CD4 Locus Research Articles

Replication Timing of CD4 and CD8 in Single-Positive Peripheral Blood Lymphocytes

The regulatory events leading to the mutually exclusive expression of CD4 and CD8 on peripheral lymphocytes are not fully understood. In particular, the association between DNA replication timing and transcriptional activity of these genes has not been previously investigated. Here, the replication kinetics of the CD4 and CD8 loci in mature single-positive T-cell populations have been examined using a novel approach to the separation of CD4+ or CD8+ lymphocytes into discrete cell cycle fractions and a competitive PCR replication timing assay. While the timing of replication of each of these loci is independent of their expression in mature CD4 or CD8 single positive T-cells, the replication of CD8, but not of CD4, shifts to a later time in S phase in transcriptionally silent HS68 fibroblast cells. These findings suggest that changes in DNA replication timing are associated with the developmentally regulated but not with the tissue-specific expression of CD4 and CD8.

Control of CD4 gene expression: connecting signals to outcomes in T cell development.

The control of CD4 gene expression is essential for proper T lymphocyte development. Signals transmitted from the T-cell antigen receptor (TCR) during the thymic selection processes are believed to be linked to the regulation of CD4 gene expression during specific stages of T cell development. Thus, a study of the factors that control CD4 gene expression may lead to further insight into the molecular mechanisms that drive thymic selection. In this review, we discuss the work conducted to date to identify and characterize the cis-acting transcriptional control elements in the CD4 locus and the DNA-binding factors that mediate their function. From these studies, it is becoming clear that the molecular mechanisms controlling CD4 gene expression are very complex and differ at each stage of development. Thus, the control of CD4 expression is subject to many different influences as the thymocyte develops.

Estimating European admixture in African Americans by using microsatellites and a microsatellite haplotype (CD4/Alu).

We have analyzed 10 unlinked microsatellites and a linked Alu deletion polymorphism at the CD4 locus in an African American population sample from Chicago (USA). Heterozygosity estimates at the microsatellite loci range from 0.727+/-0.025 (D3S1358) to 0.873+/-0.017 (D18S51), with an average of 0.794+/-0.016. These values are comparable to or higher than those reported for Europeans, with only one exception (D3S1358). The CD4/Alu haplotypic diversity (0.887+/-0.012) is comparable to diversity levels observed in sub-Saharan African populations and is higher than the diversity levels reported in European populations. No consistent pattern of within, between, or multi-locus deviations from Hardy-Weinberg expectations is observed, suggesting a low sub-heterogeneity within the sampled population. We have applied a maximum likelihood method and estimated the proportion of European admixture to the African American gene pool to be 0.26+/-0.02. The narrow confidence interval indicates that allele frequency data from multiple microsatellite loci, whether analyzed independently or as haplotypes, are particularly useful for estimating genetic admixture.

Simultaneous Determination of STR Polymorphism and a New Nucleotide Substitution in Its Flanking Region at the CD4 Locus

In the course of the investigation of a pentanucleotide repeat polymorphism at the human CD4 locus, a C-A transversion was found at the position corresponding to the 3' end of the original forward primer presented by Edwards et al. (1). In the present study, the simultaneous determination of the new sequence polymorphism and the pentanucleotide repeat polymorphism at the CD4 locus was attempted. To achieve this purpose, we adopted amplified product length polymorphism (APLP) analysis and designed some new allele-specific forward primers tagged with non-complementary nucleotides differing in length. A total of 646 DNA samples from peripheral blood of Japanese, Chinese and German populations were investigated. Although the C-A transversion was restricted to CD4*5, a new subtype allele with A and 5 repeats, designated CD4*5A, was observed at polymorphic frequencies in the three populations. The simultaneous genotyping by APLP analysis resulted in dramatically increased heterozygosity and discriminating power of the human CD4 locus.

Microsatellite profiles reveal an unexpected genetic relationship between Asian populations.

Allelic frequencies for up to five short tandem repeat systems (HumTH01. HumVWA, HumF13B, HumCD4, HumD2111) were analyzed in seven population samples from Asia using the polymerase chain reaction and gel electrophoresis. No deviations from Hardy-Weinberg equilibrium were observed. Two new alleles of the CD4 and TH01 loci were detected, and sequenced and their molecular structure is presented. A phylogenetic tree based on Thai, Han Chinese (from the northeast of China), Japanese, German and Ovambo allelic frequencies was constructed and demonstrates the close relationship of the Asian populations. Additionally, allelic frequency data for the VWA and TH01 systems were determined for the south Chinese minorities Bai, Dai and Qiang and for Koreans and compared with the above data. The Bai and Dai populations were clear outliers of the cluster of all other Asians, indicating an unexpected pattern of genetic heterogeneity of the Chinese nation. Two clusters of Asian populations could be established: the Koreans and Japanese together with the Han and Qiang Chinese, and, forming a separate cluster, the Bai and Dai populations.

