CD4 Locus Research Articles

CD8 Locus Nuclear Dynamics during Thymocyte Development

Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.

Epigenetic propagation of CD4 expression is established by theCd4proximal enhancer in helper T cells

The stability of a lineage program (cellular memory) is dependent on mechanisms that epigenetically maintain active or repressed states of gene expression (transcriptional memory). Although epigenetic silencing of genes has been clearly demonstrated from yeast to mammals, heritable maintenance of active transcription has been less clearly defined. To investigate the potential role of active transcriptional memory during lineage diversification, we employed targeted mutation of a positive-acting cis element in the Cd4 locus to determine the impact on CD4 expression and the differentiation of CD4(+) helper T cells in mice. We show that the proximal enhancer (E4(P)) of Cd4 is essential for CD4 expression in immature CD4(+)8(+) thymocytes. Furthermore, its loss resulted in reduced and unstable expression of CD4 in mature T cells. However, if the enhancer was deleted after cells had already committed to the helper T-cell lineage, CD4 expression remained high and was stable upon cell division. "Active" histone modifications, once initiated by E4(P), were also propagated independently of the enhancer. Thus, E4(P) is responsible for establishing an epigenetically inherited active Cd4 locus in the helper T-cell lineage. To our knowledge, this is the first genetic demonstration of active transcriptional memory in mammalian cells.

Lmo2 Induces T-Cell Leukemia with Epigenetic Deregulation of CD4.

The LIM domain Only-2 (Lmo2) oncogene is deregulated in the majority of human T-cell acute lymphoblastic leukemias (T-ALL). Enforced, constitutive expression of Lmo2 in transgenic mice using the T-cell-specific Cd2 promoter/enhancer induces T-ALL with median latency of 200 days and 100% penetrance. These transgenic mice have a pre-leukemic differentiation block at the double negative stage (CD4− CD8−) of T-cell differentiation. Despite this, T-ALLs in these mice arise from various stages of T-cell differentiation. To explore the relationship between T-cell development and Lmo2 oncogene function, we established cell lines from four independent T-cell tumors representing the double negative (DN), double positive (DP), and single positive (SP) stages. Two of the lines showed variegated expression of CD4 antigen. One line, 32080, was striking in that CD4 was expressed in 50% of cells whereas CD8 was uniformly present. The cell line expressed markers consistent with the intermediate single positive stage (ISP) to double positive (DP) stage transition. We sorted the 32080 line for highly CD4 positive and negative cells and observed them in culture. After one week, both sorted populations showed variegated CD4 expression. We determined CD4 expression was regulated at the transcriptional level and not due to post-transcriptional effects and that cell replication was necessary to re-establish CD4 variegation. Importantly, variegated CD4 expression was dependent on Lmo2 because its knockdown resulted in decreased CD4 expression. HDAC inhibition by trichostatin A had the reciprocal effect of inducing more CD4 expression suggesting that chromatin modification was playing a role. The CD4 locus was analyzed by chromatin immunoprecipitation and histone H3 methylation patterns consistent with silencing were found in the CD4 negative sorted cells but not in the CD4 positive population. The 32080 cell line is a striking model of ISP to DP T-cell plasticity and invokes a novel mechanism by which Lmo2 functions.

A novel genetic strategy reveals unexpected roles of the Swi–Snf–like chromatin-remodeling BAF complex in thymocyte development

We have developed a general strategy for creating littermates bearing either a tissue-specific point mutation or deletion in any target gene, and used the method to dissect the roles of Brg, the ATPase subunit of the chromatin-remodeling Brg-associated factor (BAF) complex, in early thymocyte development. We found that a point mutation that inactivates the Brg ATPase recapitulates multiple defects previously described for Brg deletion (Chi, T.H., M. Wan, P.P. Lee, K. Akashi, D. Metzger, P. Chambon, C.B. Wilson, and G.R. Crabtree. 2003. Immunity. 19:169–182). However, the point mutant helps reveal unexpected roles of Brg in CD25 repression and CD4 activation. Surprisingly, CD4 activation occurs independently of the Brg ATPase and is perhaps mediated by physical interactions between Brg and the CD4 locus. Our study thus suggests that the BAF complex harbors novel activities that can be necessary and even sufficient for stimulating transcription from an endogenous chromatin template in the absence of Brg-dependent remodeling of that template. We conclude that conditional point mutants, rarely used in mammalian genetics, can help uncover important gene functions undetectable or overlooked in deletion mutants.

