Analysis of chicken LGALSL (galectin-related protein) gene's proximal promoter and its control by Krüppel-like factors 3 and 7

Abstract

The Galectin-Related Protein (GRP), encoded by the LGALSL gene, assigned to the protein family of β-galactoside-binding Galectins, has lost carbohydrate-binding abilities. Its chicken homolog (C-GRP) occurs in the bursa of Fabricius' epithelial and B cells. Our study investigates the unknown regulatory mechanisms controlling its expression by analyzing the promoter region of the chicken (C-)LGALSL gene in chicken cells. We aimed to identify the sequence elements of the C-LGALSL gene promoter responsible for maximum activity and transcription factors (TFs) that can modulate this activity. Using luciferase reporter assays, we investigated deletion variants of the 5' region (-2480 bp to +26 bp). Through in silico analyses and site-directed mutagenesis, we explored potential transcription factor binding sites, identified crucial transcription factors through transient overexpression and tested its direct binding by ChIP. Our findings highlight that the region from -274 to -75 bp, conserved among bird species, is crucial for promoter regulation. Among other tested factors, only the chicken (ch) Krüppel-like factors, chKLF3 and chKLF7, modulate the promoter's activity. The TFs chKLF3 acts as a repressor, and chKLF7 as an activator, although direct binding could not be confirmed. In conclusion, chKLF3 and chKLF7 contribute, in contrast to other factors with binding sites in the region from -274 to -75 bp, to C-LGALSL gene promoter regulation with a balanced impact on activity.

Keywords: Galectin; Galectin-related protein; Krüppel-like factor; Promoter; Regulation; Transcription factor.