Carcinogen-induced skin tumorigenesis depends heavily on proinflammatory tumor-promoting processes. Here, we show that leukocytic Runx3 expression is central to the two-stage DMBA/TPA-induced skin tumorigenesis. Runx3-null mice were highly resistant to this process and concomitant ablation of Runx3 in dendritic and T cells fully recapitulated this resistance. Mechanistically, this resistance was associated with a shift in the skin cytokine milieu toward a tumor nonpermissive microenvironment. Specifically, leukocytic Runx3 loss substantially increased the antitumorigenic cytokine thymic stromal lymphopoietin (TSLP) and profoundly decreased two protumorigenic cytokines, interleukin-17a and osteopontin. Therefore, inflammation-mediated tumor promotion requires leukocytic Runx3 expression, as its loss creates a unique cytokine composition that polarizes the tumor microenvironment to a potent antitumorigenic state.
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.
The t(8;21) and inv(16) chromosomal aberrations generate the oncoproteins AML1-ETO (A-E) and CBF?-SMMHC (C-S). The role of these oncoproteins in acute myeloid leukemia (AML) etiology has been well studied. Conversely, the function of native RUNX1 in promoting A-E- and C-S-mediated leukemias has remained elusive. We show that wild-type RUNX1 is required for the survival of t(8;21)-Kasumi-1 and inv(16)-ME-1 leukemic cells. RUNX1 knockdown in Kasumi-1 cells (Kasumi-1(RX1-KD)) attenuates the cell-cycle mitotic checkpoint, leading to apoptosis, whereas knockdown of A-E in Kasumi-1(RX1-KD) rescues these cells. Mechanistically, a delicate RUNX1/A-E balance involving competition for common genomic sites that regulate RUNX1/A-E targets sustains the malignant cell phenotype. The broad medical significance of this leukemic cell addiction to native RUNX1 is underscored by clinical data showing that an active RUNX1 allele is usually preserved in both t(8;21) or inv(16) AML patients, whereas RUNX1 is frequently inactivated in other forms of leukemia. Thus, RUNX1 and its mitotic control targets are potential candidates for new therapeutic approaches.
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.
Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esam(hi) DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4(+)/CD11b(+) DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b(+)Esam(hi) DC. Mechanistically, loss of Runx3 alters Esam(hi) DC gene expression to a signature characteristic of WT Esam(low) DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3(-/-) Esam(hi) DC capacity to prime CD4(+) T cells, attesting to the significant role of Runx3 in specifying Esam(hi) DC identity and function.
RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MK) to characterized Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1 bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1 occupied genomic regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. Megakaryocytic specificity of Runx1/P300 bound enhancers was validated by transfection mutagenesis and Runx1/P300 co-bound regions of two key megakaryocytic genes Nfe2 and Selp were tested by in vivo transgenesis. The data provides the first example of genome wide Runx1/p300 occupancy in maturating primary FL-MK, unravel the Runx1-regulated program controlling MK maturation in vivo and identify a subset of its bona fide regulated genes. It advances our understanding of the molecular events that upon RUNX1mutations in human lead to the predisposition to familial platelet disorders and FPD-AML.
The oncogenic transcription factor Runx1 is required for the specification of definitive hematopoietic stem cells (HSC) in the developing embryo. The activity of this master regulator is tightly controlled during development. The transcription factors that upregulate the expression of Runx1 also upregulate the expression of Smad6, the inhibitory Smad, which controls Runx1 activity by targeting it to the proteasome. Here we show that Runx1, in conjunction with Fli1, Gata2, and Scl, directly regulates the expression of Smad6 in the aorta-gonad-mesonephros (AGM) region in the developing embryo, where HSCs originate. Runx1 regulates Smad6 activity via a novel upstream enhancer, and Runx1 null embryos show reduced Smad6 transcripts in the yolk-sac and c-Kit-positive fetal liver cells. By directly regulating the expression of Smad6, Runx1 sets up a functional rheostat to control its own activity. The perturbation of this rheostat, using a proteasomal inhibitor, results in an increase in Runx1 and Smad6 levels that can be directly attributed to increased Runx1 binding to tissue-specific regulatory elements of these genes. Taken together, we describe a scenario in which a key hematopoietic transcription factor controls its own expression levels by transcriptionally controlling its controller.
