CD4 Locus Research Articles

Epigenetic Reprogramming Leads to Downregulation of CD4 and Functional Changes in African Green Monkey Memory CD4+ T Cells.

African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αβ+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.

Differences in H3K4me3 and chromatin accessibility contribute to altered T-cell receptor signaling in neonatal naïve CD4 T cells.

Neonatal CD4+ T cells have reduced or delayed T-cell receptor (TCR) signaling responses compared with adult cells, but the mechanisms underlying this are poorly understood. This study tested the hypothesis that human neonatal naïve CD4+ TCR signaling and activation deficits are related to differences in H3K4me3 patterning and chromatin accessibility. Following initiation of TCR signaling using anti-CD3/anti-CD28 beads, adult naïve CD4+ T cells demonstrated increased CD69, phospho-CD3ε and interleukin (IL)-2, tumor necrosis factor-α (TNF-α), interferon-γ and IL-17A compared with neonatal cells. By contrast, following TCR-independent activation using phorbol myristate acetate (PMA)/ionomycin, neonatal cells demonstrated increased expression of CD69, IL-2 and TNF-α and equivalent phospho-ERK compared with adult cells. H3K4me3 chromatin immunoprecipitation-sequencing (ChIP-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on separate cohorts of naïve CD4+ T cells from term neonates and adults, and RNA-seq data from neonatal and adult naïve CD4+ T cells were obtained from the Blueprint Consortium. Adult cells demonstrated overall increased chromatin accessibility and a higher proportion of H3K4me3 sites associated with open chromatin and active gene transcription compared with neonatal cells. Adult cells demonstrated increased mRNA expression of the TCR-associated genes FYN, ITK, CD4, LCK and LAT, which was associated with increased H3K4me3 at the FYN and ITK gene loci and increased chromatin accessibility at the CD4, LCK and LAT loci. These findings indicate that neonatal TCR-dependent defects in activation are epigenetically regulated and provide a potentially targetable mechanism to enhance neonatal CD4+ T-cell responses.

Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus

T cell specificity and function are linked during development, as MHC-II-specific TCR signals generate CD4 helper T cells and MHC-I-specific TCR signals generate CD8 cytotoxic T cells, but the basis remains uncertain. We now report that switching coreceptor proteins encoded by Cd4 and Cd8 gene loci functionally reverses the T cell immune system, generating CD4 cytotoxic and CD8 helper T cells. Such functional reversal reveals that coreceptor proteins promote the helper-lineage fate when encoded by Cd4, but promote the cytotoxic-lineage fate when encoded in Cd8—regardless of the coreceptor proteins each locus encodes. Thus, T cell lineage fate is determined by cis-regulatory elements in coreceptor gene loci and is not determined by the coreceptor proteins they encode, invalidating coreceptor signal strength as the basis of lineage fate determination. Moreover, we consider that evolution selected the particular coreceptor proteins that Cd4 and Cd8 gene loci encode to avoid generating functionally reversed T cells because they fail to promote protective immunity against environmental pathogens.

A novel CD4 knockout mouse strain with a spontaneous frameshift mutation in the CD4 locus.

T cells express co-receptors CD4 and CD8, which are involved in the recognition of antigen presented to T cell receptors. The expression of CD4 in thymic hematopoietic cells is crucial for the thymic development and selection of T cells. In this study, we identified a novel CD4 mutant allele that emerged spontaneously in our mouse colony. The frameshift mutation led to a truncated CD4 protein which failed to reach the plasma membrane resulting in impaired development of CD4+ helper T cells. The CRISPR mediated correction of mutant allele restored the membrane CD4 expression. Further, using an adoptive transfer of T cells, we show that this model is an ideal recipient mouse for the study of CD4+ T cells.

CD4 expression in effector T cells depends on DNA demethylation over a developmentally established stimulus-responsive element

The epigenetic patterns that are established during early thymic development might determine mature T cell physiology and function, but the molecular basis and topography of the genetic elements involved are not fully known. Here we show, using the Cd4 locus as a paradigm for early developmental programming, that DNA demethylation during thymic development licenses a novel stimulus-responsive element that is critical for the maintenance of Cd4 gene expression in effector T cells. We document the importance of maintaining high CD4 expression during parasitic infection and show that by driving transcription, this stimulus-responsive element allows for the maintenance of histone H3K4me3 levels during T cell replication, which is critical for preventing de novo DNA methylation at the Cd4 promoter. A failure to undergo epigenetic programming during development leads to gene silencing during effector T cell replication. Our study thus provides evidence of early developmental events shaping the functional fitness of mature effector T cells.

