Factor Runx2 Research Articles

The Runx transcriptional co-activator, CBFβ, is essential for invasion of breast cancer cells

BackgroundThe transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFβ is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFβ is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFβ-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFβ. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFβ expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFβ. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis.ResultsWe show that CBFβ is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFβ contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFβ is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFβ is recruited to both the Osteopontin and the Galectin-3 promoters.ConclusionsCBFβ is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFβ is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFβ is essential for invasion, not all Runx2-target genes require CBFβ. We conclude that CBFβ is required for a subset of Runx2-target genes that are sufficient to maintain the invasive phenotype of the cells. These findings suggest that the interaction between Runx2 and CBFβ might represent a viable target for therapeutic intervention to inhibit bone metastasis.

TEThered to Runx: Novel binding partners for runx factors

RUNX transcription factors reside in the nuclear matrix where they integrate numerous signaling pathways to regulate gene expression and affect tissue development, regeneration, and tumorigenesis. An affinity purification and proteomic experiment was performed to identify novel Runx2 binding partners. The interactions between Runx2 and two nuclear factors (Ddx5 and CoAA) identified in this screen were previously described. Coactivator activator (CoAA) bound the DNA binding domain of Runx2 and prevented Runx-driven gene expression. The YxxQ motif in CoAA was required for Runx2 interactions. Members of the FET/TET family of proteins, including FUS/TLS and EWSR1, contain a similar motif and were hypothesized to interact with Runx2. Here, we provide evidence that FUS/TLS, EWSR1, and the Ewing's sarcoma t(12;21) fusion protein EWS–FLI bind Runx2 and alter its transcriptional activity. Potential roles of protein complexes containing FET/TET and RUNX family members during tumor formation and mesenchymal progenitor cell differentiation are discussed.

Brn3a regulates neuronal subtype specification in the trigeminal ganglion by promoting Runx expression during sensory differentiation

The transcription factor Brn3a, product of the pou4f1 gene, is expressed in most sensory neurons throughout embryogenesis. Prior work has demonstrated a role for Brn3a in the repression of early neurogenic genes; here we describe a second major role for Brn3a in the specification of sensory subtypes in the trigeminal ganglion (TG). Sensory neurons initially co-express multiple Trk-family neurotrophin receptors, but are later marked by the unique expression of TrkA, TrkB or TrkC. Maturation of these sensory subtypes is known to depend on the expression of Runx transcription factors. Newborn Brn3a knockout mice fail to express TrkC, which is associated in the TG with mechanoreceptors, plus a set of functional genes associated with nociceptor subtypes. In embryonic Brn3a-/- ganglia, the normal expression of Runx3 is never initiated in TrkC+ neurons, and Runx1 expression is greatly attenuated in TrkA+ nociceptors. These changes are accompanied by expanded expression of TrkB in neurons that abnormally express multiple Trks, followed by the loss of TrkC and TrkA expression. In transgenic embryos expressing a Brn3a-VP16 dominant transactivator, Runx3 mRNA expression is increased, suggesting that it is a direct regulatory target of Brn3a. Chromatin immunoprecipitation confirms that Brn3a binds in vivo to a conserved upstream enhancer element within histone H3-acetylated chromatin in the Runx3 locus. Together these data show that Brn3a acts upstream of the Runx factors, which then repress TrkB expression to allow establishment of the non-overlapping Trk receptor profiles and correct terminally differentiated phenotypes.

DNA Specificity Determinants Associate with Distinct Transcription Factor Functions

To elucidate how genomic sequences build transcriptional control networks, we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited, because a single factor can use degenerate sequence motifs and because related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation; thus, unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, a genome-wide approach, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and also improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T cell–specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of DNA binding motifs may enable variable transcription factor function at different genomic sites.