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.
The Ets-related gene (ERG) is an Ets-transcription factor required for normal blood stem cell development. ERG expression is down-regulated during early T-lymphopoiesis but maintained in T-acute lymphoblastic leukemia (T-ALL), where it is recognized as an independent risk factor for adverse outcome. However, it is unclear whether ERG is directly involved in the pathogenesis of T-ALL and how its expression is regulated. Here we demonstrate that transgenic expression of ERG causes T-ALL in mice and that its knockdown reduces the proliferation of human MOLT4 T-ALL cells. We further demonstrate that ERG expression in primary human T-ALL cells is mediated by the binding of other T-cell oncogenes SCL/TAL1, LMO2, and LYL1 in concert with ERG, FLI1, and GATA3 to the ERG +85 enhancer. This enhancer is not active in normal T cells but in transgenic mice targets expression to fetal liver c-kit(+) cells, adult bone marrow stem/progenitors and early CD4(-)CD8(-) double-negative thymic progenitors. Taken together, these data illustrate that ERG promotes T-ALL and that failure to extinguish activity of stem cell enhancers associated with regulatory transcription factors such as ERG can contribute to the development of leukemia.
Specific interactions of transcription factors (TFs) with their targets are crucial for specifying gene expression programs during cell differentiation. How specificity is maintained despite limited selectivity of individual TF-DNA interactions is not fully understood. RUNX1 TF is among the most frequently mutated genes in human leukemia and an important regulator of megakaryopoiesis. We used megakaryocytic cell lines to characterize the network of RUNX1 targets and cooperating TFs in differentiating megakaryocytes and demonstrated how dynamic partnerships between RUNX1 and cooperating TFs facilitated regulatory plasticity and specificity during this process. After differentiation onset, RUNX1 directly activated a large number of genes through interaction with preexisting and de novo binding sites. Recruitment of RUNX1 to de novo occupied sites occurred at H3K4me1-marked preprogrammed enhancers. A significant number of these de novo bound sites lacked RUNX motif but were occupied by AP-1 TFs. Reciprocally, AP-1 TFs were up-regulated by RUNX1 after 12-O-tetradecanoylphorbol-13-acetate induction and recruited to RUNX1-occupied sites lacking AP-1 motifs. At other differentiation stages, additional combinatorial interactions occurred between RUNX1 and its coregulators, GATA1 and ETS. The findings suggest that in differentiating megakaryocytic cell lines, RUNX1 cooperates with GATA1, AP-1, and ETS to orchestrate cell-specific transcription programs through dynamic TF partnerships.
Down syndrome, the most common genetic disorder leading to mental retardation, is caused by the presence of all or part of an extra copy of chromosome 21. At relatively early ages, Down syndrome patients develop progressive formation and extracellular aggregation of amyloid-? peptide, considered as one of the causal factors for the pathogenesis of Alzheimers disease. This neuropathological hallmark has been attributed to the overexpression of APP but could also be contributed by other HSA21 genes. BACE2 maps to HSA21 and is homologous to BACE1, a ?-secretase involved in the amyloidogenic pathway of APP proteolysis, and thus it has been hypothesized that the co-overexpression of both genes could contribute to Alzheimers like neuropathology present in Down syndrome. The aim of the present study has been to analyse the impact of the co-overexpression of BACE2 and APP, using a double transgenic mouse model. Double transgenic mice did not present any neurological or sensorimotor alterations, nor genotype-dependent anxiety-like behaviour or age-associated cognitive dysfunction. Interestingly, TgBACE2-APP mice showed deregulation of BACE2 expression levels that were significantly increased with respect to single TgBACE2 mice. Co-overexpression of BACE2 and APP did not increase amyloid-? peptide concentration in brain. Our results suggest that the in vivo effects of APP are not exacerbated by BACE2 co-overexpression but may have some protective effects in specific behavioural and cognitive domains in transgenic mice.
