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Related Collections Gene Expression Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 October 2001, Vol. 98, No. 7, pp. 2275-2278 BRIEF REPORT Distinct regulatory elements are required for faithful expression of human CD4 in T cells, macrophages, and dendritic cells of transgenic mice Zaher Hanna, Najet Rebai, Johanne Poudrier, and Paul Jolicoeur From the Laboratory of Molecular Biology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada; Departments of Medicine or Microbiology and Immunology, Université de Montréal, Montreal, Quebec, Canada; and the Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada.     Abstract Top Abstract Introduction Study design Results and discussion References To identify the regulatory elements controlling expression of the human CD4 (hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3' end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5' end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8+ dendritic cells (DCs) from lymph node and thymus were found to be different from those required for macrophage expression. The results indicate that expression of hCD4 in T cells, macrophages, and DCs is controlled by distinct regulatory elements. (Blood. 2001;98:2275-2278) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The CD4 cell surface receptor is expressed on many mouse or human cell populations, namely, on very early T-cell precursors (CD4LowCD44+CD25), on immature double-positive (DP) CD4+CD8+ T cells, on mature single-positive (SP) CD4+CD8 T cells,1,2 and on a subpopulation of CD8 dendritic cells (DCs).3,4 In humans, CD4 is also expressed in monocytes, macrophages, microglial cells, and some DCs.5,6 Several regulatory elements of the murine and human CD4 genes (mCD4, hCD4) have been identified and tested in transgenic (Tg) animal studies (reviewed by Ellmeier et al1 and Killeen and Littman2). We have exploited the structural homology, but the different patterns of expression, of the mCD4 and hCD4 genes and made chimeric mCD4/hCD4 transgenes, to map more precisely the regulatory regions of the hCD4 gene controlling its expression in macrophages and in DCs.     Study design Top Abstract Introduction Study design Results and discussion References Tg mice Constructs CD4A, CD4B, and CD4C were described previously.7 Other constructs were generated from the CD4C DNA by replacing the 2.6-kilobase pair (kbp) SacI fragment with the mouse promoter (CD4E), or the 9-kbp EcoRV-XbaI fragment with the murine silencer (0.5 kbp) (CD4F), or by deleting the 6-kbp EcoRV fragment (CD4H). Tg mice were generated as described elsewhere.7 Flow cytometry Cell suspensions were prepared from lymphoid organs and stained with antibodies, as previously described.7 Peritoneal macrophages were plated overnight. Between 80% and 95% of them stained positive for Mac-1 (Figure 2B), Mac3, and F4/80 (data not shown). DCs were isolated from lymph node (LN)8 and thymus.9     Results and discussion Top Abstract Introduction Study design Results and discussion References The Tg mice harboring 6 different DNAs were generated (Figure 1). Northern blot analysis revealed that expression was highest in the thymus but was also detectable in the spleen and LNs, and was negative in all other organs tested (liver, heart, kidney, lung, intestine, muscle, brain; data not shown). This result is consistent with previous studies indicating that the human and mouse CD4 promoter/enhancer alone or in combination is sufficient to restrict expression to hematopoietic tissues.7,10-13 As expected, the level of expression varied among different founders carrying the same construct, an effect most likely related to the site of integration of the inoculated DNA. View larger version (20K): [in this window] [in a new window]   Figure 1. Structure of the human/mouse CD4 transgenes and summary of expression data. Symbols are as follows: human (black bar) and mouse (open bar) sequences; mouse enhancer (stippled bar), human and mouse promoters (hP, mP); and human and mouse silencer (Si, mSi). Restriction sites: E, EcoRI; Bg, BglII; H, HindIII; X, XbaI; RV, EcoRV; Sc, SacI; Sl, SalI. Expression