CD4-CD8 Lineage Differentiation: Thpok-ing into the Nucleus

The mature alphabeta T cell population is divided into two main lineages that are defined by the mutually exclusive expression of CD4 and CD8 surface molecules (coreceptors) and that differ in their MHC restriction and function. CD4 T cells are typically MHC-II restricted and helper (or regulatory), whereas CD8 T cells are typically cytotoxic. Several transcription factors are known to control the emergence of CD4 and CD8 lineages, including the zinc finger proteins Thpok and Gata3, which are required for CD4 lineage differentiation, and the Runx factors Runx1 and Runx3, which contribute to CD8 lineage differentiation. This review summarizes recent advances on the function of these transcription factors in lineage differentiation. We also discuss how the "circuitry" connecting these factors could operate to match the expression of the lineage-committing factors Thpok and Runx3, and therefore lineage differentiation, to MHC specificity.

Co‐activator activator (CoAA) prevents the transcriptional activity of Runt domain transcription factors

Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells.

The human CD6 gene is transcriptionally regulated by RUNX and Ets transcription factors in T cells

CD6 is a lymphocyte surface receptor involved in lymphocyte activation and differentiation processes that is constitutively expressed on developing and mature T cells and on the B1a cells. To define the molecular basis for the tissue-specific expression of CD6 we have identified the transcription factors that control the activity of the proximal regulatory region of the human CD6 gene. The TATA-less CD6 promoter contains multiple transcriptional start sites, and its preferential activity in human T lymphocytes is dependent on RUNX- and Ets-binding sites located within a highly conserved region. RUNX and Ets-1 factors transactivated the CD6 promoter through recognition of the −215 and −230 binding sites, respectively. Chromatin immunoprecipitation assays revealed that RUNX1 constitutively occupies the CD6 promoter in vivo, and knockdown experiments demonstrated that the steady-state level of CD6 mRNA is dependent on the expression of RUNX1, RUNX3 and Ets-1 transcription factors. Therefore, RUNX1/3 and Ets1 control the expression of CD6 in human T lymphocytes, thus expanding the range of T-cell specific and developmentally regulated lymphocyte gene targets involved in T-cell activation and differentiation.

‘Runxs and regulations’ of sensory and motor neuron subtype differentiation: Implications for hematopoietic development

Runt-related (RUNX) transcription factors are evolutionarily conserved regulators of a number of developmental mechanisms. RUNX proteins often control the balance between proliferation and differentiation and alterations of their functions are associated with different types of cancer and other human pathologies. Moreover, RUNX factors control important steps during the developmental acquisition of mature phenotypes. A number of investigations are beginning to shed light on the involvement of RUNX family members in the development of the nervous system. This review summarizes recent progress in the study of the roles of mammalian RUNX proteins during the differentiation of sensory and motor neurons in the peripheral and central nervous system, respectively. The implications of those findings for RUNX-mediated regulation of hematopoietic development will also be discussed.

Involvement of notch and RUNX pathways in human breast cancer.

Abstract Abstract #6021 Background: Recent studies have demonstrated that the Notch signaling and the RUNX-mediated transcription pathways converge in mammalian hematopoiesis where they regulate proliferation and development. Evidence from in vitro experiments, mouse models and human tumors demonstrate that Notch signaling plays an oncogenic role in human breast cancer. Studies of RUNX factors involvement in human breast cancer suggest that the Core Binding Factor [CBF, a heterodimeric transcription factor composed of a RUNX DNA binding protein (RUNX1, RUNX2, or RUNX3) and a single non-DNA binding component termed CBFβ] participates in regulating the malignant phenotype of the tumor. Thus, the RUNX factors are primarily important to Notch signaling, and it seems likely that Notch:RUNX cross-talk will be important to cellular function and disease in a variety of tissue types. Here, we present our findings on cross-talk between Notch and RUNX pathways, which will lead to a better understanding of Notch and RUNX regulated transcription in breast carcinogenesis.
 Material and Methods: The expression and localization of the Notch1 NICD and RUNX2 in normal breast tissue and breast tumor were determined by immunohistochemistry on human breast tissue microarray obtained from Feist-Weiller Cancer Center. The expression profiles of Notch and RUNX were evaluated by real-time RT-PCR, Western Immunoblot, and electrophoretic mobility shift assay (EMSA).
 Results and Discussion: We found activated Notch1 and RUNX2 predominantly expressed in the cytoplasm and nucleus of cells in human normal breast tissues, respectively. However, in human breast cancer tissue samples, activated Notch1 was found in the nucleus in a high proportion of the cancer cells, while RUNX2 presence in the nucleus was much reduced and this could be due to mislocalization, over-expression or delayed degradation of the proteins. The major transcript in the breast cancer cell lines is RUNX1, but by EMSA, RUNX2 factor is the predominant biologically active and functional form of RUNX factors associated with breast cancer cell line-derived CBF. Transcript degradation and protein stability may play significant role in the regulation of RUNX factors in breast carcinogenesis. Down-regulation of CBFβ was found to affect Notch signaling in breast cancer cells and this may lead to the identification of new therapeutic targets for the successful treatment of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6021.

