ABSTRACT
Despite the fact that the negative regulatory element (NRE) within the upstream regulatory region of human IL-2 receptor alpha (IL-2Ralpha) gene has been identified two decades ago, mechanisms of the NRE function on the gene are hitherto unknown. In this paper, we report for the first time that the immunoglobulin transcription factor 2B (ITF2B) encoded by transcription factor 4 (TCF4) gene is a NRE binding protein. The full-length TCF4 cDNA clone was obtained from a HTLV-1 transformed human peripheral T cell MACHERMAKER cDNA library with NRE as the bait in yeast one-hybrid system. The NRE binding ability of ITF2B was further confirmed in chromatin-immunoprecipitation assay. Competitive RT-PCR-based promoter activity assay showed that over-expression of ITF2B protein inhibited the expression of IL-2Ralpha gene in Jurkat cells in an NRE-dependent manner. The function of ITF2B on the inhibition of both the IL-2Ralpha and the 5'LTR activity of HIV-1 shed light on the essence of NRE binding protein as a potential target for immune therapy and treatment in AIDS patients.
Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation/physiology , Receptors, Interleukin/genetics , Transcription Factors/physiology , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Blotting, Southern , DNA Primers , Electrophoretic Mobility Shift Assay , HIV Long Terminal Repeat , Humans , Immunoprecipitation , Interleukin-2 Receptor alpha Subunit , Jurkat Cells , Lymphocyte Activation , Molecular Sequence Data , Promoter Regions, Genetic , TCF Transcription Factors , Transcription Factor 4 , Transcription Factor 7-Like 2 ProteinABSTRACT
OBJECTIVE: To study the role of a BTB/POZ domain protein in the expression of hsp90alpha gene. METHODS: The eukaryotic expression plasmids of sense- and antisense-GAGA related protein (GRP) or empty vector were transfected into Jurkat cells with pREP4 episomal vector plasmids carrying the hsp90alpha promoter sequence from -1756 to +37 and control plasmids pMCAT. Total RNA was extracted. The relative promoter activity of hsp90alpha-CAT reporter gene was determined by competitive RT-PCR assay. RESULTS: GRP markly increased the relative promoter activity of hsp90alpha-CAT reporter gene during heat shock. CONCLUSION: GRP may promote the expression of hsp90alpha gene by participating in chromatin remolding.
Subject(s)
HSP90 Heat-Shock Proteins/genetics , Heat-Shock Response/genetics , Homeodomain Proteins/metabolism , Repressor Proteins/genetics , Transcription Factors/genetics , Amino Acid Motifs , Animals , Checkpoint Kinase 1 , Cloning, Molecular , DNA/metabolism , DNA, Complementary/metabolism , DNA-Binding Proteins/metabolism , Drosophila/genetics , Drosophila Proteins/genetics , Gene Expression Regulation , HSP90 Heat-Shock Proteins/biosynthesis , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Humans , Two-Hybrid System TechniquesABSTRACT
OBJECTIVE: To explore GAGA-like element binding protein in human cells. METHODS: Yeast one-hybrid system was used to screen the GAGA-like element binding proteins in HTLV-1 transformed Jurkat cell cDNA fusion library. Total RNA extracted from Jurkat cells was first labeled by reverse transcription, and was taken as cDNA probe to hybridize with the candidate positive clones. RESULTS: 9 positive clones were obtained, and 6 out of the 9 clones were positively hybridized with the cDNA probe. CONCLUSIONS: 6 candidate clones encoding for GAGA-like element binding proteins were obtained from Jurkat cells for further investigation.
