Structure and function of the CD7 molecule.

CD7 is a single-domain Ig superfamily molecule expressed on human T and NK cells, as well as on cells in the early stages of T, B, and myeloid cell differentiation. CD7 is highly expressed on malignant immature T cells and is generally absent on malignant mature T cells, such as CD4+ Sezary leukemia and HTLV-1+ adult T-cell leukemia cells. Because of lack of identification of a natural ligand and lack of a monoclonal antibody against murine CD7, the in vivo functions of CD7 have until recently remained obscure. Recent studies in CD7-deficient mice have provided new insights into CD7 function, and demonstrated key roles for CD7 in regulating peripheral T and NK cell cytokine production and sensitivity to LPS-induced shock syndromes. This article reviews recent work on the expression, structure, and function of CD7, and discusses roles the CD7 molecule might play in T and NK cell development and function.

Identification and characterization of K12 (SECTM1), a novel human gene that encodes a Golgi-associated protein with transmembrane and secreted isoforms.

The investigation of a DNase-hypersensitive site upstream of the CD7 gene on chromosome 17q25 has led to the discovery of a novel human gene designated K12 (SECTM1, the HGMW assignment). This gene spans approximately 14 kb and encodes a 1.8-kb mRNA detected at the highest levels in peripheral blood leukocytes and breast cancer cell lines. The open reading frame predicts a 248-amino-acid protein with the hydropathic characteristics of a type 1a membrane protein. Western blots show that the K12 protein exists as a cluster of bands around 27 kDa, and extractions using nonionic detergents or high pH conditions demonstrate that it behaves as an integral membrane protein. Immunofluorescence localization studies reveal that K12 is not detectable on the cell surface, but instead is found in a perinuclear Golgi-like pattern and colocalizes with a well-known Golgi marker. In addition, an approximately 20-kDa soluble form of the K12 protein derived from the N-terminal domain is specifically secreted by cells into the culture medium. Immunohistochemical analysis of peripheral blood cells shows that K12 is found in leukocytes of the myeloid lineage, with the strongest staining observed in granulocytes and no detectable expression in lymphocytes. Based on its range of expression, its broad structural characteristics that resemble cytokines and growth factors, and the chromosomal location of the gene in an area already associated with myelogenous leukemias and other malignant neoplasms, this study concludes that K12 is a novel molecule with potential importance in hematopoietic and/or immune system processes.

Gamma-globin gene promoter elements required for interaction with globin enhancers.

Normal expression of the human beta-globin domain genes is dependent on at least three types of regulatory elements located within the beta-globin domain: the locus control region (LCR), globin enhancer elements (3'beta and 3'Agamma), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated gamma-globin promoters linked to the 5'HS2 enhancer. We show that in K562 cells, 5'HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 --> +25) gamma promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5'HS2. Mutation of the gamma-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5'HS2. To determine if enhanced expression of gamma-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH gamma-globin gene promoters to 5'HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Agamma-globin gene 3' enhancer to a plasmid containing the gamma-globin gene promoter and 5'HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5'HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5', but not 3', to the gamma-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the gamma-globin promoter and the LCR/5'HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

Genomic structure and chromosomal localization of the novel ETS factor, PE-2 (ERF).

The members of the ETS family of transcription factors are grouped because they share a highly conserved DNA binding domain. These factors are involved in growth factor pathways and regulate both proliferation and differentiation. To identify ETS factors that may be involved in early hematopoietic progenitor regulation, we isolated a novel member of the ETS family by reverse transcriptase-PCR of the conserved DNA binding domain using degenerate oligonucleotides. This gene directs the synthesis of a 2704-nucleotide transcript whose largest open reading frame encodes a 548-amino-acid protein. Northern blot analysis reveals ubiquitous expression in all human tissues and cell lines tested, with highest levels in the testis, ovary, pancreas, and heart. Comparison of this gene with the available databases reveals very significant homology to the ETS factor PE-1 and probable near-identity with the recently cloned factor ERF. The PE-2 gene is composed of four exons spanning over 9 kb of genomic DNA. Sequence analysis of the promoter region reveals a GC-rich sequence without a TATA motif and with putative binding motifs for CREB, c-myb, and AP-1 factors. Using mouse-human somatic hybrids and FISH analysis, the PE-2 gene is localized to human chromosome 19q13.2, a region involved in translocations and deletions in leukemias and several solid tumors, suggesting that this novel ETS factor may play a role in carcinogenesis.

