In Vitro Characterization of a Novel Cis-acting Element (NCE) in the Cd4 Locus Alexis Grant 1 , Yordan Penev 1 , Gregory Swan 2 , Sophia Sarafova 3 Biology Department, Davidson College 1: Equal contributors; 2: Currently at NCI, Bethesda, Maryland; 3. Secondary affiliation: Immunology Department, Duke University Novel Cis-acting Element (NCE) Figure 2. NCE contains a region of high homology across vertebrate species. The positions of the known cis-elements in the Cd4 gene (abbreviations listed in Fig. 1) are indicated by colored boxes. Arrows indicate the orientation of transcriptional units in the locus. UCSC’s genome browswer 30-way Multiz alignment shows sequence conservation at the silencer and a downstream region that was knocked out along with the silencer in (6) Zou, et al. and (7) Leung, et al. 0.0 5.0 10.0 Pr + PE Pr Empty GFP rMFI SSC pdsRED 10 1 10 2 10 3 10 4 10 5 10 6 10 7 55% 300000 3650000 70000 10 1 10 2 10 3 10 4 10 5 10 6 7-AAD 300K 500K 700K FSC 30% ** * *p < 0.05 **p < 0.005 Empty Pr Pr + PE B Figure 3. Methods (modified from (8) Wildt, et al. 2007 and Wang, et al. 2010) A) Seven reporter constructs were created using PCR to amplify the known Cd4 promoter (Pr), the known proximal enhancer (PE), the 1.6 Kb region of interest (NCE) and the conserved fragments 5 and 8 from the C57BL/6 genomic DNA and ligating them into the multiple cloning site (MCS) or Afl-II site of the GFP reporter plasmid pd2eGFP-1. B) FACS analysis of transiently transfected RLM-11 cells. Five million RML-11 murine thymoma cells were transfected with 5-20 μg of one of the above reporters and 5-20 μg of pRed or CD8 using electroporation (BTX). After 18 hours, the cells were stained forCD8 and 7-AAD. CD8 and eGFP expression were analyzed using an Accurri6 flow cytometer and the FCS Express V3 software. Cells that were efficiently transfected were identified as 7-AAD-RFPhi. The Mean Fluorescence Intensity (MFI) of eGFP fluorescence of the 7-AAD-RFPhi was recorded and compared to promoter construct to calculate the relative eGFP MFI and determine the presence of promoter or enhancer activity. INTRODUCTION We thank Dr. Yuan Zhuang for hosting us in his lab during the summer, Dr. Rémy Bosselut for the generous donation of the RLM11 cell line, Dr. Sue Sharrow of the NCI for FACS analysis advice, and Dr. Alfred Singer for advice and the generous donation of antibodies. The research was supported by an Educational Enhancement Grant from North Carolina Biotechnology Center and an NIH AREA grant 1R15AI098001-01A1. ACKNOWLEDGEMENTS Figure 4. NCE enhances Cd4 promoter driven eGFP fluorescence in a developmental-stage-specific manner. NCE enhancer function was tested in 5 mouse cell lines, immortalized at different developmental stages (Fig. 1), and a CD4-SP human cell line. The proximal enhancer (PE) was used as a positive control. All bars represent at least three trials. Error bars represent the standard error of the mean. Using a one-tailed T-Test ,* = <0.05, ** = < 0.005. NCE has significant enhancer function only in the CD4+ intermediate and single positive cell lines. The decrease of proximal enhancer function with increasing maturity is readily observable, confirming that the transfection conditions used were suitable for each cell line. Figure 1. Increasing CD4 levels on developing MHC-II specific thymocytes cannot be explained by the activity of the known cis-elements. Three developmental stages of MHC-II specific T-cells are illustrated in increasing order of maturity (blue wedge) with corresponding levels of cell-surface CD4. Above are the representative cell lines used in the study. The activity of the known cis-elements in the Cd4 gene (DE: distal enhancer, PE: proximal enhancer, Pr: promoter, and Sil: silencer) is indicated by arrow thickness. Figure 6. PMA and Ionomycin stimulation and transfection with NCE. Cells from the RLM11, 2b4, and EL-4 cell lines were transfected with NCE. Half of each population was stimulated with 13nM PMA and .7uM Io 5 hours after the transfection. After 18 hours, the cells were stained for CD69 and analyzed by flow cytometry. Upregulation of CD69 indicates successful stimulation. No dramatic effect on NCE expression was observed, as determined by levels of the reporter eGFP fluorescence intensity. Figure 7. Transcription factor homology and expression. A) NCE sequence analysis. The minimal functioning