Abstract
The T helper type 2 (Th2) locus control region (LCR) regulates Th2 cell differentiation. Several transcription factors bind to the LCR to modulate the expression of Th2 cytokine genes, but the molecular mechanisms behind Th2 cytokine gene regulation are incompletely understood. Here, we used database analysis and an oligonucleotide competition/electrophoretic mobility shift assays to search for transcription factors binding to RHS5, a DNase I hypersensitive site (DHS) within the Th2 LCR. Consequently, we demonstrated that GATA-binding protein-3 (GATA-3), E26 transformation-specific protein 1 (Ets-1), octamer transcription factor-1 (Oct-1), and Oct-2 selectively associate with RHS5. Furthermore, chromatin immunoprecipitation and luciferase reporter assays showed that Oct-1 and Oct-2 bound within the Il4 promoter region and the Th2 LCR, and that Oct-1 and GATA-3 or Oct-2 synergistically triggered the transactivational activity of the Il4 promoter through RHS5. These results suggest that Oct-1 and GATA-3/Oct-2 direct Th2 cytokine gene expression in a cooperative manner.
Highlights
CD4 T cells are important for mediating adaptive immune responses against various pathogens [1]
We used a database analysis, an electrophoresis mobility shift assay (EMSA), an oligonucleotide competition assay, and an antibodysupershift assay to show that GATAbinding protein-3 (GATA-3), E26 transformation-specific protein 1 (Ets-1), octamer transcription factor-1 (Oct-1), and Oct-2 bind to RHS5
Oct-1 bound to the Il4 promoter and the Th2 locus control region (LCR), and synergistically transactivated the Il4 promoter through RHS5 in combination with GATA-3 or Oct-2
Summary
CD4 T cells are important for mediating adaptive immune responses against various pathogens [1]. Each subset of cells secretes specific sets of cytokines and has its own function in immune responses [2]. The cytokine genes, Il4, Il13, and Il5, are located in close proximity to each other within a small region, termed the Th2 cytokine locus, in human chromosome 5 and mouse chromosome 11. These genes are expressed in Th2 cells, but are silenced in other cell types. Our EMSA data revealing that Oct-1/2 and GATA-3 bind in close proximity within the RHS5 sequence (Fig 2) prompted us to examine whether these transcription factors synergistically function in Il4 gene expression. These findings imply that Oct-1 impacts the transcriptional activity of the Il4 promoter through RHS5, and acts in synergy with GATA-3 or Oct-2
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