ABSTRACT
The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4(+)CD8(+) double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRalphabeta up-regulation and CD69 expression. LacZ expression remained high ( approximately 80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, approximately 50% of the mice developed thymic lymphoblastoid tumors that were CD4(+)CD8(+/low) and mostly CD3(+). In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69(low)HSA(low) CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8(+) T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Growth Inhibitors/biosynthesis , Lymphoma, T-Cell/immunology , T-Lymphocyte Subsets/cytology , Thymus Neoplasms/immunology , Trans-Activators/biosynthesis , Trans-Activators/genetics , Animals , CD2 Antigens/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Lineage/genetics , Cell Lineage/immunology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/physiology , Down-Regulation/genetics , Down-Regulation/immunology , GATA3 Transcription Factor , Gene Expression Regulation/immunology , Growth Inhibitors/antagonists & inhibitors , Growth Inhibitors/genetics , Growth Inhibitors/physiology , Humans , Locus Control Region/immunology , Lymph Nodes/pathology , Lymphoma, T-Cell/etiology , Lymphoma, T-Cell/pathology , Lymphopenia/genetics , Lymphopenia/immunology , Mice , Mice, Transgenic , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Spleen/pathology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Thymus Neoplasms/etiology , Thymus Neoplasms/pathology , Trans-Activators/antagonists & inhibitors , Trans-Activators/physiology , Transgenes/immunology , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.