An analysis of alternatively spliced CD45 mRNA transcripts during T cell maturation in humans.

CD45 is a transmembrane protein tyrosine phosphatase found on nucleated hematopoietic cells. In humans, multiple protein isoforms of CD45 are produced by alternative mRNA splicing of exons 4, 5, and 6 coding for the extracellular portion. We measured all eight possible CD45 mRNA transcripts using RT-PCR in human thymocytes and T cell lines. We report that only six mRNA transcripts are present in T cells. The high mw CD45 mRNA transcripts containing exon 4 correlated with the stage of T cell maturation: abundant high mw transcripts (30.7% of all CD45 mRNA transcripts) were present in immature, CD3-4-8 triple-negative thymocytes which decreased (7.7%) in intermediate, CD4+8+ double-positive (DP) thymocytes and then increased (13.8% or 16.8%) in mature, CD4+8- or CD4-8+ single-positive thymocytes. In addition, there was a complex variation in the spliced mRNA transcripts coding for CD45R(O), CD45R(B), CD45R(BC), CD45R(AB), and CD45R(ABC) protein isoforms. High mw CD45 mRNA transcripts accumulated immediately prior to an important physiologic event such as thymocyte expansion. In addition, we identified linkage between RNA splicing of exons 5 and 6, and splicing of exon 5 only and exons 4, 5, and 6 in FACS-purified CD4+ and CD8+ thymocytes. These data support the developmental regulation of alternatively spliced CD45 mRNA transcripts and suggest that specific CD45 isoforms may play an important role at critical stages of T cell development.

Comparison of human and Xenopus GATA-2 promoters.

GATA proteins comprise a family of transcription factors that are required for appropriate development of hematopoietic lineages. In order to understand the transcriptional regulation of GATA genes, we have cloned the human GATA-2 gene and identified and characterized its promoter. Comparison with the Xenopus GATA-2 promoter demonstrates highly conserved CCAAT box elements, which are essential for appropriate Xenopus expression. Unlike the Xenopus gene, the human GATA-2 gene lacks GATA binding motifs within the first 800 bp of 5' flanking sequence. In addition, the human GATA-2 promoter has two highly conserved ets sites that resemble the binding site for a recently described ets repressor factor, ERF. These conserved DNA sequence motifs provide strong candidate regions for the regulation of GATA-2.

Complex regulation of human c-kit transcription by promoter repressors, activators, and specific myb elements.

The c-kit proto-oncogene is expressed in several tissues during development. To understand the mechanisms controlling the expression of this gene, we characterized the human c-kit promoter. Expression is controlled transcriptionally. The 5'-flanking DNA was used to make promoter deletion-reporter constructs that were tested in cells that were either positive or negative for endogenous c-Kit. The results demonstrate that DNA, to at least position -4100, directs transcription well in both positive and negative cells. Addition of DNA from position -4100 to -5500 causes a reduction in expression to near-basal levels in c-Kit-negative cells but has little effect in c-Kit-positive cells. The DNA from -4100 to -5500 was tested for repressor function. It inhibits transcription from some heterologous promoters in c-Kit-negative cells. Likewise, this segment inhibits transcription from the homologous proximal promoter in a cell-specific manner, but the entire promoter is necessary for complete repression in c-Kit-negative cells. Two Myb binding motifs were also identified, and their role in regulating transcription was examined by mutation and functional testing. One, MYB1, acts as a partial repressor, whereas the other, MYB2, is a positive element that appears essential for expression. Binding proteins to both sites were characterized by several methods. MYB1 binds and responds functionally to c-Myb, but MYB2 does not. The results of these studies indicate that the regulation of c-kit transcription is complex, involving interactions among several activators and repressors.

The mouse CD7 gene: identification of a new element common to the human CD7 and mouse Thy-1 promoters.

