ABSTRACT
Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8(III) enhancer, which directs expression in double-positive (DP) thymocytes, and E8(II), which is active in DP cells and CD8(+) T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8(III) enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8(III) in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8(II) is also deleted. These results indicate that E8(III) is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development.
Subject(s)
CD4 Antigens/biosynthesis , CD8 Antigens/biosynthesis , CD8 Antigens/genetics , Enhancer Elements, Genetic/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Thymus Gland/immunology , Thymus Gland/metabolism , Animals , Binding Sites/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line , Dendritic Cells/immunology , Dendritic Cells/metabolism , E-Box Elements/genetics , Gene Deletion , Gene Expression Regulation , Gene Targeting , Humans , Intestinal Mucosa/cytology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-myb/metabolism , T-Lymphocyte Subsets/cytology , Thymus Gland/cytologyABSTRACT
The sequence containing 'upstream Sal repeats' (USR) from the Arabidopsis thaliana ribosomal DNA intergenic region (IGR) was tested for its influence on the in vivo activity of nearby protein coding genes. On average, the presence of the IGR fragment leads to a four-fold increase in the expression of a reporter gene, beta-glucuronidase, under control of the strong CaMV 35S promoter. With the help of the site-specific cre-lox recombination system, we have also obtained pairs of transgenic lines with or without the USR-containing fragment, both integrated at the same chromosomal position. Results with these transgenic lines, which contain an NPT II (kanamycin resistance) gene under control of the nos promoter as a test gene, confirmed the results obtained with the CaMV 35S-driven GUS gene. Moreover, they show that the IGR sequence can oppose tendencies of gene silencing. We hypothesize that the described effect relates to features of the chromatin structure in the proximity of the upstream Sal repeats.