Regulation of expression of the human lymphocyte activation gene-3 (LAG-3) molecule, a ligand for MHC class II.

The lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II ligand evolutionarily related to CD4, is expressed exclusively in activated T and NK lymphocytes and seems to play a role in regulating the evolving immune response. We first determined that surface LAG-3 expression on activated human T cells is upregulated by certain cytokines (IL-2, IL-7, IL-12) and not by others (IL-4, IL-6, IL-10, TNF-alpha, TNF-beta, IFN-gamma). Surface LAG-3 expression correlated with intracellular IFN-gamma production in both CD4+ and CD8+ T-cell subsets. We then analyzed the 5' transcription control sequences of LAG-3. A DNase I hypersensitive site induced in T cells following cellular activation was found in the region including the transcriptional start site, showing that DNA accessibility is a mechanism which restricts LAG-3 expression to activated T cells. Transcription is initiated at three sites. A GC box, 80 base pairs (bp) upstream of the major transcription start site, forms a minimal promoter which is regulated by two upstream regions containing positive and negative regulatory elements with multiple protein binding sites as shown by footprinting analysis. In particular, a GATA/c-Ets motive was identified in a short segment homologous to the mouse CD4 distal enhancer, suggesting that LAG-3, which is embedded in the CD4 locus, may be controlled by some CD4 regulatory elements. Finally, a 100 bp region downstream of the transcription start site was shown to be involved in the cell-specific control of LAG-3 expression. Understanding this highly regulated expression may help to determine the intriguing role of this activation-induced MHC class II ligand.

Likelihood Analysis of Disequilibrium Mapping, and Related Problems

In this paper a theory is developed that provides the sampling distribution of alleles at a diallelic marker locus closely linked to a low-frequency allele that arose as a single mutant. The sampling distribution provides a basis for maximum-likelihood estimation of either the recombination rate, the mutation rate, or the age of the allele, provided that the two other parameters are known. This theory is applied to (1) the data of Hästbacka et al., to estimate the recombination rate between a locus associated with diastrophic dysplasia and a linked RFLP marker; (2) the data of Risch et al., to estimate the age of a presumptive allele causing idiopathic distortion dystonia in Ashkenazi jews; and (3) the data of Tishkoff et al., to estimate the date at which, at the CD4 locus, non-African lineages diverged from African lineages. We conclude that the extent of linkage disequilibrium can lead to relatively accurate estimates of recombination and mutation rates and that those estimates are not very sensitive to parameters, such as the population age, whose values are not known with certainty. In contrast, we also conclude that, in many cases, linkage disequilibrium may not lead to useful estimates of allele age, because of the relatively large degree of uncertainly in those estimates.

Genomic organization of the human LAG-3/CD4 locus.

Genomic organization of the human LAG-3/CD4 locus.

Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene

Regulated expression of recombinant genes in CD4+ cells is an important objective for gene therapy of AIDS, as these cells represent the principal target for viral replication of human immunodeficiency virus (HIV). We report here that specific combinations of CD4 cell-specific and viral regulatory elements can enhance expression of an antiviral gene product. Different viral regulatory elements were incorporated into a previously reported CD4 locus control region to increase the expression of reporter genes in T and monocytic cell lines. The CD4-specific regulatory elements were included to enhance expression in CD4 cells, and viral regulatory regions, including the cytomegalovirus immediate-early (CMV IE) upstream enhancer, which contains the kappa B and Ap1 regulatory elements and a Tat-responsive element of the HIV type 1 long terminal repeat, were used to increase gene expression and modulate its activity in response to viral infection. In transient transfection assays, this vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone. Expression of an inhibitory form of the Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4+ peripheral blood mononuclear cells. The combination of CD4 lineage-specific and viral regulatory elements will facilitate the development of more effective antiviral genetic strategies for AIDS.

Molecular haplotyping of genetic markers 10 kb apart by allele-specific long-range PCR.

