that MEF overexpression in stably transfected cells increased lysozyme mRNA and protein expression. We conclude that MEF is required for epithelial cell transactivation of lysozyme.

Transcriptional regulation of the lysozyme gene in airway gland serous cells

Lysozyme is expressed in serous, but not mucous, cells of the tracheobronchial glands and thereby constitutes a marker of the serous cell lineage in these glands. To identify DNA regulatory elements and transcription factors mediating the commitment of progenitor cells to the serous cell lineage, we have characterized the regulatory activity and DNA‐protein interactions of the 5′‐flanking region of the bovine lysozyme gene lys 5a. Results obtained from these studies indicate that although approximately 94 bp of 5′ flanking DNA are necessary for high level expression in transient transfection assays, an evolutionarily conserved promoter within 66 bp of the transcription start site is sufficient to confer serous cell‐specific expression. Farther upstream, within 6.1 kb of the 5′ flanking region, are 4 silencers. Analysis of the serous cell‐specific lysozyme promoter by electrophoretic mobility shift assay (EMSA) revealed the presence of binding sites for 3 serous cell nuclear proteins, designated LSF1, LSF2 and LSF3. Binding of LSF2 and LSF3 was localized to a 20‐mer subdomain (−50/−30) of the cell‐specific promoter using binding competition assays. More accurate identification of the protein binding site(s) was achieved through the use of mutagenesis, which implicated the motif 5′AAGGAAT 3′ (−46/−40) in both protein binding and serous cell‐specific transcriptional activity. This motif has previously been identified as a binding site for ets protein transcription factors, suggesting that serous cell‐specific regulation of lys 5a transcription is partly controlled by the binding of ets‐like protein(s) to the motif 5′AGGAAGT3′.