Human CD7 (hCD7) is a 40 000 Mr member of the immunoglobulin gene superfamily that is expressed early in natural killer (NK) and T-lymphocyte development. CD7 is involved in lymphocyte activation, as ligation of CD7 activates NK and TCRgammadelta T lymphocytes, and ligation of CD7 on TCRalphabeta T lymphocytes induces a non-mitogenic calcium flux. We have previously cloned and characterized the gene for human CD7 (hCD7) and have described its expression in transgenic mice. Recently a mouse cDNA homologous to hCD7 was reported, which we mapped to the corresponding mouse chromosomal location as hCD7. We now report the identification and characterization of a mouse CD7 (mCD7) genomic clone. We demonstrated that the mCD7 gene was similar both in size and structural organization to hCD7. Comparison of the 5' flanking sequences of the mCD7 and hCD7 genes revealed two regions of sequence similarity. Electrophoretic mobility shift assay confirmed both of these regions to be sites of tissue-restricted protein binding in vitro. The more 3' similarity region also shared sequence with a region in the mouse Thy-1 gene 5' flanking region, suggesting that this sequence may be a cis-acting regulatory element common to all three genes. Thus, the promoter regions and exonic organization were similar in the human CD7, mouse CD7, and mouse Thy-1 genes.

Characterization of human CD7 transgenic mice.

CD7 is a 40-kDa transmembrane glycoprotein member of the lg gene superfamily expressed on most peripheral blood T lymphocytes and NK cells. CD7 is also expressed on myeloid, NK, B, and T cell precursors during adult hematopoiesis. Because Thy-1 is absent in human thymocytes and peripheral blood T cells and shows structural similarities to the human CD7 gene, we have suggested that human CD7 may be a functional homologue in humans of mouse Thy-1. To study the tissue-specific expression of the CD7 gene utilizing its own promoter, we constructed transgenic mice that contained both the coding and flanking regions of the human CD7 gene. We found that human CD7 was expressed in transgenic mice in T, B, NK, and myeloid lineages and was induced with T cell activation. Unlike the expression of CD7 in humans, the CD7 transgene was present on mature B lymphocytes and macrophages. Like mouse Thy-1, transgenic human CD7 was expressed in immature and mature T cells and in Sca-1+ bone marrow mononuclear cells. Unlike mouse Thy-1, the human CD7 transgene was not expressed in mouse brain or fibroblasts. The human CD7 transgene was expressed during fetal development before mouse Thy-1 in fetal liver mononuclear cells. Expression of the human CD7 transgene did not alter mouse thymopiesis or Thy-1 expression. Taken together, these data demonstrated that the CD7 transgene contained sufficient regulatory regions to direct hematopoietic expression and mitogenic induction. The pattern of CD7 transgene expression more closely resembled that of CD7 in humans than that of mouse Thy-1.

The c-kit proto-oncogene receptor is expressed on a subset of human CD3-CD4-CD8- (triple-negative) thymocytes.

The c-kit receptor is a tyrosine-kinase transmembrane receptor first identified as an oncogene in the HZ4-feline leukemia virus and later found to be important in hematopoiesis in mice. The ligand for this receptor (Steel factor) can stimulate hematopoiesis both in vitro and in vivo. To study the pattern of c-kit receptor expression in normal human hematopoietic progenitor cells, we prepared a monoclonal antibody (9B9) against human c-kit receptor by using a synthetic peptide (amino acids 476-501) from the extracellular domain of c-kit receptor to immunize Balb/c mice. Monoclonal antibody 9B9 bound to recombinant c-kit protein, the erythroleukemic line HEL, the megakaryocytic line MEG-01, and the murine mast cell line P815. Monoclonal antibody 9B9 also bound to the surface of the CD7+CD3-CD4-CD8- T cell lymphoid cell lines DU.528 and HSB2T, and also to 1 to 4% of normal bone-marrow cells. The majority (67 +/- 6%) of CD34+ bone-marrow progenitor cells coexpressed c-kit receptor. Flow-cytometry analysis of immature CD3-CD4-CD8- (triple-negative) thymocytes indicated 30 +/- 9.5% expressed the c-kit receptor, and thymidine incorporation assay revealed that the receptor is functional. Indirect fluorescent microscopy of human thymic tissue, using a monoclonal antibody against Steel factor, revealed its presence on scattered mononuclear cells within the intralobular septae and the subcapsular cortex, which are regions where the triple-negative thymocytes are also localized. These data provide evidence that the c-kit receptor is present on human hematopoietic bone marrow and intrathymic T cell progenitor cells, and that it likely plays a role in early T cell lymphopoiesis.

A novel beta-globin mutation, beta Durham-NC [beta 114 Leu-->Pro], produces a dominant thalassemia-like phenotype.