Runx3 protein products that are translated from the distal (P1)- and proximal (P2)-promoter transcripts appear on Western blots as a 47-46kDa doublet corresponding to full-length proteins bearing the P1- and P2-N-termini respectively. An additional 44kDa protein band, the origin and nature of which was unclear, is also detected. Transfection of full-length Runx3 cDNA bearing the P2 N-terminus (P2-cDNA) into HEK293 cells resulted in expression of both 46 and 44kDa proteins. Sequence analysis of the P2-cDNA revealed an in-frame ATG 90bp downstream (+90ATG) of the proximal +1ATG. Insertion of an N-terminal HA-tag into P2-cDNA immediately downstream of the +1ATG produced HA-tagged 46kDa and untagged 44kDa proteins, consistent with the possibility that the latter was translated through initiation at the internal +90ATG site. Deleting or blocking the activity of the +1ATG, the natural cap-dependent translation initiation site in P2-cDNA, abrogated production of the 46kDa Runx3 protein while facilitating production of the 44kDa product. These findings supported the notion that Runx3 44kDa protein resulted from internal translation initiation at the +90ATG. Northern blot and RT-PCR analyses performed on RNA from P2-cDNA transfected cells showed a single transcript and product respectively, of the expected size, ruling out the possibility that the 44kDa protein was translated from transcripts originating at a cryptic promoter or produced by alternative splicing. Taken together, the data indicate that the 44kDa protein results from translation initiation at the internal ATG and that Runx3, like its family members Runx1 and Runx2, contains a mechanism for internal mRNA translation initiation.
RUNX proteins are heterodimeric factors that play crucial roles during development and differentiation of cells of the immune system. The RUNX3 transcription factor controls lineage decisions during thymopoiesis and T-cell differentiation, and modulates myeloid cell effector functions. We now report the characterization of the human RUNX3/p33 isoform, generated by splicing out a Runt DNA-binding domain-encoding exon, and whose transcriptional activities differ from those of the prototypic RUNX3/p44 molecule. Unlike RUNX3/p44, RUNX3/p33 is induced upon maturation of monocyte-derived dendritic cells (MDDC), and is unable to transactivate the regulatory regions of the CD11a, CD11c and CD49e integrin genes. Overexpression of RUNX3/p33 in myeloid cell lines led to diminished expression of genes involved in inflammatory responses. Moreover, overexpression of RUNX3/p33 down-modulated the basal level of IL-8 production from immature monocyte-derived dendritic cells (MDDC). Besides, siRNA-mediated knock-down of RUNX3 led to diminished levels of IL-8 RNA in immature MDDC, and modulated the neutrophil-recruiting capacity of myeloid cell line supernatants. Since IL-8 promotes neutrophil chemotaxis and degranulation during inflammatory responses, and exerts mitogenic and angiogenic actions within tumor microenvironment, our results imply that myeloid RUNX3 expression regulates the recruitment of leukocytes towards inflammatory foci and might also contribute to human cancer progression.
The transcription factor Runx1 is a key regulator of definitive hematopoiesis in the embryo and the adult. Lineage-specific expression of Runx1 involves transcription and post-transcription control through usage of alternative promoters and diverse 3UTR isoforms, respectively. We identified and mapped microRNA (miR) binding sites on Runx1 3UTR and show that miR-27a, miR-9, miR-18a, miR-30c, and miR-199a* bind and post-transcriptionally attenuate expression of Runx1. miR-27a impacts on both the shortest (0.15 kb) and longest (3.8 kb) 3UTRs and, along with additional miRs, might contribute to translation attenuation of Runx1 mRNA in the myeloid cell line 416B. Whereas levels of Runx1 mRNA in 416B and the B cell line 70Z were similar, the protein levels were not. Large amounts of Runx1 protein were found in 70Z cells, whereas only minute amounts of Runx1 protein were made in 416B cells and overexpression of Runx1 in 416B induced terminal differentiation associated with megakaryocytic markers. Induction of megakaryocytic differentiation in K562 cells by 12-o-tetradecanoylphorbol-13-acetate markedly increased miR-27a expression, concomitantly with binding of Runx1 to miR-27a regulatory region. The data indicate that miR-27a plays a regulatory role in megakaryocytic differentiation by attenuating Runx1 expression, and that, during megakaryopoiesis, Runx1 and miR-27a are engaged in a feedback loop involving positive regulation of miR-27a expression by Runx1.
Activation of naive CD8(+) T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) gamma expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-gamma, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.