TET-mediated regulation of effector CD4 T cells

Abstract Functionally distinct subsets of pathogen-specific CD4 helper T cells, including T follicular helper (Tfh) and T helper type 1 (Th1) subsets, are critical for orchestrating the induction and maturation of innate and adaptive immune responses. Proper genetic programming of Tfh and Th1 differentiation is required for regulating immune responses to clear primary infections and promote long-lived memory CD4 T cell-mediated protection against pathogen re-exposures. Genetic approaches to study CD4 T cell differentiation have been largely focused on transcriptional programming, but gene regulation is also critically tied to epigenetic modifications, which represents a major knowledge gap in our understanding of CD4 T cell biology. Ten-eleven-translocation (TET) family enzymes facilitate DNA demethylation to influence chromatin accessibility and promote gene expression. Prior work has shown that TET2 activity has been linked to optimal Th1 responses in vitro. TET1 and TET3 proteins mediate Cd4 locus demethylation and are required for stable CD4 expression during thymocyte development. However, the precise role of either TET1 or TET3 activity to mediate CD4 T cell fate and function in vivo has not been directly investigated. Thus, we hypothesized that TET1 and TET3 activity in peripheral, mature CD4 T cells is required for regulating T helper cell differentiation and function. We used conditional genetic and chimeric approaches, coupled with robust systems of infectious disease immunology, to directly test our hypothesis. Our data show substantial CD4 T cell developmental skewing towards Tfh fates in the absence of specific TET family members. This suggests that TET1 and TET3 enzymes play a role in governing Tfh and Th1 CD4 cell fate.

The histone chaperone CAF-1 cooperates with the DNA methyltransferases to maintain Cd4 silencing in cytotoxic T cells.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development

The inheritance of gene expression patterns is dependent on epigenetic regulation, but the establishment and maintenance of epigenetic landscapes during T cell differentiation are incompletely understood. Here we show that two stage-specific Cd4 cis-elements, the previously characterized enhancer E4p and a novel enhancer E4m, coordinately promote Cd4 transcription in mature thymic MHC-II-specific T cells, in part through the canonical Wnt pathway. Specifically, E4p licenses E4m to orchestrate DNA demethylation by TET1 and TET3, which in turn poises the Cd4 locus for transcription in peripheral T cells. Cd4 locus demethylation is important for subsequent Cd4 transcription in activated peripheral T cells wherein these cis-elements become dispensable. By contrast, in developing thymocytes the loss of TET1/3 does not affect Cd4 transcription, highlighting an uncoupled event between transcription and epigenetic modifications. Together our findings reveal an important function for thymic cis-elements in governing gene expression in the periphery via a heritable epigenetic mechanism.

Requirement for intron structures in activating the Cd8a locus

During differentiation of CD4+CD8+ double-positive (DP) thymocytes into the CD4-CD8+ single-positive (CD8SP) thymocytes committed to the cytotoxic T cell lineage, Cd8a transcription is temporally terminated after positive selection and is subsequently reinitiated, a process known as coreceptor reversal. Despite the identification of a transcriptional enhancer in the Cd8a gene that directs reporter transgene expression specifically in CD8SP thymocytes, the molecular mechanisms controlling reactivation of the Cd8a gene are not fully understood. Here, we show that, after positive selection, hCD2 reporter expression from the Cd8a locus, which was generated by insertion of hCD2 cDNA into the first exon of the Cd8a gene, requires the incorporation of intron sequences into the hCD2 transcript. The presence of polyadenylation signals after hCD2 cDNA inhibited hCD2 expression in mature CD8+ T cells, whereas hCD2 expression in DP thymocytes recapitulated the Cd8a expression. Incorporation of the endogenous short intron structure and heterologous intron structure of the Cd4 locus restored hCD2 expression in mature CD8+ T cells in a variegated manner. Interestingly, stage-specific DNA demethylation was impaired in Cd8a reporter alleles that failed to express hCD2 in CD8+ T cells, and intron sequences lacking RNA splicing signals still restored hCD2 expression. These observations indicate that "intron-mediated enhancement" is involved in a stage-specific reactivation of the Cd8a locus harboring hCD2 cDNA. However, the Cd8a gene was transcribed in mature CD8+ T cells, albeit at a lower level, from a mutant Cd8a locus lacking intron structures, suggesting that protein-coding sequences in transcripts affect sensitivity to intron-mediated enhancement.