Haplotypes, combinations of polymorphic markers in a chromosome, are critical for genome diversity research. However, their utility in population samplings is compromised by uncertain linkage phase determinations from unrelated individuals. Molecular haplotyping accomplishes direct phase determination by generation of hemizygous templates from diploid genomic samples. We report molecular haplotyping by allele-specific long-range PCR of two markers 9.5 kb apart at the CD4 locus: a bi-allelic Alu deletion and a multi-allelic repeat. We verified CD4 molecular haplotypes by classical Mendelian analysis. Molecular haplotyping should prove useful in mapping disease genes and in establishing founder effects.

Sequence determination of an allelic ladder for the STR polymorphism at the CD4 locus and application of the ladder in testing an Austrian Caucasian population sample

The short tandem repeat (STR) polymorphism at the CD4 locus, designated HUMCD4. was examined by PCR, native polyacrylamide electrophoresis and subsequent silver staining using an allelic ladder of eight distinguishable alleles occurring in an Austrian Caucasian population sample as a standard size marker. The ladder was produced by pooling equal concentrations of eluted, separately amplified and sequenced alleles, which were previously identified by their different electrophoretical migration. Components of the ladder are in regular intervals of five basepairs. Alleles 4 to 8 were designated according to the number of AAAAG repeat units. The four longer alleles 8′ to 11 showed a stable A to G transition in one of the repeat units and were designated counting the AAAGG unit for a AAAAG. Allele 8′ was not included in the ladder because it showed the same electrophoretic mobility as allele 8. This ladder proved to be a precise and reliable tool in the analysis of 600 chromosomes of the Austrian population. The population investigated showed no deviation from Hardy-Weinberg equilibrium ( P = 0.23).

Global patterns of linkage disequilibrium at the CD4 locus and modern human origins.

Haplotypes consisting of alleles at a short tandem repeat polymorphism (STRP) and an Alu deletion polymorphism at the CD4 locus on chromosome 12 were analyzed in more than 1600 individuals sampled from 42 geographically dispersed populations (13 African, 2 Middle Eastern, 7 European, 9 Asian, 3 Pacific, and 8 Amerindian). Sub-Saharan African populations had more haplotypes and exhibited more variability in frequencies of haplotypes than the Northeast African or non-African populations. The Alu deletion was nearly always associated with a single STRP allele in non-African and Northeast African populations but was associated with a wide range of STRP alleles in the sub-Saharan African populations. This global pattern of haplotype variation and linkage disequilibrium suggests a common and recent African origin for all non-African human populations.

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

The coordinated expression of CD4 and CD8 during T-cell development is tightly coupled with the maturation state of the T cell. Additionally, the mutually exclusive expression of these receptors in mature T cells is representative of the functional T-cell subclasses (CD4+ helper T cells versus CD8+ cytotoxic T cells). We have studied the regulation CD4 gene transcription during T-cell development in an attempt to gain an understanding of the molecular mechanisms involved in T-cell development and differentiation. Here we present the identification of a second transcriptional enhancer in the murine CD4 locus 24 kb upstream of the CD4 promoter. This enhancer is active in mature T cells and is especially active in CD4+ helper T cells. A number of nuclear proteins bind to elements in the minimal CD4 enhancer that includes consensus sites for AP-1, Sp1, Gata, and Ets transcription factor families. We find that the Ets consensus site is crucial for enhancer activity and that the recently identified Ets factor, Elf-1, which is expressed at high levels in T cells and involved in the regulation of several other T-cell-specific genes, is a dominant protein in T-cell nuclear extracts that binds to this site.

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

The coordinated expression of CD4 and CD8 during T-cell development is tightly coupled with the maturation state of the T cell. Additionally, the mutually exclusive expression of these receptors in mature T cells is representative of the functional T-cell subclasses (CD4+ helper T cells versus CD8+ cytotoxic T cells). We have studied the regulation CD4 gene transcription during T-cell development in an attempt to gain an understanding of the molecular mechanisms involved in T-cell development and differentiation. Here we present the identification of a second transcriptional enhancer in the murine CD4 locus 24 kb upstream of the CD4 promoter. This enhancer is active in mature T cells and is especially active in CD4+ helper T cells. A number of nuclear proteins bind to elements in the minimal CD4 enhancer that includes consensus sites for AP-1, Sp1, Gata, and Ets transcription factor families. We find that the Ets consensus site is crucial for enhancer activity and that the recently identified Ets factor, Elf-1, which is expressed at high levels in T cells and involved in the regulation of several other T-cell-specific genes, is a dominant protein in T-cell nuclear extracts that binds to this site.