Regulation of the CD56 promoter and its association with proliferation, anti-apoptosis and clinical factors in multiple myeloma

Multiple myeloma (MM) is an incurable B-cell malignancy characterised by uncontrolled growth and accumulation of malignant plasma cells in the bone marrow. Aberrant expression of CD56 in patients with MM is thought to contribute to a worsened disease course and metastasis. We therefore investigated the regulation of the CD56 promoter in relation to typical clinical factors. We used qPCR and FACS to measure the expression levels of CD56, and potential regulatory factors in patients with MM and related these with MM progression/prognosis. The transcription factors BTBD3, Pax5, RUNX1 and MMSET were positively associated with CD56 expression, as was CYCLIN D1, which is involved in disease progression, anti-apoptosis and proliferation. RUNX1 was negatively associated with the survival of stem-cell transplanted patients. Our findings propose four potential activators of the CD56 promoter and for CD56 to be involved in proliferation and anti-apoptosis, leading to disease progression in MM.

Protein-deoxyribonucleic acid interactions linked to gene expression: DNase I digestion.

AbstractMapping DNase I hypersensitive sites can often localize the control regions in a eukaryotic gene. It is generally believed that chromosomal regions with loose or more open conformation are sensitive to DNase I cleavage. By comparing the patterns of hypersensitive sites obtained by digestion of silent genes with those obtained when the genes are actively transcribed, regions that participate in gene regulation (either induction or suppression) can be identified. A major strength of this method is that it is an “unbiased” approach to characterize the location of control sequences for the regulation of a gene. A good example of the power of DNase I hypersensitivity mapping is the rat Osteocalcin gene. Osteocalcin gene expression is detected only in bone tissues and in fully mature osteoblasts. DNase I cleavage is not observed in the OC gene in nonbone tissues or in immature osteoblasts (where OC is not transcribed). However, two DNase I hypersensitive sites are detected in the OC gene in bone tissues and in mature osteoblasts where the gene is actively transcribed. The first DNase I region encompasses the basal promoter regions that contain regulatory sequences for the RUNX, C/EBP, activator protein-1, and TFIID transcription factors. The second DNase I hypersensitive domain is mapped to a region 0.5 kilobases upstream of the transcription initiation site. This region contains a vitamin D enhancer element and regulatory sequences for RUNX factors. The sequences at these hypersensitive sites are both sufficient and necessary for basal and bone-tissue specific expression of the Osteocalcin gene.KeywordsTranscription Initiation SiteIsoamyl AlcoholDounce HomogenizerMature OsteoblastHypersensitive SiteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structural Coupling of Smad and Runx2 for Execution of the BMP2 Osteogenic Signal

Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-beta (TGFbeta) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of Runx2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in Runx2 null cells. These cells do not respond to BMP2 signal in the absence of Runx2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFbeta signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity.

TheC. elegansCBFβ homologue BRO-1 interacts with the Runx factor, RNT-1, to promote stem cell proliferation and self-renewal

In this report, we investigate the C. elegans CBFbeta homologue, BRO-1. bro-1 mutants have a similar male-specific sensory ray loss phenotype to rnt-1 (the C. elegans homologue of the mammalian CBFbeta-interacting Runx factors), caused by failed cell divisions in the seam lineages. Our studies indicate that BRO-1 and RNT-1 form a cell proliferation-promoting complex, and that BRO-1 increases both the affinity and specificity of RNT-1-DNA interactions. Overexpression of bro-1, like rnt-1, leads to an expansion of seam cell number and co-overexpression of bro-1 and rnt-1 results in massive seam cell hyperplasia. Finally, we find that BRO-1 appears to act independently of RNT-1 in certain situations. These studies provide new insights into the function and regulation of this important cancer-associated DNA-binding complex in stem cells and support the view that Runx/CBFbeta factors have oncogenic potential.