Exploring RhoA G17V-Mediated T-Cell Dysfunction

The RhoA G17V mutation occurs in 60% of tumors from patients with Angioimmunoblastic T-cell Lymphomas (AITL) as well as in a subset of Peripheral T-cell Lymphoma, not otherwise specified (PTCL-NOS). The non-malignant counterparts of AITL cells are posited to be follicular-helper T-cells (TFH), and RhoA G17V mutation is strongly associated with a TFH gene expression program in both AITL and PTCL-NOS cases. The G17V substitution is predicted to lock the small GTPase RhoA in an inactive conformation, generating a protein that can bind guanine exchange factors more avidly than unmutated RhoA, likely producing neomorphic effects by inhibiting other small GTPases and G-coupled protein receptors.We have generated mice that express RhoA G17V under the control of regulatory elements from the murine CD4 locus, including two separate founder lines with essentially identical developmental and functional phenotypes. These mice demonstrate a severe reduction in thymic and splenic T cells beginning at the thymic CD4/CD8 double-positive stage, consistent with effects reported from loss-of-function (LOF) RhoA models. RhoA G17V mice also show distinctive effects in peripheral T-cell compartments, including a marked reduction in splenic naïve T-cells with a relative increase in the proportion of activated and/or memory T-cells among those that remain. Notably, there is an increase in the proportion of TFHcells among RhoA G17V mice, suggesting that the mutant protein skews differentiation towards this lineage. Also, unlike reports from RhoA LOF models, we observe increased activation of RhoA G17V T cells when sub-optimal T-cell receptor (TCR)-pathway stimulation is performed with CD3 crosslinking, consistent with a neomorphic activity.We have also observed a striking inflammatory tail fibrosis that occurs with essentially complete penetrance by 8-10 weeks of age in RhoA G17V mice but not in littermate controls. Development of fibrosis is preceded by a cellular infiltrate of hematopoietic cells. including a prominent population of CD4+cells, suggesting that CD4-specific RhoA G17V expression promotes autoimmunity.Finally, we crossed RhoA G17V mice with mice lacking expression of TET2 in the hematopoietic compartment (via Vav-Cre-mediated deletion) and the ovalbumin-specific OT-II transgenic TCR. These mice developed spontaneous T-cell lymphomagenesis comprised of Bcl6-expressing CD4 cells and involving the liver, spleen, and peripheral lymph nodes. Further immunization of these mice with NP40-ovalbumin and alum resulted in Bcl-6-expressing T cell lymphomas within 1-4 months after initial immunization. Thus, the same genotype observed in >50% of AITLs (i.e., RhoA G17V/TET2 loss), combined with TCR stimulation rapidly produces T cell lymphomas with TFHphenotypes.In conclusion, T-cells with RhoA G17V mutations may overcome regulatory pathways before and after malignant transformation, including those that control activation and prevent autoreactivity. These phenotypes also provide a platform from which to determine the specific biochemical effects mediated by RhoA G17V that contribute to T-cell hyperactivation and transformation. DisclosuresWeinstock:Novartis: Consultancy, Research Funding.

Abstract A073: Epigenetic regulation of the Cd4 locus

Abstract CD4 helper and CD8 cytotoxic T cells originate from progenitor cells with binary potential. Transcriptional regulatory elements are key in establishing heritable epigenetic mechanisms to ensure gene-expression programs that maintain lineage identity. We show that, during thymocyte development, the co-receptor CD4 is regulated by a stage-specific regulatory element that ensures commitment to the CD4 lineage. Sustained CD4 expression in post-selection thymocytes requires this intronic element situated 3' to a silencer element previously shown to repress cd4 in CD8 cytotoxic T cells. Despite normal CD4 expression before selection, thymocytes lacking this intronic element failed to maintain CD4 expression following positive selection and were redirected to the CD8 lineage after MHC class II–restricted selection. We find that this regulatory element is key for propagating TET enzyme–mediated locus demethylation in CD4 T cells, a process that is likely initiated by another cis-element controlling CD4 expression at the double positive stage, before lineage commitment. This locus demethylation event is critical in ensuring heritable lineage identity and disruption of this process results in unstable CD4 expression and altered gene expression profiles. Cis and trans element interactions and molecular mechanisms governing heritable expression in response to distinct signals that dictate lineage ‘choice’ during T cell development will be presented. Citation Format: Priya Issuree, Kenny Day, Dan R. Littman. Epigenetic regulation of the Cd4 locus [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A073.

First Demonstration of Antigen Induced Cytokine Expression by CD4-1+ Lymphocytes in a Poikilotherm: Studies in Zebrafish (Danio rerio).

