An ensemble of regulatory elements controls Runx3 spatiotemporal expression in subsets of dorsal root ganglia proprioceptive neurons.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs-dubbed R1, R2, and R3-that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs' ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs.

Runx3 in Immunity, Inflammation and Cancer.

In this chapter we summarize the pros and cons of the notion that Runx3 is a major tumor suppressor gene (TSG). Inactivation of TSGs in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago it was suggested that RUNX3 is involved in gastric cancer development, a postulate extended later to other epithelial cancers portraying RUNX3 as a major TSG. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. In contrast, RUNX3 is overexpressed in a significant fraction of tumor cells in various human epithelial cancers and its overexpression in pancreatic cancer cells promotes their migration, anchorage-independent growth and metastatic potential. Moreover, recent high-throughput quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models have unequivocally demonstrated that RUNX3 is not a bona fide cell-autonomous TSG. Importantly, accumulating data demonstrated that RUNX3 functions in control of immunity and inflammation, thereby indirectly influencing epithelial tumor development.

Runx3 at the interface of immunity, inflammation and cancer.

Inactivation of tumor suppressor genes (TSG) in normal cells provides a viability/growth advantage that contributes cell-autonomously to cancer. More than a decade ago claims arose that the RUNX3 member of the RUNX transcription factor family is a major TSG inactivated in gastric cancer, a postulate extended later to other cancers. However, evidence that Runx3 is not expressed in normal gastric and other epithelia has challenged the RUNX3-TSG paradigm. Here we critically re-appraise this paradigm in light of recent high-throughput, quantitative genome-wide studies on thousands of human samples of various tumors and new investigations of the role of Runx3 in mouse cancer models. Collectively, these studies unequivocally demonstrate that RUNX3 is not a bona fide cell-autonomous TSG. Accordingly, RUNX3 is not recognized as a TSG and is not included among the 2000 cancer genes listed in the "Cancer Gene Census" or "Network for Cancer Genes" repositories. In contrast, RUNX3 does play important functions in immunity and inflammation and may thereby indirectly influence epithelial tumor development.

Runx3 prevents spontaneous colitis by directing the differentiation of anti-inflammatory mononuclear phagocytes.

Mice deficient in the transcription factor Runx3 develop a multitude of immune system defects, including early onset colitis. This paper demonstrates that Runx3 is expressed in colonic mononuclear phagocytes (MNP), including resident macrophages (RM) and dendritic cell subsets (cDC2). Runx3 deletion in MNP causes early onset colitis due to their impaired maturation. Mechanistically, the resulting MNP subset imbalance leads to up-regulation of pro-inflammatory genes as occurs in IL10R-deficient RM. In addition, RM and cDC2 display a marked decrease in expression of anti-inflammatory/TGF ß-regulated genes and ß-catenin signaling associated genes, respectively. MNP transcriptome and ChIP-seq data analysis suggest that a significant fraction of genes affected by Runx3 loss are direct Runx3 targets. Collectively, Runx3 imposes intestinal immune tolerance by regulating maturation of colonic anti-inflammatory MNP, befitting the identification of RUNX3 as a genome-wide associated risk gene for various immune-related diseases in humans, including gastrointestinal tract diseases such as Crohn's disease and celiac.

Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis.

BACKGROUND: Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development. RESULTS: We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors. CONCLUSION: The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the significance of alternative promoter usage in Runx1 biology.

Dynamic expression of Runx1 in skin affects hair structure.

The three mammalian Runx transcription factors, some of which are known to be involved in human genetic diseases and cancer, are pivotal players in embryo development and function as key regulators of cell fate determination and organogenesis. Here, we report the expression of Runx1 during the development of hair and other skin appendages in the mouse and describe the effect of Runx1 on the structural hair output. In hair follicles, where the three Runx proteins are expressed, Runx1 expression is most prominent in both mesenchymal and epithelial compartments. The epithelial expression includes the hair keratin forming layers of the hair shaft and the bulge, where interestingly, Runx1 is co-expressed with keratin 15, a putative hair follicle stem cell marker. In the hair mesenchyme, during early stages of hair morphogenesis, Runx1 is expressed in a discrete dermal sub-epithelial layer, while at later stages it is found in a hair cycle dependent pattern in the dermal papilla. To elucidate the function of Runx1 in the hair follicle we have generated a Runx1 epidermal conditional knockout and found that the mutant mice display a remarkable structural deformation of the zigzag hair type. The data delineate Runx1 as a novel specific marker of several hair follicle cell types and sheds light on its role in hair morphogenesis and differentiation.

Transcription factor Runx3 regulates interleukin-15-dependent natural killer cell activation.