Correction for Yu et al. , Nucleoprotein structure of the CD4 locus: Implications for the mechanisms underlying CD4 regulation during T cell development

Correction for Yu <i>et al.</i> , Nucleoprotein structure of the CD4 locus: Implications for the mechanisms underlying CD4 regulation during T cell development

Nucleoprotein structure of the CD4 locus: Implications for the mechanisms underlying CD4 regulation during T cell development

The CD4 gene is regulated in a stage-specific manner during T cell development, being repressed in CD4(-)CD8(-) double-negative (DN) and CD8 cells, but expressed in CD4(+)CD8(+) double-positive (DP) and CD4 cells. Furthermore, the expression/repression pattern is reversible in developing (DN and DP) thymocytes, but irreversible in mature (CD4 and CD8) T cells. Here, we explored the molecular mechanisms underlying this complex mode of regulation by examining the nucleoprotein structure of the CD4 locus throughout T cell development and in DN cells lacking the CD4 silencer. In DN cells, the CD4 enhancer is preloaded with multiple transcription activators, but p300 recruitment is impaired by the silencer that is associated with the repressor Runx1. DP cells achieve high-level CD4 expression via a combination of CD4 derepression and true activation, but Runx1 remains bound to the silencer that retains an open chromatin configuration. In CD4 cells, Runx1 dissociates from the silencer that has become less accessible, and CD4 transcription appears to be achieved via a mechanism distinct from that operating in DP cells. In CD8 cells, the CD4 promoter becomes incorporated into heterochromatin-like structure. Our data shed light on the molecular basis of CD4 regulation and provide a conceptual framework for understanding how the same regulatory elements can mediate both reversible and irreversible CD4 regulation.

Antagonistic Interactions between Ikaros and the Chromatin Remodeler Mi-2β Determine Silencer Activity and Cd4 Gene Expression

Lineage commitment is induced by changes in gene expression dictated by the intimate interaction between transcription factors and chromatin regulators. Here, we revealed the antagonistic interplay between Ikaros and its associate the chromatin remodeler Mi-2beta during T cell development, as exemplified by the regulation of Cd4 expression. Loss of Ikaros or Mi-2beta led to activation or repression, respectively, of the Cd4 locus at inappropriate stages of development. Their combined mutation reverted to normal CD4 expression. In double-negative thymocytes, Ikaros binding to the Cd4 silencer contributed to its repressive activity. In double-positive thymocytes, concomitant binding of Mi-2beta with Ikaros to the Cd4 silencer caused silencer inactivation, thereby allowing for CD4 expression. Mi-2beta facilitated recruitment of histone acetyl transferases to the silencer. This recruitment possibly antagonized Ikaros and associated repressive activities. Thus, concomitant interactions between functionally opposing chromatin-regulating machineries are an important mode of gene regulation during lineage determination.

Role of the transcription factor Th‐POK in CD4:CD8 lineage commitment

The molecular basis of CD4:CD8 lineage commitment, in particular the mechanism by which the precise correlation between lineage choice and T-cell receptor (TCR) specificity toward class I or II major histocompatibility complex is achieved, remains controversial. Both stochastic/selective and instructive models in various forms have been proposed to explain this correlation. The two main experimental approaches previously employed to elucidate this process have focused on the beginning and end of the process, i.e. the influence of TCR signaling and the alternate transcriptional control of the CD4 and CD8 loci during commitment. The recent finding that the transcription factor Th-POK is necessary and sufficient for CD4 commitment has now provided a direct entry point for studying the intracellular pathways that govern lineage commitment. Here, we review data leading to the identification and characterization of this factor and discuss the implications of these studies in the context of current models of lineage commitment.

Molecular Genetic Analysis of TPO and CD4 Loci Among Two Endogamous Ethnic Groups of Maharashtra in Western India

Molecular genetic polymorphism study was undertaken in two endogamous ethnic groups of Maharashtra in Western India at two microsatellites i.e., TPO, a tetranucleotide repeat locus and CD4, a pentanucleotide repeat locus. The two ethnic groups studied were Konkanastha Brahmins and Marathas belonging to Indo-European language family. Eighty-two random, unrelated individuals were genotyped for the locus TPO, whereas for CD4 locus, 79 individuals were genotyped.

