1. CD4 is a transmembrane glycoprotein expressed on the cell surface of thymocytes and mature T- lymphocytes with helper function. T-cells develop in the thymus and their stages of development are defined by the level of CD4 and CD8 expression. Briefly, T-cell progenitors enter the thymus as DN cells, expressing no CD4 or CD8 on their cell surface. Upon successful rearrangement of TCR  they develop into DP thymocytes, which express both CD4 and CD8. Finally, positively selected DP thymocytes differentiate into CD4 or CD8 single positive cells. The DP enhancer is necessary for the expression of CD4 protein in DP cells. We speculate that there could be a different, unknown promoter region that is “turned on” by this DP enhancer instead of or in addition to the promoter used for CD4 expression in mature T-helper cells. The diagram below shows the Cd4 gene and the transcriptional elements that regulate CD4 expression. It also shows how a potential secondary promoter could express the same protein. A Potential Second Promoter in the Cd4 Gene that Functions at the Double Positive Stage of Development Walker Shaw and Sophia Sarafova Biology Department, Davidson College, Davidson, NC 28036 Background Information We identified a second transcription start site for the murine Cd4 gene, located in the first intron, that functions at the DP stage of T cell development. We think it may represent a second promoter in the Cd4 gene, similar to the one described in humans. To confirm the presence, identify, and understand the function of this transcriptional control element we will Repeat current work with a FACS sorted pure population of DP cells. Repeat the analysis using CD4 SP cells to determine if the alternate transcription start site is unique to DP cells. Purify and sequence the DNA segments from the 5’ RACE to determine the potential alternate transcription start site(s) and help look for a potential promoter. Identify the putative second promoter of the Cd4 gene by transient transfection into AKR1G1 double positive cell line of constructs containing DNA from upstream of the newly identified transcription start site. Results Our FACS stain data showed that PNA panning of thymocytes decreased the CD4 cell contamination by 78% (compare A and B) yielding a 95% pure DP cell population (B), which was used for RNA isolation and 5’ RACE. Cd4 gene transcription products were visualized by RT followed by Rapid Amplification of cDNA 5’ Ends (5’ RACE) and agarose gel electrophoresis (C). Normally, a 320bp product is expected if the known Cd4 promoter is used in the DP cells. Products of larger size are unexpected and can be explained only if Cd4 transcription is initiated from a different promoter in DP cells (occurring in intron 1). We observed both the 320bp and a larger product (lanes 3 and 4 gray and black arrows), indicating that an additional transcription initiation site exists in DP cells. The bands in lanes 1 and 2 represent DNA contamination. Conclusions and Future Work 84.37% 6.91% 95.58% 1.52% NO cDNA (Control) NO RT (Control) Liquid RT RT on Beads 320bp Methods Thymus cells CD8 antibody Tagged with FITC CD4 antibody Tagged with PE TTTTTTT-3’ 3’-AAAAAAA-----5’ 5’-primerTTTT 3’AAAA------5’ 5’-primer TTTT------------CCC-3’ PNA Cell Panning Cells FloatCells Stick to Plate Immunostaining of Cells Check purity By FACS Isolate Panned Cells’ RNA with Trizol® Reserve for FACS staining mRNA bound mRNA in solution Bind mRNA to BEADS 5’ RACE on Beads 5’ RACE In Solution CD4 Locus … Known mRNA Product Ex1SilEx2 ATG Ex3Ex4Ex5 ? TATA Ex1Ex2 ATG Ex3Ex4Ex5 Potential DP mRNA Product Ex2 ATG Ex3Ex4Ex5 CD4 … … … PDPe 5’-primer TTTT------------------CCC-3’ exon3----capfinder 3’-GGG-capfinder 5’ primer TTTTTTT 5’-primer BEADS Avidin-coated beads biotin 3’-primer AAAA -----------------GGG-capfinder-5’ Reverse transcription of the first strand; Cs added on 3’ end by the reverse transcriptase. Analyze PCR products on agarose gel (see Results) (same products, not attached to beads) Reverse transcription of the second strand using capfinder cDNA products PNA 5’-primer TTTT--------------------------------CCC-3’ 3’-primer AAAA -------------------------------GGG-capfinder-5’ GGG-capfinder exon3 PCR using Cd4 specific exon3 primer A B Two color immunostaining and flowcytometry of fresh B10.A thymocytes Two color immunostaining and flowcytometry of PNA-panned B10.A thymocytes C 500bp 1 2 3 4 Primer dimers 100 200 300 400 500 600 700 800 900 1000 Abstract Cd4 gene expression is regulated by several different known transcriptional elements, including a silencer, a promoter, an immature enhancer, a distal enhancer, and a proximal enhancer. Different combinations of these elements are employed to regulate Cd4 expression in mature CD4 cells versus immature double positive (DP) cells. In humans an additional transcriptional control element has been described that serves as a second promoter for Cd4 and is proposed to be responsible for Cd4 expression in macrophages and some dendritic cells. Since mice do not have cell surface expression of CD4 in the macrophage, the presence of a second promoter and its potential function during T cell development has not been investigated so far. To determine if a second promoter is present in the first intron of the Cd4 gene and utilized during the DP stage of T cell development, we looked for new transcription start sites in RNA from murine DP cells by 5’RACE. In addition to the expected 320bp band that indicates transcription from the known promoter, we observed a strong 500bp band, indicating that there is another transcription start site in the Cd4 gene. We are in the process of cloning and sequencing all the Cd4 transcription products and identifying the second promoter. Whether the putative second promoter is used in mature CD4 cells remains to be determined. Acknowledgements We would like to thank Amy Becton for maintaining our mouse colony, Susan Sharrow (NCI, EIB) for antibodies and FACS advice, and Terry Guinter (NCI, EIB) for help with the PNA panning protocol.