Mutations within exon 3 of the beta-globin gene are relatively uncommon, and many of these mutations produce a dominant thalassemia-like phenotype. We describe a novel thalassemic hemoglobinopathy caused by a single nucleotide substitution (CTG-->CCG) at codon 114 resulting in a leucine to proline substitution and designate it beta Durham-NC [beta 114 Leu-->Pro]. The mutation producing this thalassemic hemoglobinopathy is located near to the beta Showa-Yakushiji mutation (beta 110 Leu-->Pro). Both of these hemoglobinopathies share similar phenotypic features with moderately severe microcytic anemia. Using computer imaging of the hemoglobin molecule, we examined several reported point mutations within exon 3 of the beta-globin gene. These point mutations cause a single amino acid substitution in the G helix, and result in a thalassemic and/or hemolytic phenotype. Computer imaging of nine separate examples suggests that amino acid substitutions affecting side chains that project into the heme pocket may destabilize the heme moiety within the beta-globin chain, resulting in a thalassemic phenotype. Hemolytic phenotypes may be the result of decreased alpha 1 beta 1 interactions. The beta Durham-NC mutation further characterizes a novel group of thalassemias/hemoglobinopathies that are clinically difficult to identify and require accessory laboratory testing.

Two new polymorphisms but no mutations of the KIT gene in patients with myelodysplasia at positions corresponding to human FMS and murine W locus mutational hot spots.

The KIT proto-oncogene encodes a tyrosine kinase receptor which plays a critical role in haemopoiesis. We have screened genomic DNA from bone marrow mononuclear cells of 46 patients with myelodysplasia (MDS) for mutations/deletions of exons 6, 13, 17, and 21 of the KIT gene (stem cell factor receptor) using polymerase chain reaction (PCR), polyacrylamide gel electrophoresis, and autoradiography to detect single-stranded conformational polymorphisms (SSCP). These exons include positions analogous to those mutated in the FMS gene (colony-stimulating factor-1 receptor) in myelodysplastic syndrome (MDS) and mutated/deleted in the Dominant White Spotting mouse (W locus) which results in macrocytic anaemia. Two different gel running conditions were used for each exon. Polymorphisms were identified only at 4 degrees C in exon 17 (three out of 44 MDS samples and two of 21 DNA samples from normal subjects), and in the non-coding region of exon 21 (five out of 34 MDS samples and seven out of 19 normals). Direct sequencing identified a G to A base change at nucleotide 3169 within exon 21, and a C to T change at position 2415 in exon 17. No conformational changes suggestive of mutations or deletions have been found to date, although we cannot rule out low frequency clonal abnormalities undetectable by our method, which has a sensitivity in our hands of approximately 5%. Polymorphisms occur frequently in the KIT gene. Together with this study, a total of five have been described.

Genotypic divergence precedes clinical dissemination in a case of synchronous bilateral B-cell malignant lymphoma of the testes.

Malignant lymphoma of the testis occurs bilaterally more often than any other tumor type. We report the case of a 62-year-old man who presented with synchronous, bilateral, testicular malignant lymphomas without clinical or radiologic evidence of extratesticular disease. The patient received no therapy other than bilateral orchiectomy and subsequently developed widespread disease 6 months later. Southern blot DNA analysis was performed on the initial orchiectomy samples for immunoglobulin (Ig) gene rearrangements. These genotypic analyses showed different clonal rearrangements in the Ig heavy chain JH region but identical clonal rearrangements in the Ig light chain C Kappa region. To our knowledge this is the first genotypic demonstration of a common clonal origin in synchronous, bilateral, testicular malignant lymphomas. We interpret these findings as molecular evidence that the patient's malignant lymphoma was already disseminated at initial presentation, although it was clinically undetectable at that time.

Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain.

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

Structure of the CD59-encoding gene: further evidence of a relationship to murine lymphocyte antigen Ly-6 protein.

The gene for CD59 [membrane inhibitor of reactive lysis (MIRL), protectin], a phosphatidylinositol-linked surface glycoprotein that regulates the formation of the polymeric C9 complex of complement and that is deficient on the abnormal hematopoietic cells of patients with paroxysmal nocturnal hemoglobinuria, consists of four exons spanning 20 kilobases. The untranslated first exon is preceded by a G+C-rich promoter region that lacks a consensus TATA or CAAT motif. The second exon encodes the hydrophobic leader sequence of the protein, and the third exon encodes the amino-terminal portion of the mature protein. The fourth exon encodes the remainder of the mature protein, including the hydrophobic sequence necessary for glycosyl-phosphatidylinositol anchor attachment. The structure of the CD59 gene is very similar to that encoding Ly-6, a murine glycoprotein with which CD59 has some structural similarity. The striking similarity in gene structure is further evidence that the two proteins belong to a superfamily of proteins that may also include the urokinase plasminogen-activator receptor and a squid glycoprotein of unknown function.