Regulatory factors for initial T lymphocyte lineage specification

Initiation of T lymphocyte development depends on balanced regulatory inputs from multiple essential transcription factors. This review highlights contributions of E2A, hematopoietic transcription factor PU.1, growth factor independence (Gfi)-1, T cell factor (TCF)-1, and Runx factors and their interactions with the Notch pathway to promote T cell development. E2A and Runx family factors have been implicated in establishing competent precursors in which Notch signaling can induce the T cell program. An early role was also indicated for PU.1. Later PU.1 activities are antagonistic to pro-T cell factors, however, including E proteins, Myb, Gfi-1, and TCF-1. Diversion to a non-T lineage can be promoted by PU.1, CCAAT/enhancer binding protein, or even GATA and TCF, but these diversion mechanisms are blocked by Notch signaling. An emergent gene network summarizes the cross-regulatory relationships among these factors. Entry into the T-cell pathway is controlled by a dynamic balance among essential regulatory factors that depend on Notch signaling not only to trigger initiation of the T-cell program but also to maintain the lineage fidelity of their collective action.

Epigenetic silencing of potentially functional KIR2DL5 alleles: Implications for the acquisition of KIR repertoires by NK cells

NK cells detect altered patterns of HLA expression in infections and tumors using a variegated repertoire of killer cell Ig-like receptors (KIR). Each clone surveys different HLA molecules by expressing a limited subset of the KIR encoded in its genome, which is maintained throughout cell divisions by epigenetic mechanisms (methylation of the nonexpressed genes). How KIR repertoires are acquired remains, however, unexplained. Human KIR2DL5 is a useful model for studying KIR expression because it has alleles with similar coding regions, but drastically divergent expression - whilst some are transcribed in a typically clonal manner, others, with distinctive promoter polymorphisms, are nonexpressed. Here we investigate the relationship between the sequence diversity of KIR2DL5, including three novel alleles, and its variable transcription. The promoters of the transcribed alleles recruit the transcriptional regulator RUNX3, whilst a mutation shared by all silent alleles precludes this binding. However, all promoters are functional in vitro, and pharmacological DNA demethylation of NK cells rescues the transcription of silent alleles, indicating that only epigenetic mechanisms prevent their inclusion in a normal KIR repertoire. Our results are consistent with a model in which RUNX factors could function as switch elements in the acquisition of KIR repertoires by NK cell precursors.

Drosophila haematopoiesis

Like in vertebrates, Drosophila haematopoiesis occurs in two waves. It gives rise to three types of haemocytes: plasmatocytes (phagocytosis), crystal cells (melanization) and lamellocytes (encapsulation of parasites). A first population of haemocytes, specified during embryogenesis, gives rise to an invariant number of plasmatocytes and crystal cells. A second population of haemocytes is specified during larval development in a specialized haematopoietic organ, the lymph gland. All three types of haemocytes can be specified in this organ, but lamellocytes only differentiate in response to parasitism. Thus, larval in contrast to embryonic haematopoiesis can be modulated by physiological constraints. Molecular cascades controlling embryonic haematopoiesis are relatively well established and require transactivators such as GATA, FOG and Runx factors, which are also co-opted in mammalian haematopoiesis. Mechanisms involved during larval haematopoiesis are less well understood although a number of chromatin remodelling factors and signalling pathways (JAK/STAT, Toll, Hedgehog, Notch) are required. In healthy larvae a pool of progenitors is maintained within the lymph gland, under the control of a signalling centre which expresses Collier, Serrate, Antennapedia and Hedgehog, and controls haemocyte homeostasis. Its key role in haemocyte homeostasis is reminiscent of interactions described in vertebrates between haematopoietic stem cells and their microenvironment (niche).

