Preliminary Study On Screening Of IL-2R? Gene Negative Regulatory Element (NRE) Related Binding Proteins And Their Function

To explore NRE binding protein (NRE-BP) in human Jurkat cells, yeast one-hybrid systerm was used to screen the NRE-BP in HTLV-1 transformed human peripheral T cell Matchermaker cDNA fusion library. 11 positive clones were obtained, and 9 out of the 11 clones recovered His~+ and β-gal activity after transformed YMNRE reporter strain . All of the 9 clones positively hybridized with Jurkat cell cDNA probe. Sequence analysis and BLASTN, BLASTP analysis indicated that the positive clones cDNA were 98-99% homologues to proteins edcoded by TCF4 gene , TCF3 gene , ribosomal 3A, COUP family and HTLV-I pX. Protein edcoded by TCF4 and TCF3 also named E protein, which had typical bHLH domain and bound specific E box (CANNTG). TCF4 positive clone, which could bind specifically with NRE, were confirmed by electrophoretic mobility shift assay (EMSA). E protein had important impact on regulation of many kinds of gene expression.To study the effect of ITF2B protein on the IL-2Rα gene and HIV LTR gene expression, we use a competitive RT-PCR to quantify promoter activity of them, CAT reporter genes driven by 5' upstream sequences of human IL-2Rα gene or the regulatory fragment of HIV LTR was co-transfected with ITF2B cDNA expression plasmid into Jurkat cells followed by competitive RT-PCR based CAT assay. The results indicated that ITF2B protein inhibited the expression of IL-2Rα gene constitutively and after PHA activated when NRE and NIRS exsit. Truncation experiment showed that NRE element must exist when ITF2B inhibit the expression of IL-2Rα gene. Also ITF2B protein inhibited the expression of HIV gene, which worked most on chromatin level. The results also suggested the necessity of the research of gene regulation on chromatin level.We also constructed NIRS reporter plasmids used in yeast one-hybrid systerm, which can help further research on IL-2Rα gene NIRS binding protein.