Reconstitution of the subclass-specific expression of CD4 in thymocytes and peripheral T cells of transgenic mice: identification of a human CD4 enhancer.

Abstract

StlmmaryDuring thymic maturation, CD4-CD8-TCR-immature thymocytes differentiate through a CD4+CD8+TCR 1~ intermediate into two functionally distinct mature T cell subsets: helper T cells expressing CD4 and a major histocompatibility complex (MHC) class II-restricted T cell receptor (TCR), and cytotoxic T cells expressing CD8 and an MHC class I-restricted TCR. The mutually exclusive expression of CD4 and CD8 is maintained in the periphery during expansion of these mature T cell subsets. To elucidate the mechanisms controlling CD4 and CD8 expression on differentiating thymocytes and mature peripheral T ceils, we have examined the expression of human CD4 gene constructs in the lymphoid tissues of transgenic mice. Our analyses demonstrate that sequences contained within or closely linked to the human CD4 gene are sufficient to reconstitute the appropriate regulation of human CD4 expression on all thymocyte and mature peripheral T cell subsets. Specifically, appropriate developmental regulation was dependent on two sets of sequences, one contained within a 1.3-kb restriction fragment located 6.5 kb upstream of the human CD4 gene, and the other present within or immediately flanking the gene. Nucleotide sequence analysis identified the 1.3-kb restriction fragment as the likely human homologue of an enhancer found 13 kb upstream of the mouse CD4 transcription initiation site. The human CD4 transgenic mice provide a useful system for the identification and characterization of additional sequence elements that participate in human CD4 gene regulation and for the elucidation of regulatory mechanisms governing the developmental program mediating the maturation of the CD4 + and CD8 + peripheral T cell subsets. Mature CD4+CD8 -class II-restricted and CD4-CD8 + class I-restricted lymphocytes differentiate intrathymically from CD4-CD8-TCR-cells that originate from fetal liver or bone marrow precursors (1). The major intermediate in the T cell differentiation pathway is the doublepositive thymocyte, which expresses both CD4 and CD8 and low levels of the od3 TCR. Antibody blocking experiments (2, 3), as well as more recent studies in gene knockout mice (4,5), have demonstrated that CD4 and CD8 are essential for the differentiation of double-positive thymocytes into mature class II-and class I-restricted T cells, respectively. On mature T cells, CD4 and CD8 participate in Ag recognition and T cell activation by binding to monomorphic determinants on MHC class II and class I proteins, respectively (6-12). Coengagement of an Ag-MHC complex by an c~/3 TCR and either CD4 or CD8 is believed to be a critical component of the T cell activation pathway (13-17). The lymphocytespecific tyrosine kinase, p56 lck, is physically associated with the cytoplasmic domains of both CD4 and CD8 and can be activated by antibody crosslinking of either of these glycoproteins (18-21). Thus, coengagement of Ag-MHC by the TCR and CD4 or CD8 may trigger T cell activation by transducing transmembrane signals via p56 lck (22,23). By binding MHC, CD4 and CD8 m...

Elf-1 binds to a critical element in a second CD4 enhancer.

“…We were unable to detect enhancer activity in large restriction fragments that contain the previously identified, proximal enhancer when assayed in D10, AKRlG1, or Jurkat cells (Fig. 1B and (2,22,31).…”

“…Previous studies by our laboratory and others on CD4 gene regulation have identified and characterized CD4 promoter and enhancer elements in the mouse and human CD4 loci that are capable of stimulating transcription of reporter genes in transient transfections of cultured cells as well as in transgenic mice (2,49,51,54). To date, however, reconstitution of the developmental pattern and T-cell subclass-specific expression of CD4 has been accomplished only through the use of large cosmid constructs that contain many kilobases of genomic DNA sequences within and around the human CD4 locus (2,14,31). Clearly other elements in the CD4 locus are required in addition to the promoter and enhancer elements already identified.…”

“…Previous studies by our laboratory and others on CD4 gene regulation have identified and characterized CD4 promoter and enhancer elements in the mouse and human CD4 loci that are capable of stimulating transcription of reporter genes in transient transfections of cultured cells as well as in transgenic mice (2,49,51,54). To date, however, reconstitution of the developmental pattern and T-cell subclass-specific expression of CD4 has been accomplished only through the use of large cosmid constructs that contain many kilobases of genomic DNA sequences within and around the human CD4 locus (2,14,31 We also determined that an Ets family transcription factor, Elf-1, is a dominant protein in T-cell nuclear extracts that binds to this site in vitro. Elf-1 has previously been shown to be required for the activation induced transcription of the T-cell-specific interleukin-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes as well as in the regulation of the T-cell-specific human immunodeficiency virus type 2 (HIV-2) and human T-cell leukemia virus type I (HTLV-I) enhancers (4,17,37,64).…”

The Epigenetic Landscape of Lineage Choice: Lessons From the Heritability of Cd4 and Cd8 Expression

“…DHS mapping revealed several putative regulatory elements in the locus (Adlam and Siu, 2003;Sands and Nikolic-Zugic, 1992;Sawada and Littman, 1991), and eventually led to the identification of three enhancers: the distal (E4 D ) and proximal enhancers (E4 P ) at ~24kb and ~13kb upstream of the Cd4 TSS, respectively, and the thymocyte enhancer (E4 T ) at ~36 kb downstream (Adlam and Siu, 2003;Sawada and Littman, 1991;Wurster et al, 1994) (Reviewed in detail by (Taniuchi et al, 2004)). In combination with the Cd4 promoter, E4 P drives T cell specific transgene expression in multiple mouse lines (Blum et al, 1993;Hanna et al, 1994;Killeen et al, 1993). The developmental window during which E4 P shows activity, however, was not entirely clear: while some studies found it to be active in DN and pre-selection DP cells through to mature helper and cytotoxic T cells (Blum et al, 1993;Hanna et al, 1994;Killeen et al, 1993;Manjunath et al, 1999;Sawada et al, 1994;Siu et al, 1994), others indicated that E4 P activity begins post-positive selection (Adlam et al, 1997).…”

“…In combination with the Cd4 promoter, E4 P drives T cell specific transgene expression in multiple mouse lines (Blum et al, 1993;Hanna et al, 1994;Killeen et al, 1993). The developmental window during which E4 P shows activity, however, was not entirely clear: while some studies found it to be active in DN and pre-selection DP cells through to mature helper and cytotoxic T cells (Blum et al, 1993;Hanna et al, 1994;Killeen et al, 1993;Manjunath et al, 1999;Sawada et al, 1994;Siu et al, 1994), others indicated that E4 P activity begins post-positive selection (Adlam et al, 1997). In addition, it appears that, in the context of transgenes, E4 P may lose activity in mature T cells following TCR stimulation (Manjunath et al, 1999).…”