Cloning and structural analysis of the human c-kit gene.

The recent identification of the mouse White spotting and Steel loci as genes encoding the c-kit receptor and its ligand, respectively, has shed light on the importance of this ligand and receptor in embryogenesis, melanogenesis and hematopoiesis. In order to determine if the c-kit proto-oncogene is involved in human disease, we isolated seven overlapping lambda recombinants, using a fetal brain cDNA, and characterized the normal human gene (KIT). The longest mapped transcript is 5230 bp, is alternatively spliced and includes 21 exons that span more than 70 kb of DNA. From the exon-intron structure, we have localized an alternative splice site to the 3' end of exon 9. The overall c-kit gene structure closely resembles that found in the CSF-1R gene (c-fms). This similarity includes a large first intron, the same number of exons containing translated sequence and very similar exon-intron boundaries. Using pulsed-field gel electrophoresis, we have linked KIT to the platelet-derived growth factor receptor A gene, with both residing on a 700-kb BssHI fragment. These data will allow investigation into the control of KIT expression and the potential to identify mutations or altered expression of this gene in human disease.

Isolation and characterization of the genomic human CD7 gene: structural similarity with the murine Thy-1 gene.

The human CD7 molecule is a 40-kDa member of the immunoglobulin gene superfamily that is expressed on T-lymphoid and myeloid precursors in fetal liver and bone marrow. CD7 is also expressed on T lymphocytes in multiple stages of T-cell development, including a major subset of mature peripheral T cells. In this paper we report the isolation and characterization of the human CD7 gene and 5' flanking region. Sequence analysis revealed that the CD7 gene comprises four exons that span 3.5 kilobases. The 5' flanking region (506 base pairs) has a high G + C content and no "TATA" or "CCAAT" elements. DNase I sensitivity analysis of chromatin from the CD7+ progenitor cell leukemia line, DU528, and the CD7-, CD4+, CD8+, TCR alpha beta + T-cell line, DU980 (where TCR is the T-cell receptor), revealed two distinct hypersensitive sites 5' of the CD7 gene. Hypersensitive site 1, present in the DU980 T-cell line, was located 4.5 kilobases upstream of the presumed CD7 transcription initiation site. Only DNase I hypersensitive site 2, which mapped to the promoter region, was found in the DU528 line. Comparison of the organization of the CD7 gene with that of other members of the immunoglobulin gene superfamily revealed that the human CD7 gene most closely resembles the murine Thy-1 gene. Both CD7 and Thy-1 are encoded by small genes with four exons, contain TATA-less promoters, and have a similar functional organization. These structural similarities suggest that human CD7 and murine Thy-1 may be functional homologues.

Dissociation of thymidine incorporation and transferrin receptor expression from cell growth and c-myc accumulation in alpha-interferon-treated cells.

Alpha-interferon is capable of altering the pattern of growth of both normal and neoplastic cells, but the pathways essential to sensitivity and resistance to alpha-interferon are unknown. To explore the growth inhibition induced by alpha-interferon, we examined the interferon-sensitive cell line Daudi and the resistant cell line HL-60. In Daudi, alpha-interferon induced a fall in c-myc mRNA accumulation at 24 h, inhibited tritiated thymidine ([3H]Thd) uptake at 48-72 h, and inhibited proliferation at 72-96 h. The half-life of c-myc mRNA was shortened from 31 to 13 min by alpha-interferon treatment. In HL-60, no alteration in c-myc accumulation or cell growth was observed, but [3H]Thd uptake was inhibited by 49%. Exogenous thymidine partially reversed the effects of alpha-interferon on [3H]Thd incorporation. The number of transferrin receptors, as measured by immunofluorescence, was unaffected by alpha-interferon in both cell lines. We conclude that the growth inhibitory effects of alpha-interferon are neither dependent upon inhibition of thymidine metabolism nor on expression of the transferrin receptor, but may be linked to control of c-myc.