Epigenetic gene silencing by Runx proteins.

Runx family proteins have the potential for either activating or suppressing gene expression in a context-dependent manner. There are several mechanisms by which transcriptional repression can occur. A wide range of locus inactivation, that is often called gene silencing, is thought to be achieved by chromatin modifications. Recently, Runx family proteins were found to have an essential role in either temporal transcriptional repression or irreversible epigenetic silencing at the CD4 locus through binding to a CD4 silencer at different stages of development. These findings link Runx function to epigenetic gene regulation, and shed new light on the mechanisms by which Runx represses target gene expression.

The CD4⧸CD8 Lineage Choice: New Insights into Epigenetic Regulation during T Cell Development

Close examination of how the CD4 and CD8 genes are regulated during T lymphocyte development, not only in the thymus but also in secondary lymphoid organs, has provided with valuable insight into mechanisms of lineage specification. Studies in this area promise to uncover how helper versus cytotoxic T cells differentiate, and, possibly, even how effector T cells give rise to subsets of long-lived memory T cells with specialized activation properties. In a more general sense, examination of these loci has provided a unique opportunity to study epigenetic regulation in vertebrate development. The CD4 locus remains the only vertebrate gene currently shown to undergo developmentally regulated epigenetic silencing, and as such, it presents an ideal system for analyzing how heterochromatin is established during developmental processes in higher eukaryotes.

Frequencies at CD4, FES, and F13A1 Microsatellite Loci in Central-Southern Sardinia (Italy)

Abstract DNA samples were extracted and purified by salting out procedure, precipitated by absolute ethanol and quantified by 1% agarose gel. The DNAs were amplified by Perkin Elmer PCR system 2400 using standard PCR protocols (2-4). Amplified products were separated in denaturing acrylamide gel using an Automated Laser Fluorescent apparatus as previously described (5). HardyWeinberg tests and analysis of genotypic disequilibrium between unlinked loci were carried out by using the Genepop software (6). The results obtained are shown in Table 1. It is to be noted that in all single-locus and multilocus tests, the p-value was statistically insignificant. Furthermore, we calculated the linkage disequilibrium between genotypes of independent loci. Among the possible three pairs of loci, only one case of statistically significant p-value was obtained (FES/F13A1; p = 0.024). Complete data are available at the e-mail address of the corresponding author upon request.

Genetic Differentiation of the Population of Central Asia Inferred from Autosomal Markers

The gene pool of five ethnic groups of the Central Asian population was characterized using nine human-specific polymorphic insertion/deletion loci (ACE, PLAT, APOA1, PV92, F13B, A25, B65, CD4, Mt-Nuc). It has been shown for the first time that at the CD4 locus, the frequency of Alu(–) is inversely related to the Mongoloid component of the population. For the Central Asian populations, the lowest and highest frequencies of the Alu deletion at locus CD4 were recorded respectively in Dungans (0.04), immigrants from China, and Tajiks (0.15). The coefficient of gene differentiation in the Central Asian populations for all the genes was 2.8%, which indicates a relatively low level of population genetic subdivision in this region. The unity of the gene pool of the Central Asian Caucasoids was shown.

Population data of five STRs in three regions from Portugal

Allele and haplotype frequencies of five chromosome STR loci (CD4, TPO, FES, TH01 and VWA) were determined for unrelated males throughout Portugal. This report presents STR data for three separate regions of Portugal, being the first time that data on the south of the country is presented. This study reveals that the three regions from Portugal are not genetically homogeneous. The north of Portugal presents significant differences in the CD4 locus, when compared with the other two populations. When compared with Madeira and Açores, the three regions show a different behavior at TPO and VWA loci.

Linking CD4 gene expression and 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 selection process are believed to be linked to the regulation of CD4 gene expression during specific stages of T cell development. Thus, the control CD4 gene expression is an ideal model system for studying the molecular mechanisms that drive T cell development. Here, I discuss the characterization of transcriptional control elements in the CD4 locus and the factors that mediate their function. The study of these elements has led to significant insights into the mechanisms in which the T lymphocyte develops it mature functional characteristics.

Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage.