A transcriptional silencer controls the developmental expression of the CD4 gene.

The appropriate expression of the CD4 glycoprotein is required for T-cell function and development. Here we define the transcriptional control elements in the CD4 locus that convey CD(4+)-specific expression of a marker gene in transgenic mice. Using nuclear run-on experiments, we have determined that the major mechanism for CD4 expression control during development is transcriptional. We have identified a developmental stage- and tissue-specific negative regulatory element in the first intron of the murine CD4 gene that has the characteristics of a transcriptional silencer. The CD4 silencer functions to inhibit marker gene expression at two different stages of T-cell development, as well as in non-T hematopoietic cells, and thus is the critical controlling element responsible for T-cell-specific, as well as developmental- and subclass-specific, expression.

Cis-dominant regulation of CD4 and CD8 gene expression in rat/mouse T cell heterohybridomas.

To determine whether expression of CD4 and CD8 molecules on T cells is determined-solely by transacting regulators, we examined heterohybridomas derived from the fusion of a rat CD4+ T cell line and the CD4- CD8- mouse thymoma BW5147. The majority of hybrid offspring expressed rat CD4. However, a fraction of the cell lines obtained expressed not only rat CD4 but also various amounts of mouse CD4 and CD8 molecules from both species. Cloning of two of these heterogeneous lines revealed that expression of all four Ag varied not only between different clones but also within clonal populations. The expression of Ag not present on the parental cells suggested an alteration in the normally stable regulatory mechanisms present in those cells. Moreover, a lack of concordant expression between the rat and mouse loci was observed, indicating that active and silent homologous loci can exist together in single nuclei. Expression of CD4 and CD8 in these cells, therefore, cannot be solely mediated by trans-acting diffusible regulators but must also depend on cis-dominant effects on the loci themselves. The phenotypic heterogeneity of clonal populations was found to result from unpredictable shifts, both positive and negative, in the expression of CD4 and CD8 over time, indicating that the cis-dominant effects were only quasistable. Preliminary examinations of the density of 5-methylcytosine within the CD4 and CD8 loci in various phenotypic populations separated by FACS from within heterogeneous clones revealed a correlation between surface expression of the mouse CD8 protein and a lack of methylation around the mouse CD8 gene. In contrast, the CD4 gene remained extensively methylated regardless of its surface expression.

T cell-specific protein-DNA interactions occurring at the CD4 locus: identification of possible transcriptional control elements of the murine CD4 gene.

The cis-acting transcriptional control elements of the murine CD4 gene were investigated within 75 kb of chromatin associated with the CD4 locus. DNase I hypersensitive (DH) sites were identified in several T and non-T cell lines, and in freshly isolated thymocytes. A total of 22 DH sites were found, seven of which are present only in T cells expressing CD4 or CD8. The T cell-specific DH sites are located in four regions: (i) 5' of the first exon of CD4, (ii) in the first intron, (iii) near the second and third exons, and (iv) 3' of the CD4 gene. Some of these sites inversely correlate to the CD4 expression at defined stages of T cell development, suggesting a role for these sites in repression of this gene. The T cell-specific DH sites were subcloned and analyzed for protein-DNA interactions using the electrophoretic mobility shift assay. All T cell-specific DH sites analyzed appear to be a consequence of T cell-specific protein-DNA interactions. We have also identified the nuclear matrix attachment regions (MARs) and repetitive elements associated with the CD4 gene. Two nuclear MARs, separated by a region of highly repetitive DNA, are located 5' of the gene. Another region of highly repetitive DNA exists within the third intron. We discuss the implications of our results for the developmental regulation of CD4 expression.

Gene targeting at the human CD4 locus by epitope addition.

Homologous recombination at the CD4 locus in a human T-cell line has been achieved by an approach called epitope addition. The endogenous CD4 gene provided transcription, translation, and leader sequences to a crippled introduced Thy-1 gene, resulting in the expression of murine Thy-1 epitopes on the surface of the human cells. Thy-1+ cells were selected using the Fluorescence Activated Cell Sorter (FACS). An estimated 700-fold enrichment for homologous versus nonhomologous integration events was obtained, such that 70% of cells scoring positive for Thy-1 were derived from gene targeting. Three of the Thy-1+ cell lines expressed protein only from the targeted allele; thus, these cells were functionally CD4-.