conserved sequence of NCE was analyzed for sequence identity with transcription factor binding sites using NCBI’s Blast tool. Sequences with the greatest homology were ranked & mapped using color-coding. B) Real-time PCR confirmation of transcription factor expression levels. RNA was extracted from cells of the AKR (DP), RLM11 (Int.), and 2B4 (SP) cell lines using Trisol reagent. CDNA was synthesized using qScript TM cDNA Reaction Mix from Quanta Biosciences. QPCR primers were designed to span exon boundaries (with the exception of NFAT5) and ordered using the PrimeTime qPCR tool by Integrated DNA Technologies. Results were quantified via SYBR green real time eppendorf PCR and compared to previously reported data on the Gene Skyline database from the Immunological Genome Project. No notable differences were observed. The experiment was repeated 3 times in triplicate. Transcription factor expression levels were normalized to the expression levels of a housekeeping gene, GAPDH. A B Figure 5. Fragment 5 is a functional component of NCE. A. Enzyme digest of plasmids and map of digestion sites. Plasmids with fragment 5 and two instances of fragment 5 (5x2) were digested using the enzymes KpnI, SpeI, BglII, and Bam HI because the digestion sites for these enzymes have been mapped in each plasmid (5B). For each digest, the observed band size correlated to the expected size of single-stranded DNA fragments according to the digestion sites of the plasmid, confirming the identities of both fragments. B. GFP expression relative to promoter. NCEx2 and fragment 5 showed a two-fold increase in GFP expression compared to promoter. Fragment 5x2 showed a three-fold increase from the promoter. Fragment 5x2 showed a three-fold increase from the promoter. Results are based on one trial. • We have isolated and characterized the minimum functional conserved sequence of a novel positive regulatory element (NCE) in the Cd4 locus. • NCE exhibits developmental stage specificity in murine cells as it functions in the intermediate developmental stage cell line RLM11 and the CD4+ single positive developmental cell lines EL-4 and 2B4, but not in the CD8/CD4 double positive stage cell lines AKR1G1 and VL3-3M2. • The assignment of developmental stage to the cell lines matches expected levels of characteristic transcription factor expression, with the possible exception of ThPok in the 2B4 cell line. • Based on a single assay, we conclude no correlation between TCR stimulation and NCE function, suggesting that the element might operate in a TCR- independent manner. CONCLUSIONS METHODS RESULTS 1. Ellmeier W, Sawada S, Littman DR. The regulation of CD4 and CD8 coreceptor gene expression during T cell development. Annu Rev Immunol. 1999; 17: 523-554. 2. Singer A, Adoro S, Park JH. Lineage fate and intense debate: Myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat Rev Immunol. 2008 Oct; 8(10): 788-801. 3. Kioussis D, Ellmeier W. Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Nat Rev Immunol. 2002 Dec; 2(12): 909-19. 4. Siu G. Controlling CD4 gene expression during T cell lineage commitment. Semin Immunol. 2002 Dec; 14(6): 441-51. 5. Sarafova SD, Van Laethem F, Adoro S, Guinter T, Sharrow SO, Feigenbaum L, Singer A. Upregulation of CD4 expression during MHC class II-specific positive selection is essential for error-free lineage choice. Immunity. 2009 Sep 18; 31(3): 480-90. 6. Zou YR, Sunshine MJ, Taniuchi I, Hatam F, Killeen N, Littman DR. Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage. Nat Genet. 2001 Nov; 29(3): 332-336. 7. Leung RK, Thomson K, Gallimore A, Jones E, Van den Broek M, Sierro S, Alsheikhly AR, McMichael A, Rahemtulla A. Deletion of the CD4 silencer element supports a stochastic mechanism of thymocyte lineage commitment. Nat Immunol. 2001 Dec; 2(12): 1167-1173. 8. Wildt KF, Sun G, Grueter B, Fischer M, Zamisch M, Ehlers M, Bosselut R. The transcription factor Zbtb7b promotes CD4 expression by antagonizing Runx-mediated activation of the CD4 silencer. J Immunol. 