Adaptive immunity in homeotherms depends greatly on CD4+ Th cells which release cytokines in response to specific antigen stimulation. Whilst bony fish and poikilothermic tetrapods possess cells that express TcR and CD4-related genes (that exist in two forms in teleost fish; termed CD4-1 and CD4-2), to date there is no unequivocal demonstration that cells equivalent to Th exist. Thus, in this study we determined whether CD4-1+ lymphocytes can express cytokines typical of Th cells following antigen specific stimulation, using the zebrafish (Danio rerio). Initially, we analyzed the CD4 locus in zebrafish and found three CD4 homologues, a CD4-1 molecule and two CD4-2 molecules. The zfCD4-1 and zfCD4-2 transcripts were detected in immune organs and were most highly expressed in lymphocytes. A polyclonal antibody to zfCD4-1 was developed and used with an antibody to ZAP70 and revealed double positive cells by immunohistochemistry, and in the Mycobacterium marinum disease model CD4-1+ cells were apparent surrounding the granulomas typical of the infection. Next a prime-boost experiment, using human gamma globulin as antigen, was performed and revealed for the first time in fish that zfCD4-1+ lymphocytes increase the expression of cytokines and master transcription factors relevant to Th1/Th2-type responses as a consequence of boosting with specific antigen.

LMO2 induces T-cell leukemia with epigenetic deregulation of CD4

In this study, we present a remarkable clonal cell line, 32080, derived from a CD2-Lmo2- transgenic T-cell leukemia with differentiation arrest at the transition from the intermediate single positive to double positive stages of T-cell development. We observed that 32080 cells had a striking variegated pattern in CD4 expression. There was cell-to-cell variability, with some cells expressing no CD4 and others expressing high CD4. The two populations were isogenic and yet differed in their rates of apoptosis and sensitivity to glucocorticoid. We sorted the 32080 line for CD4-positive or CD4-negative cells and observed them in culture. After 1 week, both sorted populations showed variegated CD4 expression, like the parental line, showing that the two populations could interconvert. We determined that cell replication was necessary to transit from CD4(+) to CD4(-) and CD4(-) to CD4(+). Lmo2 knockdown decreased CD4 expression, while inhibition of intracellular NOTCH1 or histone deacetylase activity induced CD4 expression. Enforced expression of RUNX1 repressed CD4 expression. We analyzed the CD4 locus by Histone 3 chromatin immunoprecipitation and found silencing marks in the CD4(-) cells and activating marks in the CD4(+) population. The 32080 cell line is a striking model of intermediate single positive to double positive T-cell plasticity and invokes a novel mechanism for LMO2's oncogenic functions.

Genetic diversity and relationships of populations of northern Eurasia by polymorphic Alu insertions

Autosomal gene pools of 27 populations representing 12 ethnic groups of Siberia, Central Asia, and the Far East have been characterized for the first time using a set of eight polymorphic Alu insertions. The results of our analysis indicate a significant level of genetic diversity in populations of northern Eurasian and the considerable differentiation of their gene pool. It was shown that the frequency of the Alu (−) allele at the CD4 locus was inversely related to the magnitude of the Mongoloid component of the gene pool: the lowest and highest frequencies of the CD4 Alu deletion were recorded in Eskimos (0.012) and in Russians and Ukrainians (0.35), respectively. A gene flow analysis showed that Caucasoid populations (Russians, Tajiks, and Uzbeks), as well as Turkic ethnic groups of southern Siberia (Altaians and Tuvans), Khanty, and Mansi populations, in contrast to ethnic groups of eastern Siberia and the Far East, have been recipients of a considerable gene flow. A correlation analysis showed that genetic distances determined using polymorphic Alu insertions were correlated with the anthropological characteristics of the populations studied.

Modulating effect of Val66Met polymorphism of brain-derived neurotrophic factor gene on clinical and psychological characteristics of patients with schizophrenia

We for the first time have examined the autosomal gene pool of the Siberia, Central Asian and the Far East populations (27 populations of 12 ethnic groups) using a set of polymorphic Alu insertions in the human genome. The results of the analysis testify (i) to a significant level of genetic diversity in the Northern Eurasian populations and (ii) to a considerable differentiation of gene pool in the population of this region. It has been shown that at the CD4 locus, the frequency of Alu (-) is inversely related to the Mongoloid component of the population, the lowest and highest frequencies of the Alu deletion at locus CD4 were recorded respectively in Eskimo (0.012) and Russian and Ukrainian (0.35). The analysis of gene flow proved Caucasoid populations (Russian, Tajik and Uzbek), as well as those of Turkic ethnic groups from the Southern Siberia (Altaians and Tuvinians) and Khanty and Mansy populations to be the recipients of a considerable gene flow from the outside of the concerned population system, as compared with the East Siberian and the Far East ethnic groups. The results of the correlation analysis received with use polymorphic Alu insertion testify to the greatest correlation of genetic distances with anthropological characteristics of populations.