in different subsets of cells is shown as + for expression,  for no expression, and ± for expression on a low number of cells. Double-positive (DP) CD4+CD8+ thymocytes; single-positive (SP) peripheral lymphocytes CD4+ and CD8+; B cells (B); macrophages (MAC); dendritic cells (DC); myeloid (M) and lymphoid (L) DCs; nd, not done. Expression of hCD4 in DP CD4+CD8+ T cells Fluorescent-activated cell sorter (FACS) analysis showed that DP and SP CD4+ thymocytes of CD4E, CD4F, and CD4H Tg mice expressed the hCD4 reporter at high levels, similar to CD4C Tg mice7 (data not shown). Deletion of most of intron 1 (CD4F) or substitution of the human for the mouse promoter (CD4E) did not significantly change expression in the thymic T cells. Therefore, the CD4E and CD4F transgenes contain all the regulatory elements required for promoting CD4 expression in DP T cells. This result is in agreement with data from other groups who used similar human14,15 or mouse10 constructs. In contrast, other Tg mice harboring apparently similar mCD416,17 or hCD418,19 constructs did not express their reporter gene in the CD4+CD8+ DP T cells, while retaining expression in the SP CD4+ T cells. These discrepancies may reflect the presence or not of intronic element(s), just in front of exon 2, required for the expression in DP T cells and identified by Rushton and colleagues20 as the proximal promoter. Silencing of the CD4 promoter in SP CD8+ T cells In all Tg lines, including in most new lines (CD4E, F, H), expression of hCD4 in spleen and LNs was not detected on B cells and was restricted to T cells, almost exclusively on mature CD4+CD8 T cells (80%-90%, Table 1, data not shown). The SP CD8+ T cells from CD4E and CD4H (about 10%-18%), but not from CD4F (<3%) Tg mice expressed hCD4 (Figure 2A, Table 1, data not shown), indicating that switching the human with the mouse promoter (CD4E) or deleting a large portion of the intron 1 (CD4H, CD4F) had no effect on the expression on CD4+ T cells. These results also showed that the mouse silencer,10,11 in the context of the human promoter (CD4F), appears to be more effective in shutting off expression in CD8+ T cells than the human silencer14 (CD4H, CD4C). The human silencer may require additional collaborative sequences, not yet identified, to be fully operative.                                View this table: [in this window] [in a new window]   Table 1. hCD4 reporter gene expression on peritoneal macrophages and T cells from lymph nodes of hCD4 Tg mice View larger version (55K): [in this window] [in a new window]   Figure 2. FACS analysis of hCD4 expression. (A) LN cells. Double staining with antihuman CD4 (hCD4) and antimouse CD4 (mCD4) or mouse CD8 (mCD8) monoclonal antibodies was carried out. The percentage of T cells expressing hCD4 in a representative mouse for each line is shown. Note that the leakiness of the hCD4 expression on the mature CD8+ T cells was absent in the CD4F Tg line. (B) Peritoneal macrophages. Double staining of plated peritoneal macrophages was done with anti-hCD4 and anti-Mac-1 antibodies. The percentage of macrophages expressing Mac-1 and hCD4 is shown. The quadrant settings are based on unstained controls. (C) DCs. Triple staining of enriched LN DCs. Cells that were gated for CD11c+CD11b/Mac-1+ (myeloid) and CD11c+CD8+ (lymphoid) expression (i) show expression of hCD4 (ii). The thymic DCs were all CD11c+CD8+. The bars in each panel in ii show the staining of hCD4 on DCs from non-Tg control mice. Transgene expression in peritoneal macrophages In CD4A, CD4C, and CD4H Tg mice, hCD4 was expressed on over 85% of peritoneal macrophages (Mac-1+)7 (Figure 2B, Table 1). However, macrophages from the CD4E and CD4F Tg mice showed very low or undetectable levels of expression. These results suggest that expression in macrophages requires the concomitant presence of 2 hCD4 sequences: one located in the 2.6-kbp promoter and a second (about 3.5 kbp) in the 5' intron 1 region (Figure 1). Transgene expression in DCs Because macrophages and DCs can be derived from the same myeloid precursor and because the 2 major mouse subpopulations of DCs ("myeloid" CD11c+CD11b+CD8 