The role of growth factors in chondrogenesis and osteogenesis

Purpose of review During endochondral ossification mesenchymal cells aggregate and form cartilage elements. Chondrocytes in the center of these elements differentiate successively into columnar, proliferating and hypertrophic chondrocytes, which are subsequently replaced by bone. This review gives an overview of signals that coordinate different steps of this process. Recent findings Considerable progress has been made in analyzing the function of four signaling systems, that of Indian hedgehog, Wnt proteins, bone morphogenetic proteins and fibroblast growth factors. Together with the transcription factors Sox9 and Runx2 they control chondrocyte and osteoblast differentiation. Summary Chondrocytes and osteoblasts are derived from a common precursor, the osteo-chondro progenitor. New findings indicate that the level of Wnt signaling is critical to direct osteo-chondro progenitor cells towards the chondrogenic or osteogenic fate. Subsequently, Indian hedgehog signaling directly regulates the differentiation of distal into columnar chondrocytes, whereas parathyroid hormone-related protein acts downstream of Indian hedgehog to control the onset of hypertrophic differentiation and the size of the zone of columnar chondrocytes. Moreover, Indian hedgehog induces osteoblast differentiation in the perichondrium. Subsequently, Wnt signaling commits the differentiation of osteoprogenitors into mature osteoblasts. These findings complement our knowledge of how the interplay of different growth factors controls chondrocyte and osteoblast differentiation.

“Runx”ing towards Sensory Differentiation

Somatosensory stimuli are encoded by molecularly and anatomically diverse classes of dorsal root ganglia (DRG) neurons. In this issue of Neuron, three papers demonstrate that the Runx transcription factors, Runx1 and Runx3, respectively regulate the molecular identities and spinal terminations of TrkA+ nociceptive neurons and TrkC+ proprioceptive neurons. These findings emphasize the importance of intrinsic genetic programs in generating the diversity of DRG neurons and specifying the circuits into which they incorporate.

Graded Activity of Transcription Factor Runx3 Specifies the Laminar Termination Pattern of Sensory Axons in the Developing Spinal Cord

Different functional classes of dorsal root ganglion sensory neurons project their axons to distinct target zones within the developing spinal cord. To explore the mechanisms that link sensory neuron subtype identity and axonal projection pattern, we analyzed the roles of Runx and ETS transcription factors in the laminar targeting of sensory afferents. Gain- and loss-of-function studies in chick embryos reveal that the status of Runx3 expression is a major determinant of the dorso-ventral position of termination of proprioceptive and cutaneous sensory axons. In addition, the level of expression and/or activity of Runx3 in individual proprioceptive sensory neurons appears to specify whether their axons terminate in intermediate or ventral regions. Our findings suggest that the selectivity of Runx3 expression, and its level of activity, control sensory afferent targeting in the developing spinal cord.

At the crossroads: diverse roles of early thymocyte transcriptional regulators

Transcriptional regulation of T-cell development involves successive interactions between complexes of transcriptional regulators and their binding sites within the regulatory regions of each gene. The regulatory modules that control expression of T-lineage genes frequently include binding sites for a core set of regulators that set the T-cell-specific background for signal-dependent control, including GATA-3, Notch/CSL, c-myb, TCF-1, Ikaros, HEB/E2A, Ets, and Runx factors. Additional regulators in early thymocytes include PU.1, Id-2, SCL, Spi-B, Erg, Gfi-1, and Gli. Many of these factors are involved in simultaneous regulation of non-T-lineage genes, T-lineage genes, and genes involved in cell cycle control, apoptosis, or survival. Potential and known interactions between early thymic transcription factors such as GATA-3, SCL, PU.1, Erg, and Spi-B are explored. Regulatory modules involved in the expression of several critical T-lineage genes are described, and models are presented for shifting occupancy of the DNA-binding sites in the regulatory modules of pre-Talpha, T-cell receptor beta (TCRbeta), recombinase activating genes 1 and 2 (Rag-1/2), and CD4 during T-cell development. Finally, evidence is presented that c-kit, Erg, Hes-1, and HEBAlt are expressed differently in Rag-2(-/-) thymocytes versus normal early thymocytes, which provide insight into potential regulatory interactions that occur during normal T-cell development.