Runx3 is one of the three mammalian Runt domain transcription factors comprising the deeply conserved RUNX gene family. While the three proteins recognize the same DNA-motif, the functional overlaps are minor; each Runx has a distinct subset of biological functions. This lack of functional redundancy is the consequence of a tightly regulated spatio/temporal expression of the genes by transcriptional and post-transcriptional control mechanisms. Over the years several groups created Runx3-deficient mouse models. Analysis of these mice revealed various phenotypic features that result from loss of cell autonomous function of Runx3. Here we summarize the phenotypic similarities and dissimilarities between two of the Runx3-deficient mouse strains, discuss the basis of the discrepancies and highlight the crux of the dispute.
The formation of functional connectivity in the nervous system is governed by axon guidance that instructs nerve growth and branching during development, implying a similarity between neuronal subtypes in terms of nerve extension. We demonstrate the molecular mechanism of another layer of complexity in vertebrates by defining a transcriptional program underlying growth differences between positionally different neurons. The rate of axon extension of the early subset of embryonic dorsal root ganglion sensory neurons is encoded in neurons at different axial levels. This code is determined by a segmental pattern of axial levels of Runx family transcription factor Runx3. Runx3 in turn determines transcription levels of genes encoding cytoskeletal proteins involved in axon extension, including Rock1 and Rock2 which have ongoing activities determining axon growth in early sensory neurons and blocking Rock activity reverses axon extension deficits of Runx3(-/-) neurons. Thus, Runx3 acts to regulate positional differences in axon extension properties apparently without affecting nerve guidance and branching, a principle that could be relevant to other parts of the nervous system.
Down syndrome (DS) leads to complex phenotypes and is the main genetic cause of birth defects and heart diseases. The Ts65Dn DS mouse model is trisomic for the distal part of mouse chromosome 16 and displays similar features with post-natal lethality and cardiovascular defects. In order to better understand these defects, we defined electrocardiogram (ECG) with a precordial set-up, and we found conduction defects and modifications in wave shape, amplitudes, and durations in Ts65Dn mice. By using a genetic approach consisting of crossing Ts65Dn mice with Ms5Yah mice monosomic for the App-Runx1 genetic interval, we showed that the Ts65Dn viability and ECG were improved by this reduction of gene copy number. Whole-genome expression studies confirmed gene dosage effect in Ts65Dn, Ms5Yah, and Ts65Dn/Ms5Yah hearts and showed an overall perturbation of pathways connected to post-natal lethality (Coq7, Dyrk1a, F5, Gabpa, Hmgn1, Pde10a, Morc3, Slc5a3, and Vwf) and heart function (Tfb1m, Adam19, Slc8a1/Ncx1, and Rcan1). In addition cardiac connexins (Cx40, Cx43) and sodium channel sub-units (Scn5a, Scn1b, Scn10a) were found down-regulated in Ts65Dn atria with additional down-regulation of Cx40 in Ts65Dn ventricles and were likely contributing to conduction defects. All these data pinpoint new cardiac phenotypes in the Ts65Dn, mimicking aspects of human DS features and pathways altered in the mouse model. In addition they highlight the role of the App-Runx1 interval, including Sod1 and Tiam1, in the induction of post-natal lethality and of the cardiac conduction defects in Ts65Dn. These results might lead to new therapeutic strategies to improve the care of DS people.
Runx transcription factor family proteins have essential roles during T-cell development by either activating or repressing target genes. For instance, lineage- and stage-specific expression of Cd4 and ThPOK is controlled by a transcriptional silencer embedded in each locus, whose activity requires bindings of Runx complexes. The evolutionarily conserved VWRPY penta-peptide sequences in Runx proteins have been shown to be responsible for repressive function as a platform to recruit Groucho/TLE transcriptional corepressors. However, it remains elusive whether requirement for the VWRPY motif differs among Runx target genes. By examining mice lacking VWRPY motifs in both Runx1 and Runx3 proteins, here, we show a full and partial derepression of Cd4 and ThPOK in CD8-linegae T cells, respectively. Thus, whereas Cd4 silencing completely depends on the VWRPY motif, both VWRPY-dependent and -independent mechanisms operate to repress ThPOK gene. These results indicate that Runx proteins utilize different modes to repress expression of different target genes.
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