and "lymphoid" CD11c+CD11bCD8+) are thought to fulfill different functions in vivo,21 it was of interest to compare transgene expression in these DC subpopulations. This comparative analysis was carried out on purified DCs from peripheral LNs and from thymus. The data are shown for DCs expressing either a myeloid (CD11c+ CD11b+) or a lymphoid (CD11c+ CD8+) phenotype. In CD4A, CD4C, CD4E, and CD4H Tg mice, expression of hCD4 was detected in high proportion (80%) on both CD11b+ and CD8+ LN DCs, as well as on thymic CD8+ DC (Figure 2C). The CD4F Tg DC express the reporter gene at low or undetectable levels on few (1%-9%) myeloid DCs and on a significant proportion (11%-40%; depending on founder lines) of lymphoid DCs (Figure 2C). Thus, some DC regulatory elements appear to reside in the promoter, the larger remaining fragment in CD4F DNA. The mouse enhancer appears to be dispensable for expression in DC (CD4A), as in macrophages.7 The fact that the same hCD4 regulatory sequences allow expression in both myeloid and lymphoid murine DCs contrasts with the mCD4 expression that appears to be restricted to CD8 subpopulations both in the spleen4 and in LNs.3 Interestingly, the murine promoter, which was unable to direct expression in macrophages by itself, could nevertheless promote expression in DCs (CD4E). This result indicates a distinct requirement of these 2 populations for expression of the hCD4 gene and suggests that the myeloid-specific element is most likely present in the 3.5-kbp 5' end intron 1 fragment remaining in CD4H. This work confirms earlier studies in lymphoid T cells on the importance of some regulatory elements within the CD4 gene, the distal promoter,22-24 the proximal promoter,20 the proximal and distal enhancers,13,25,26 and the silencer,10,11,14 and extends them significantly for macrophages and DCs.     Acknowledgments We thank Ginette Massé, Nathalie Gauthier, Michel Ste-Marie, Michel Robillard, Stéphane Gagnon, and Karina Lamarre for their excellent technical assistance and Nathalie Tessier for her valuable help in the FACS analysis.     Footnotes Submitted November 8, 2000; accepted May 31, 2001. Supported by grants from the Medical Research Council to P.J. and Z.H. and from the National Cancer Institute to P.J. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Paul Jolicoeur, Laboratory of Molecular Biology, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, QUE, H2W 1R7, Canada; e-mail: jolicop{at}ircm.qc.ca; or Zaher Hanna, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec, H2W 1R7, Canada; e-mail: hannaz{at}ircm.qc.ca.     References Top Abstract Introduction Study design Results and discussion References 1. Ellmeier W, Sawada S, Littman DR. The regulation of CD4 and CD8 coreceptor gene expression during T cell development. Ann Rev Immunol. 1999;17:523-554[CrossRef][Medline] [Order article via Infotrieve]. 2. Killeen N, Littman DR. The regulation and function of the CD4 coreceptor during T lymphocyte development. Curr Top Microbiol Immunol. 1996;205:89-106[Medline] [Order article via Infotrieve]. 3. Salomon B, Cohen JL, Masurier C, Klatzmann D. Three populations of mouse lymph node dendritic cells with different origins and dynamics. J Immunol. 1998;160:708-717[Abstract/Free Full Text]. 4. Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol. 2000;164:2978-2986[Abstract/Free Full Text]. 5. Wood GS, Warner NL, Warnke RA. Anti-Leu-3/T4 antibodies react with cells of monocyte/macrophage and Langerhans lineage. J Immunol. 1983;131:212-216[Abstract]. 6. Kazazi F, Mathijs JM, Foley P, Cunningham AL. Variations in CD4 expression by human monocytes and macrophages and their relationships to infection with the human immunodeficiency virus. J Gen Virol. 1989;70:2661-2672[Abstract/Free Full Text]. 7. Hanna Z, Simard C, Jolicoeur P. Specific expression of the human CD4 gene in mature CD4+CD8 and immature CD4+CD8+T cells, and in macrophages of transgenic mice. Mol Cell Biol. 1994;14:1084-1094[Abstract/Free Full Text]. 8. Berney C, Herren S, Power CA, Gordon S, Martinez-Pomares L, Kosco-Vilbois MH. A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein. J Exp Med. 1999;190:851-860[Abstract/Free Full Text]. 