Natural killer cells belong to the family of innate lymphoid cells comprising the frontline defense against infected and transformed cells. Development and activation of natural killer cells is highly dependent on interleukin-15 signaling. However, very little is known about the transcription program driving this process. The transcription factor Runx3 is highly expressed in natural killer cells, but its function in these cells is largely unknown. We show that loss of Runx3 impaired interleukin-15-dependent accumulation of mature natural killer cells in vivo and under culture conditions and pregnant Runx3(-/-) mice completely lack the unique population of interleukin-15-dependent uterine natural killer cells. Combined chromatin immunoprecipitation sequencing and differential gene expression analysis of wild-type versus Runx3-deficient in vivo activated splenic natural killer cells revealed that Runx3 cooperates with ETS and T-box transcription factors to drive the interleukin-15-mediated transcription program during activation of these cells. Runx3 functions as a nuclear regulator during interleukin-15-dependent activation of natural killer cells by regulating the expression of genes involved in proliferation, maturation, and migration. Similar studies with additional transcription factors will allow the construction of a more detailed transcriptional network that controls natural killer cell development and function.

Runx3-mediated transcriptional program in cytotoxic lymphocytes.

The transcription factor Runx3 is highly expressed in CD8(+) T and NK cytotoxic lymphocytes and is required for their effective activation and proliferation but molecular insights into the transcription program regulated by Runx3 in these cells are still missing. Using Runx3-ChIP-seq and transcriptome analysis of wild type vs. Runx3(-/-) primary cells we have now identified Runx3-regulated genes in the two cell types at both resting and IL-2-activated states. Runx3-bound genomic regions in both cell types were distantly located relative to gene transcription start sites and were enriched for RUNX and ETS motifs. Bound genomic regions significantly overlapped T-bet and p300-bound enhancer regions in Runx3-expressing Th1 helper cells. Compared to resting cells, IL-2-activated CD8(+) T and NK cells contain three times more Runx3-regulated genes that are common to both cell types. Functional annotation of shared CD8(+) T and NK Runx3-regulated genes revealed enrichment for immune-associated terms including lymphocyte activation, proliferation, cytotoxicity, migration and cytokine production, highlighting the role of Runx3 in CD8(+) T and NK activated cells.

Absence of Runx3 expression in normal gastrointestinal epithelium calls into question its tumour suppressor function.

The Runx3 transcription factor regulates cell fate decisions during embryonic development and in adults. It was previously reported that Runx3 is strongly expressed in embryonic and adult gastrointestinal tract (GIT) epithelium (Ep) and that its loss causes gastric cancer. More than 280 publications have based their research on these findings and concluded that Runx3 is indeed a tumour suppressor (TS). In stark contrast, using various measures, we found that Runx3 expression is undetectable in GIT Ep. Employing a variety of biochemical and genetic techniques, including analysis of Runx3-GFP and R26LacZ/Runx3(Cre) or R26tdTomato/Runx3(Cre) reporter strains, we readily detected Runx3 in GIT-embedded leukocytes, dorsal root ganglia, skeletal elements and hair follicles. However, none of these approaches revealed detectable Runx3 levels in GIT Ep. Moreover, our analysis of the original Runx3(LacZ/LacZ) mice used in the previously reported study failed to reproduce the GIT expression of Runx3. The lack of evidence for Runx3 expression in normal GIT Ep creates a serious challenge to the published data and undermines the notion that Runx3 is a TS involved in cancer pathogenesis.

Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.

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.

Groucho/transducin-like Enhancer-of-split (TLE)-dependent and -independent transcriptional regulation by Runx3.

Regulation of gene expression by tissue-specific transcription factors involves both turning on and turning off transcription of target genes. Runx3, a runt-domain transcription factor, regulates cell-intrinsic functions by activating and repressing gene expression in sensory neurons, dendritic cells (DC), and T cells. To investigate the mechanism of Runx3-mediated repression in an in vivo context, we generated mice expressing a mutant Runx3 lacking the C-terminal VWRPY, a motif required for Runx3 interaction with the corepressor Groucho/transducin-like Enhancer-of-split (TLE). In contrast with Runx3(-/-) mice, which displayed ataxia due to the death of dorsal root ganglia TrkC neurons, Runx3(VWRPY-/-) mice were not ataxic and had intact dorsal root ganglia neurons, indicating that ability of Runx3 to tether Groucho/TLE is not essential for neurogenesis. In the DC compartment, the mutant protein Runx3(VWRPY-) promoted normally developed skin Langerhans cells but failed to restrain DC spontaneous maturation, indicating that this latter process involves Runx3-mediated repression through recruitment of Groucho/TLE. Moreover, in CD8(+) thymocytes, Runx3(VWRPY-) up-regulated alphaE/CD103-like WT Runx3, whereas unlike wild type, it failed to repress alphaE/CD103 in CD8(+) splenocytes. Thus, in CD8-lineage T cells, Runx3 regulates alphaE/CD103 in opposing regulatory modes and recruits Groucho/TLE to facilitate the transition from activation to repression. Runx3(VWRPY-) also failed to mediate the epigenetic silencing of CD4 gene in CD8(+) T cells, but normally regulated other pan-CD8(+) T cell genes. These data provide evidence for the requirement of Groucho/TLE for Runx3-mediated epigenetic silencing of CD4 and pertain to the mechanism through which other Runx3-regulated genes are epigenetically silenced.

