Identification and characterization of a chicken major histocompatibility complex class II[beta] gene promoter

Abstract

<p>The major histocompatibility complex (MHC) contains a variety of genes. Among them, the class II genes encode proteins involved in antigen presentation to helper T cells, a key step in initiating immune responses. The regulation of class II gene expression has been extensively studied in mammals. Such studies have been lacking in the avian species, despite the well-established association of the avian MHC with disease resistance and production traits. The objective of this study was to functionally analyze a putative chicken MHC class II gene promoter;Using the chloramphenicol acetyltransferase (CAT) reporter system, a functional chicken MHC class II[beta] gene promoter has been identified. A 0.7 kb DNA fragment from the 5[superscript]' flanking region of a class II gene was cloned into the vectors upstream of the CAT structural gene, and transfected into the MQ-NCSU chicken macrophage cell line that expresses MHC class II antigens. Three transfection methods were evaluated to establish a system for DNA transfection into the cell line. The calcium phosphate method had the highest transfection efficiency. The CAT results indicated that the 0.7 kb chicken DNA fragment contains a functional promoter. Promoter activity was relatively weak, which may be explained by the low level of MHC class II expression in this cell line, or by a requirement for additional enhancer sequences. Deletion analysis of this 0.7 kb DNA revealed a short fragment in the 3[superscript]' end that was crucial for the promoter function, and identified negative regulatory elements further upstream. Deletion of the conserved MHC class II X and Y boxes did not have a significant influence on promoter activity. Sequence analysis of the 0.7 kb class II gene upstream region suggested possible involvement of interferon, ETS-related proteins, and other factors in regulating this promoter. An interferon-rich chicken T cell line culture supernatant increased surface expression of MHC class II antigens and class II[beta] promoter activity in this macrophage cell line. This induction effect was inhibited by glucocorticoids. This first functional characterization of a chicken MHC class II gene promoter will aid in understanding the regulatory mechanisms that control the expression of these immune function-related genes.</p>