Cell differentiation involves activation and silencing of lineage-specific genes. Here we show that the transcription factor Runx3 is induced in T helper type 1 (T(H)1) cells in a T-bet-dependent manner, and that both transcription factors T-bet and Runx3 are required for maximal production of interferon-gamma (IFN-gamma) and silencing of the gene encoding interleukin 4 (Il4) in T(H)1 cells. T-bet does not repress Il4 in Runx3-deficient T(H)2 cells, but coexpression of Runx3 and T-bet induces potent repression in those cells. Both T-bet and Runx3 bind to the Ifng promoter and the Il4 silencer, and deletion of the silencer decreases the sensitivity of Il4 to repression by either factor. Our data indicate that cytokine gene expression in T(H)1 cells may be controlled by a feed-forward regulatory circuit in which T-bet induces Runx3 and then 'partners' with Runx3 to direct lineage-specific gene activation and silencing.

The RUNX transcription factors are important regulators of lineagespecific gene expression. RUNX are bifunctional, acting both as activators and repressors of tissue-specific target genes. Recently, we have demonstrated that Runx3 is a neurogenic transcription factor, which regulates development and survival of proprioceptive neurons in dorsal root ganglia. Here we report that Runx3 and Runx1 are highly expressed in thymic medulla and cortex, respectively, and function in development of CD8 T cells during thymopoiesis. Runx3-deficient (Runx3 KO) mice display abnormalities in CD4 expression during lineage decisions and impairment of CD8 T cell maturation in the thymus. A large proportion of Runx3 KO peripheral CD8 T cells also expressed CD4, and in contrast to wild-type, their proliferation ability was largely reduced. In addition, the in vitro cytotoxic activity of alloimmunized peritoneal exudate lymphocytes was significantly lower in Runx3 KO compared with WT mice. In a compound mutant mouse, null for Runx3 and heterozygous for Runx1 (Runx3 ؊/؊ ;Runx1 ؉/؊ ), all peripheral CD8 T cells also expressed CD4, resulting in a complete lack of single-positive CD8 ؉ T cells in the spleen. The results provide information on the role of Runx3 and Runx1 in thymopoiesis and suggest that both act as transcriptional repressors of CD4 expression during T cell lineage decisions.T he mammalian RUNX3͞AML2 gene resides on human chromosome 1p36.1 and mouse chromosome 4, respectively (1-4). It belongs to the RUNX family of transcription factors, which contains three genes. The two other family members, RUNX1 and RUNX2, play fundamental roles in hematopoietic and osteogenic lineage-specific gene expression, and when mutated, are associated with human diseases (5, 6). The three RUNX genes are regulated at the transcriptional level by two promoters, and at the translational level by internal ribosome entry site (IRES)-and cap-dependent translation control (7-14). The gene products of RUNX bind to the same DNA motif and activate or repress transcription of target genes through recruitment of common transcriptional modulators (15)(16)(17)(18). Despite this occurrence, each of the Runx genes has well defined biological functions reflected in a different expression pattern of the genes (19-23) and distinct phenotypes of the corresponding knockout mice (6,(24)(25)(26)(27).During mouse embryogenesis Runx3 is expressed in hematopoietic organs, epidermal appendages, developing bones, and sensory ganglia (20). Studies in knockout (KO) mice revealed that Runx3 is a neurogenic-specific transcription factor required for development and survival of TrkC neurons in the dorsal root ganglia. In the absence of Runx3 these neurons die, leading to disruption of the stretch reflex neuronal circuit, and consequently to severe ataxia (25,26). Intriguingly, in one strain of Runx3 KO, the gastric mucosa of newborn mice exhibits hyperplasia due to stimulated proliferation and suppressed apoptosis of stomach epithelial cells (27).It has previously been reporte...

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Runx3 transcription factor regulates cell lineage decisions in thymopoiesis and neurogenesis. Here we report that Runx3 knockout (KO) mice develop spontaneous eosinophilic lung inflammation associated with airway remodeling and mucus hypersecretion. Runx3 is specifically expressed in mature dendritic cells (DC) and mediates their response to TGF-b. In the absence of Runx3, DC become insensitive to TGF-b-induced maturation inhibition, and TGF-b-dependent Langerhans cell development is impaired. Maturation of Runx3 KO DC is accelerated and accompanied by increased efficacy to stimulate T cells and aberrant expression of b2-integrins. Lung alveoli of Runx3 KO mice accumulate DC characteristic of allergic airway inflammation. Taken together, abnormalities in DC function and subset distribution may constitute the primary immune system defect, which leads to the eosinophilic lung inflammation in Runx3 KO mice. These data may help elucidate the molecular mechanisms underlying the pathogenesis of allergic airway inflammation in humans.

The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

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The human chromosome 21 AMLI gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AMLI is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AMLI. The 5' genomic region of AMLi was cloned and sequenced. The tions of AMLi by the chimeric proteins (2). Interestingly, we and others identified in normal blood a number of AMLI mRNA species that encode shortened proteins similar in size to the AMLJ segment in the fused t(8;21) and t(3;21) products (3, 11-13). When transfected into cells, they dominantly suppress activity of the full-length AMLi protein (2, 14, 15). When overexpressed due to gene dosage, these mRNAs may have a significant role in the Down syndrome-associated AML, in which the chromosomal defect is trisomy 21 (16, 17).AMLI is expressed mainly in the hematopoietic system as a complex pattern of mRNAs ranging in size between 2 and 8 kb (2,3,6). Several of these transcripts differ in their 5' and 3' untranslated regions (UTRs) (13,18). There are two distinct 5' UTRs that exhibit different expression patterns in hematopoietic tissues and in response to mitogenic stimulation (13). These data raise the possibility that AMLI expression is transcriptionally regulated by two discrete promoter regions and prompted us to investigate the genomic regions upstream of these 5' UTRs. Here we describe the structural/functional analysis of these two regions and demonstrate that they harbor the AML1 mRNA start sites and serve as transcriptional promoters that regulate expression of the AMLI gene.t MATERIALS AND METHODSLibrary Screening, Phage DNA Mapping, and Sequencing. A genomic library enriched for human chromosome 21 was screened as described (6). Phages containing the 5' region of AML1 were isolated, fragments of their inserts were subcloned in pBluescript (Stratagene), and both strands were sequenced by the automated Taq dideoxynucleotide chain-termination method on a model 373A Applied Biosystem apparatus.Primer-Extension Analysis. Two primers were used for each 5' UTR. The reverse primers for the proximal region (Fig. 1BI) span nucleotides 74-104 and 121-150 relative to + 1 of the AMLl-a cDNA (6), while those used for the distal region (Fig. 1BII) Fig. 2 were derived by using convenient restriction sites, from the Xho I/Avr II fragment