Loss of Runx3 function in leukocytes is associated with spontaneously developed colitis and gastric mucosal hyperplasia.

RUNX transcription factors are key regulators of lineage-specific gene expression and might be involved in autoimmune diseases. Runx3 plays a role during the development of sensory neurons and T cells and regulates transforming growth factor beta (TGF-beta) signaling in dendritic cells. Here, we report that at 4 weeks of age, Runx3 knockout (KO) mice spontaneously develop inflammatory bowel disease (IBD) characterized by leukocyte infiltration, mucosal hyperplasia, formation of lymphoid clusters, and increased production of IgA. Additionally, at a considerably older age (8 months), the KO mice also develop progressive hyperplasia of the gastric mucosa associated with disturbed epithelial differentiation and cellular hyaline degeneration. Analysis of cytokines in the colonic mucosa of Runx3 KO mice revealed a mixed T helper 1/T helper 2 response. By using immunohistochemistry and RNA in situ hybridization, Runx3 expression in the gastrointestinal tract is detected in lymphoid and myeloid populations but not in the epithelium. The data indicate that loss of leukocytic cell-autonomous function of Runx3 results in IBD and gastric lesion in the KO mice. IBD in humans is viewed as a complex genetic disorder. Several susceptibility loci were identified on different human chromosomes including the chromosomal region 1p36 where RUNX3 resides. It is thus tempting to speculate that mutations in RUNX3 may constitute an IBD risk factor in humans.

Runx3 regulates mouse TGF-beta-mediated dendritic cell function and its absence results in airway inflammation.

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-beta. In the absence of Runx3, DC become insensitive to TGF-beta-induced maturation inhibition, and TGF-beta-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 beta2-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.

Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis.

The RUNX transcription factors are important regulators of lineage-specific 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.

The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons.

The RUNX transcription factors are important regulators of linage-specific gene expression in major developmental pathways. Recently, we demonstrated that Runx3 is highly expressed in developing cranial and dorsal root ganglia (DRGs). Here we report that within the DRGs, Runx3 is specifically expressed in a subset of neurons, the tyrosine kinase receptor C (TrkC) proprioceptive neurons. We show that Runx3-deficient mice develop severe limb ataxia due to disruption of monosynaptic connectivity between intra spinal afferents and motoneurons. We demonstrate that the underlying cause of the defect is a loss of DRG proprioceptive neurons, reflected by a decreased number of TrkC-, parvalbumin- and beta-galactosidase-positive cells. Thus, Runx3 is a neurogenic TrkC neuron-specific transcription factor. In its absence, TrkC neurons in the DRG do not survive long enough to extend their axons toward target cells, resulting in lack of connectivity and ataxia. The data provide new genetic insights into the neurogenesis of DRGs and may help elucidate the molecular mechanisms underlying somatosensory-related ataxia in humans.

Phylogenesis and regulated expression of the RUNT domain transcription factors RUNX1 and RUNX3.

The RUNX transcription factors are key regulators of lineage specific gene expression in developmental pathways. The mammalian RUNX genes arose early in evolution and maintained extensive structural similarities. Sequence analysis suggested that RUNX3 is the most ancient of the three mammalian genes, consistent with its role in neurogenesis of the monosynaptic reflex arc, the simplest neuronal response circuit, found in Cnidarians, the most primitive animals. All RUNX proteins bind to the same DNA motif and act as activators or repressors of transcription through recruitment of common transcriptional modulators. Nevertheless, analysis of Runx1 and Runx3 expression during embryogenesis revealed that their function is not redundant. In adults both Runx1 and Runx3 are highly expressed in the hematopoietic system. At early embryonic stages we found strong Runx3 expression in dorsal root ganglia neurons, confined to TrkC sensory neurons. In the absence of Runx3, knockout mice develop severe ataxia due to the early death of the TrkC neurons. Other phenotypic defects of Runx3 KO mice including abnormalities in thymopoiesis are also being investigated.