2007 Oct 1; 179(7): 4405-14. REFERENCES Conditions like HIV infection, chemotherapy, and congenital defects will deplete patients’ CD4 T-cells. CD4 T-cells are the coordinators of the adaptive immune response, and the strength of a person’s immune system depends on the amount and timing of production of functional CD4 T-cells. In the process of developing into CD4 T-cells, the progenitor cells in the thymus (thymocytes) go from the double positive (DP) stage (CD4+CD8+) to the intermediate (INT) stage (CD4+CD8lo), to the single positive (SP) stage (CD4+CD8-), with progressive increase of CD4 expression that is essential for making the correct lineage choice based on the specificity of their T cell receptor (TCR) (1,5). The alternative lineage that stems from the INT stage is the CD8 cytotoxic T cell, which is responsible for killing host cells infected with intracellular pathogens (2). Our overall goal is to understand how the upregulation of CD4 expression is regulated during development. Previous studies have identified a promoter, two enhancers, and a silencer responsible for Cd4 gene expression (4). However, the activity of the known enhancer elements decreases with maturation, which is inconsistent with the simultaneous increase in CD4 levels as the cells develop from DP to SP (3). Thus, current knowledge of the function of the regulatory elements involved in Cd4 expression is not sufficient to explain its tight regulation during the intermediate phase between the double positive and single positive (CD4+) stages of T cell development (Figure 1). To address this inconsistency, we are investigating the newly identified cis-transcriptional regulatory element (NCE) for enhancer function dependent on developmental stage. Previous experiments have shown that NCE, a highly conserved sequence on intron 1 of the Cd4 locus (Figure 2), functions as an enhancer independent of its position and orientation. We used cells from the AKR, RLM11, EL-4 and 2B4 cell lines to represent the double positive, intermediate, and single positive stages of thymocyte development. Here we describe the differences in NCE function between developmental stages based on these cell line models. Additionally, we show our initial attempts at identifying the minimum functional unit of NCE and the transcription factors that bind it, as well as the element ’s T-cell receptor responsiveness. 300bp Fragment 5 Fragment 5x2 1kb ladder Kpn1 Spe1 BamHI BglII Uncut 1kb 3kb 10kb 4kb 1kb ladder 10kb 1kb ladder Kpn1 Spe1 BamHI BglII Uncut 300bp 1kb 3kb 4kb Key KpnI (K) SpeI (S) BamHI (BH) BglII (B) GFP BH S S B K 450bp K 540bp GFP BH S S S S K K 540bp 1.6kb 450bp 450bp 0 0.5 1 1.5 2 2.5 3 3.5 Pr NCEx1 NCEx2 Frag 5 Frag 5x2 GFP expression relative to promoter Afl- II Pr eGFP SV40 Poly A 1 PE Pr eGFP SV40 Poly A PE Pr 2 6 Pr eGFP SV40 Poly A 4 Pr eGFP SV40 Poly A 3 Pr eGFP SV40 Poly A NCE NCE NCE NCE 7 Pr eGFP SV40 Poly A eGFP 5 Pr eGFP SV40 Poly A NCE with conserved region (red) Cd4 Promoter (550bp) Cd4 Proximal Enhancer (300bp) Enhanced Green Fluorescent Protein Pr PE + Pr NCEx1 + Pr NCEx2 + Pr Fragment 5 + Pr Fragment 5x2 + Pr Fragment 8 + Pr A CD69 RLM11 EL4 2B4 Cell line eGFP CD69 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 388 776 1163 1551 GFP-A 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 19 39 58 77 GFP-A 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 3 6 9 12 GFP-A 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 57 114 170 227 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 1097 2194 3290 4387 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 1101 2202 3303 4404 Not stimulated Stimulated Isotype control TTCCCAGGGGTGACGTACACAGGATGCTGCGCTTACTCTTTATGAGATATAGC GCCTCTGGGGAGGCTGAGGATGTGGGAATCAAAGGCCAGGTCTCTGGGGT AGCAGCGGAAACCTGAAGCACCAATGCAGGCTGTGGCTGGAAACCCAC Binding sites: 1)KLFS: Kruppel like transcription factors 2)IKRS: Ikaros zinc family finger 3)ETSF: ETS1 factors (ELK-1) 4) RBPF: RBPJ – kappa 5) LEFF: LEF1/TCF 6) C2H2 zinc finger transcription factors 2 (ThPOK) 7) CATT: CCAAT binding factors 8) CEBP: Ccaat/Enhancer Binding Protein 9) ETSF: Human and murine ETS1 factors 10) NFAT: Nuclear factor of activated T-cells 60 70 80 90 100 110 120 130 140 150 1 2 10 20 30 40 50 3 4 5 9 1 3 7 1 0 6 8 0 100 200 300 400 DP Int. SP Relative expression normalized to GAPDH NFAT5 0 100 200 300 400 DP Int. SP Relative expression normalized to GAPDH RBPJκ 0 200 400 600 800 DP Int. SP Relative expression normalized to GAPDH ThPok 0 100 200 300 400 DP Int. SP Relative expression normalized to GAPDH CEBPζ Expected Observed 0 1 2 3 4 5 6 7 8 9 AKR1G1 VL3-3M2 RLM11 EL-4 2B4 Jurkat GFP rMFI Pr Pr + PE Pr + NCE Pr + NCEx2 ** ** ** * * * * * * DP (mouse) Int (mouse) SP (mouse) SP (human) * *