A Study of Genomic Diversity of Three Tribal Populations of Chhattisgarh, India

The diverse linguistic and ethnic tribal populations harboring state Chhattisgarh is a hotspot for human genomic diversity research. Genetic analysis of individual variation at the DNA level is widely used to investigate the extent of genetic diversity and to understand the population structure among different populations. The present study was conducted to find out the extent of affinities and diversities among three tribal populations of Chhattisgaarh, India namely Halba, Gadba and Oraon. They belong to different language families. Halba belongs to Indo-European, Gadba belongs to Austro-Asiatic while Oraon belongs to Dravidian language family. Analysis on seven widely distributed polymorphic insertion/deletion loci (Alu APO, Alu ACE, Alu D1, Alu PV92, Alu FXIIIB, Alu CD4 del and Alu PLAT) in 160 unrelated individuals of both sexes were done. All loci were highly polymorphic except Alu CD4 del locus. All the three tribes showed lowest heterozygosity for CD4 locus. GST value was recorded lowest for Alu PLAT locus (0.0004) while the highest was recorded for Alu PV92 locus (0.1029). The unrooted Neighbor-Joining (NJ) tree showed Gadba and Oraon form one cluster while Halba tribe differentiates from them.

Stage-specific epigenetic gene silencing during thymocyte lineage commitment

During lineage commitment, precursor cells must establish their signature gene expression programs to endow them with the requisite set of cellular functions. To maintain cellular identity, the gene expression program must be inherited stably by progeny of lineage-committed cells. Epigenetic regulation serves as a central mechanism to maintain such cellular memory. Although a lot of progress has been made in the last decade towards defining the spectrum of epigenetic modifications on histones and DNA, as well as the relevant enzymatic machinery, the mechanisms by which these modifiers are controlled during development remain poorly understood. Gene regulation at the Cd4 and Thpok loci provides ideal models for developmentally regulated gene silencing. A single transcriptional silencer at each locus establishes heritable, irreversible epigenetic silencing only in lineage-committed cells, whereas the same silencer elements establish a reversible repressive state in precursor cells. The dynamic versus permanent silencing of developmentally regulated loci by the stage-specific functions of silencers will be discussed in the context of cell lineage commitment.

Identification of a novel, developmental-stage-specific enhancer in the Cd4 gene (P4442)

Abstract CD4 helper T cells coordinate the immune response and are highly dependent on the expression of the Cd4 gene for proper development and function. The function of a promoter, proximal enhancer, and a silencer have been well documented and together explain how the Cd4 gene gets turned on in CD4 T cells and off in CD8 T cells. However once turned on, the amount and timing of Cd4 expression varies during T cell development and activation. This modulation of CD4 surface levels is essential for proper lineage specification and T cell function. Yet, how subtle changes of CD4 expression are regulated remains unclear. We have recently identified a novel positive cis-acting transcriptional regulatory element (NCE) in the Cd4 locus. Here we demonstrate that NCE enhances Cd4 promoter function in position and orientation independent manner using a transient transfection assay with an eGFP reporter construct in RLM11 murine thymoma cell line. Using cell lines at different developmental stages, we determined that NCE functions well at the intermediate, but not the DP stage of development. This formally demonstrates that NCE functions as an enhancer and may have developmental specificity. We are in the process of confirming these findings in vivo by generating BAC transgenic mice that lack the NCE in the Cd4 locus.

Coreceptor gene imprinting governs thymocyte lineage fate

Immature thymocytes are bipotential cells that are signalled during positive selection to become either helper- or cytotoxic-lineage T cells. By tracking expression of lineage determining transcription factors during positive selection, we now report that the Cd8 coreceptor gene locus co-opts any coreceptor protein encoded within it to induce thymocytes to express the cytotoxic-lineage factor Runx3 and to adopt the cytotoxic-lineage fate, findings we refer to as 'coreceptor gene imprinting'. Specifically, encoding CD4 proteins in the endogenous Cd8 gene locus caused major histocompatibility complex class II-specific thymocytes to express Runx3 during positive selection and to differentiate into CD4(+) cytotoxic-lineage T cells. Our findings further indicate that coreceptor gene imprinting derives from the dynamic regulation of specific cis Cd8 gene enhancer elements by positive selection signals in the thymus. Thus, for coreceptor-dependent thymocytes, lineage fate is determined by Cd4 and Cd8 coreceptor gene loci and not by the specificity of T-cell antigen receptor/coreceptor signalling. This study identifies coreceptor gene imprinting as a critical determinant of lineage fate determination in the thymus.

RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation

SummaryT cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.