9. Vremec D, Zorbas M, Scollay R, et al. The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells. J Exp Med. 1992;176:47-58[Abstract/Free Full Text]. 10. Sawada S, Scarborough JD, Killeen N, Littman DR. A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development. Cell. 1994;77:917-929[CrossRef][Medline] [Order article via Infotrieve]. 11. Siu G, Wurster AL, Duncan D, Soliman TM, Hedrick SM. A transcriptional silencer controls the developmental expression of the CD4 gene. EMBO J. 1994;13:3570-3579[Medline] [Order article via Infotrieve]. 12. Gillespie FP, Doros L, Vitale J, Blackwell C, Gosselin J, Snyder BW. Tissue-specific expression of human CD4 in transgenic mice. Mol Cell Biol. 1993;13:2952-2958[Abstract/Free Full Text]. 13. Blum MD, Wong GT, Higgins KM, Sunshine MJ, Lacy E. Reconstitution of subclass-specific expression of CD4 in thymocytes and peripheral T cells of transgenic mice: identification of a human CD4 enhancer. J Exp Med. 1993;177:1343-1358[Abstract/Free Full Text]. 14. Donda A, Schulz M, Burki K, De Libero G, Uematsu Y. Identification and characterization of a human CD4 silencer. Eur J Immunol. 1996;26:493-500[Medline] [Order article via Infotrieve]. 15. Uematsu Y, Donda A, De Libero G. Thymocytes control the CD4 gene differently from mature T lymphocytes. Int Immunol. 1997;9:179-187[Abstract/Free Full Text]. 16. Duncan DD, Adlam M, Siu G. Asymmetric redundancy in CD4 silencer function. Immunity. 1996;4:301-311[CrossRef][Medline] [Order article via Infotrieve]. 17. Adlam M, Duncan DD, Ng DK, Siu G. Positive selection induces CD4 promoter and enhancer function. Int Immunol. 1997;9:877-887[Abstract/Free Full Text]. 18. Salmon P, Boyer O, Lores P, Jami J, Klatzmann D. Characterization of an intronless CD4 minigene expressed in mature CD4 and CD8 T cells, but not expressed in immature thymocytes. J Immunol. 1996;156:1873-1879[Abstract]. 19. Boyer O, Zhao JC, Cohen JL, et al. Position-dependent variegation of a CD4 minigene with targeted expression to mature CD4+ T cells. J Immunol. 1997;159:3383-3390[Abstract]. 20. Rushton JJ, Zorich GP, Stolc V, Neudorf SM. Characterization of a promoter within the first intron of the human CD4 gene. Eur J Biochem. 1997;245:768-773[Medline] [Order article via Infotrieve]. 21. Shortman K. Burnet Oration: Dendritic cells: multiple subtypes, multiple origins, multiple functions. Immunol Cell Biol. 2000;78:161-165[CrossRef][Medline] [Order article via Infotrieve]. 22. Siu G, Wurster AL, Lipsick JS, Hedrick SM. Expression of the CD4 gene requires a Myb transcription factor. Mol Cell Biol. 1992;12:1592-1604[Abstract/Free Full Text]. 23. Duncan D, Stupakoff A, Hedrick SM, Marcu KB, Siu G. A Myc-associated zinc finger protein binding site is one of four important functional regions in the CD4 promoter. Mol Cell Biol. 1995;15:3179-3186[Abstract]. 24. Salmon P, Giovane A, Wasylyk B, Klatzmann D. Characterization of the human CD4 gene promoter: transcription from the CD4 gene core promoter is tissue-specific and is activated by Ets proteins. Proc Natl Acad Sci U S A. 1993;90:7739-7743[Abstract/Free Full Text]. 25. Wurster AL, Siu G, Leiden JM, Hedrick SM. Elf-1 binds to a critical element in a second CD4 enhancer. Mol Cell Biol. 1994;14:6452-6463[Abstract/Free Full Text]. 26. Sawada S, Littman DR. Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene. Mol Cell Biol. 1991;11:5506-5515[Abstract/Free Full Text]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: E. Priceputu, Z. Hanna, C. Hu, M.-C. Simard, P. Vincent, S. Wildum, M. Schindler, F. Kirchhoff, and P. Jolicoeur Primary Human Immunodeficiency Virus Type 1 Nef Alleles Show Major Differences in Pathogenicity in Transgenic Mice J. Virol., May 1, 2007; 81(9): 4677 - 4693. [Abstract] [Full Text] [PDF] X. Li, E. Calvo, M. Cool, P. Chrobak, D. G. Kay, and P. Jolicoeur Overexpression of Notch1 Ectodomain in Myeloid Cells Induces Vascular Malformations through a Paracrine Pathway Am. J. Pathol., January 1, 2007; 170(1): 399 - 415. [Abstract] [Full Text] [PDF] E. Priceputu, I. Bouallaga, Y. Zhang, X. Li, P. Chrobak, Z. S. Hanna, J. 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