The process of thymocyte development culminates in the maturation of helper (CD4+) and cytotoxic (CD8+) T cells from their common precursors, the CD4+CD8+ double-positive cells. A crucial step during lineage specification is the termination of expression of either the CD4 or the CD8 coreceptor. A silencer element within the first intron of the CD4 gene is sufficient for CD4 transcriptional repression in cells of the cytotoxic lineage, as well as in thymocytes at earlier stages of differentiation. Here we show that the function of the CD4 silencer is required only at distinct stages of development. Its deletion before the initiation of lineage specification resulted in CD4 derepression throughout thymocyte differentiation. By contrast, once cells committed to the cytotoxic CD8+ lineage, the CD4 locus remained silent through subsequent mitoses, even when the silencer element was excised. The epigenetic inheritance of the silenced CD4 locus was not affected by the inhibition of DNA methylation or histone deacetylation, and may thus involve other mechanisms that ensure a stable state of gene expression.

Negative regulation of CD4 gene expression by a HES-1-c-Myb complex.

Expression of the CD4 gene is tightly controlled throughout thymopoiesis. The downregulation of CD4 gene expression in CD4(-) CD8(-) and CD4(-) CD8(+) T lymphocytes is controlled by a transcriptional silencer located in the first intron of the CD4 locus. Here, we determine that the c-Myb transcription factor binds to a functional site in the CD4 silencer. As c-Myb is also required for CD4 promoter function, these data indicate that depending on the context, c-Myb plays both positive and negative roles in the control of CD4 gene expression. Interestingly, a second CD4 silencer-binding factor, HES-1, binds to c-Myb in vivo and induces it to become a transcriptional repressor. We propose that the recruitment of HES-1 and c-Myb to the silencer leads to the formation of a multifactor complex that induces silencer function and repression of CD4 gene expression.

The Accuracy of Statistical Methods for Estimation of Haplotype Frequencies: An Example from the CD4 Locus

Haplotype analysis has become increasingly important for the study of human disease as well as for reconstruction of human population histories. Computer programs have been developed to estimate haplotype frequencies statistically from marker phenotypes in unrelated individuals. However, there currently are few empirical reports on the accuracy of statistical estimates that must infer linkage phase. We have analyzed haplotypes at the CD4 locus on chromosome 12 that consist of a short tandem-repeat polymorphism and an Alu insertion/deletion polymorphism located 9.8 kb apart, in 398 individuals from 10 geographically diverse sub-Saharan African populations. Haplotype frequency estimates obtained using gene counting based on molecularly haplotyped (phase-known) data were compared with haplotype frequency estimates obtained using the expectation-maximization algorithm. We show that the estimated frequencies of common haplotypes do not differ significantly with the use of phase-known versus phase-unknown data. However, rare haplotypes are occasionally miscalled when their presence/absence must be inferred. Thus, for those research questions for which the common haplotypes are most important, frequency estimates based on the phase-unknown marker-typing results from unrelated individuals will be sufficient. However, in cases where knowledge of rare haplotypes is critical, molecular haplotyping will be necessary to determine linkage phase unambiguously.

Northwest African distribution of the CD4/Alu microsatellite haplotypes

We have analysed a linked microsatellite/Alu polymorphism at the CD4 locus (CD4/Alu) in 666 chromosomes from samples of the Iberian Peninsula, Northwest Africa, and West sub‐Saharan Africa. The Iberian Peninsula differs from other European populations by its higher levels of haplotype diversity (0.75), and weaker association between the microsatellite allele 90 and Alu(−) chromosomes. These results are explainable by a substantial gene flow from Northwest Africa. Significant geographic clines for the five major haplotypes suggest a south to north migration from sub‐Saharan Africa into Northwest Africa. In spite of this, the consistent presence of haplotype 110(−) in this area is congruent with an ancient and autochthonous human presence in Northwest Africa.

Northwest African distribution of the CD4/Alu microsatellite haplotypes.

We have analysed a linked microsatellite/Alu polymorphism at the CD4 locus (CD4/Alu) in 666 chromosomes from samples of the Iberian Peninsula, Northwest Africa, and West sub-Saharan Africa. The Iberian Peninsula differs from other European populations by its higher levels of haplotype diversity (0.75), and weaker association between the microsatellite allele 90 and Alu(-) chromosomes. These results are explainable by a substantial gene flow from Northwest Africa. Significant geographic clines for the five major haplotypes suggest a south to north migration from sub-Saharan Africa into Northwest Africa. In spite of this, the consistent presence of haplotype 110(-) in this area is congruent with an ancient and autochthonous human presence in Northwest Africa.