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The level of CD40 expression on dendritic cells (DCs) plays a decisive role in disease protection during Leishmania donovani (LD) infection. However, current understanding of the molecular regulation of CD40 expression remains elusive. Using molecular, cellular and functional approaches, we identified a role for Runx1 and Runx3 transcription factors in the regulation of CD40 expression in DCs. In response to lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα) or antileishmanial drug sodium antimony gluconate (SAG), both Runx1 and Runx3 translocated to the nucleus, bound to the CD40 promoter and upregulated CD40 expression on DCs. These activities of Runx proteins were mediated by the upstream phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Notably, LD infection attenuated LPS- or TNFα-induced CD40 expression in DCs by inhibiting PI3K-Akt-Runx axis via protein tyrosine phosphatase SHP-1. In contrast, CD40 expression induced by SAG was unaffected by LD infection, as SAG by blocking LD-induced SHP-1 activation potentiated PI3K-Akt signaling to drive Runx-mediated CD40 upregulation. Adoptive transfer experiments further showed that Runx1 and Runx3 play a pivotal role in eliciting antileishmanial immune response of SAG-treated DCs in vivo by promoting CD40-mediated type-1 T cell responses. Importantly, antimony-resistant LD suppressed SAG-induced CD40 upregulation on DCs by blocking the PI3K-Akt-Runx pathway through sustained SHP-1 activation. These findings unveil an immunoregulatory role for Runx proteins during LD infection., Author summary Visceral leishmaniasis (VL), caused by Leishmania donovani, is the second most deadly parasitic disease in the world after malaria. The protective immunity against this disease largely depends on the level of CD40 (a cell surface protein) expression on a specialized group of immune cells called “dendritic cells” (DCs), which play a central role in initiating antileishmanial immune responses. Accordingly, to develop an improved immunological approach for the treatment/prevention of VL, it is important to understand the molecular events that favor the induction of CD40 expression on DCs. However, the molecular mechanisms controlling CD40 expression are not yet fully understood. Our work describes, for the first time, that the transcription factors Runx1 and Runx3 promote CD40 expression on DCs, which thereby potentiate the ability of DCs to induce antileishmanial immune responses in vivo. Our study thus provides new insights into the molecular mechanism required for an immune response to combat VL.

Sanggenon C (SC), which is a natural flavonoid found in the stem bark of Cortex Mori, has been discovered to have the antioxidant, anti-inflammatory, and antitumor properties. However, its effect in osteoporosis has not yet been reported. In this research, the effect of SC on the proliferation of MC3T3-E1 cells was evaluated by using the MTT assay. Alkaline phosphatase (ALP) activity and the mRNA expression of Runx2, Collagen I, OPG, and RANKL were examined. TRAP-positive cell counting and bone resorption pits were adopted to observe the effect of SC on the formation and function of osteoclasts. Next, the mRNA level of TRAP, CTSK, NFATc1, and TRAF6 of osteoclasts were measured by real-time qPCR. In addition, the anti-osteoporosis activity of SC in vivo was evaluated in the zebrafish model. Our study indicated that SC exhibited a significant stimulatory effect on MC3T3-E1 cell proliferation at 1 to 10 &mu, M and caused an increase in ALP activity at 0.3 to 10 &mu, M. It could upregulate the expression of Runx2, Collagen I, and increases the OPG/RANKL ratio. Furthermore, SC was found to inhibit the formation and function of osteoclasts, which is demonstrated by a lower number of TRAP-positive multinuclear cells and a fewer area of bone resorption pits compared to the control group. TRAP, CTSK, and NFATc1 were downregulated in 0.3 to 10 &mu, M SC treated groups. In addition, 3 to 10 &mu, M SC also inhibited the expression of TRAF6 mRNA. When prednisone-induced zebrafish was treated with 0.3, 1, 3, and 10 &mu, M SC, higher mineralization of vertebrate column was discovered in a dose-dependent pattern, which suggests that SC could reverse the bone loss of zebrafish caused by prednisone. In summary, these findings indicated that SC has the potential to prevent or treat osteoporosis.

R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes, including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell-enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo. Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among patients with breast cancer. Thus, we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers., Facultad de Ciencias Médicas, Centro de Investigaciones Inmunológicas Básicas y Aplicadas

In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of RUNX transcription factor paralogs with apparent functional redundancy. Here we asked what cell-type-specific biologies might be supported by the selective expression of RUNX paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional nonequivalence between RUNX paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain and evolutionary reconstruction suggested convergence of RUNT domain residues toward submaximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors., Author summary In the development of the mammalian growth plate, while several transcription factors are individually well known for their key roles in regulating phases of chondrocyte differentiation, there is little information on how they interact and cooperate with each other. We took an unbiased genome wide approach to identify the transcription factors and signaling pathways that play dominant roles in the chondrocyte differentiation cascade. We developed a searchable library of differentially expressed genes, GP-DGEL, which has fine spatial resolution and global transcriptomic coverage for discovery of processes, pathways and disease candidates. Our work identifies a novel regulatory mechanism that integrates the action of three transcription factors, SOX9, GLI and FOXA. SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon entry into prehypertrophy, FOXA competes with SOX9, and control of hypertrophy passes to FOXA, RUNX, AP1 and MEF2 factors.

Recent advances in single-cell transcriptomics techniques have opened the door to the study of gene regulatory networks (GRNs) at the single-cell level. Here, we studied the GRNs controlling the emergence of hematopoietic stem and progenitor cells from mouse embryonic endothelium using a combination of single-cell transcriptome assays. We found that a heptad of transcription factors (Runx1, Gata2, Tal1, Fli1, Lyl1, Erg and Lmo2) is specifically co-expressed in an intermediate population expressing both endothelial and hematopoietic markers. Within the heptad, we identified two sets of factors of opposing functions: one (Erg/Fli1) promoting the endothelial cell fate, the other (Runx1/Gata2) promoting the hematopoietic fate. Surprisingly, our data suggest that even though Fli1 initially supports the endothelial cell fate, it acquires a pro-hematopoietic role when co-expressed with Runx1. This work demonstrates the power of single-cell RNA-sequencing for characterizing complex transcription factor dynamics.

Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm.

CD4 + helper T cells and CD8 + cytotoxic T cells form the two major subsets of peripheral T lymphocytes. Helper T cells fulfill crucial roles in the activation and coordination of the immune response, while cytotoxic T cells kill virus-infected or tumor cells. Recent data suggest that the lineage identify of helper T cells is not fixed and that CD4 + T cells under certain physiological conditions can be reprogrammed to express CD8 lineage genes and to develop into intestinal intraepithelial CD4 + cytotoxic T lymphocytes that lack the expression of the key helper T cell lineage commitment factor ThPOK. Moreover, the analysis of mice with a conditional deletion of the transcription factor ThPOK or the histone deacetylases HDAC1 and HDAC2 indicated that CD8 lineage genes are actively repressed in CD4 + T cells in order to maintain the lineage integrity of helper T cells. In this review I summarize recent studies that indicate plasticity of CD4 + T cells towards a CTL program and that demonstrate that ThPOK and HDAC1–HDAC2 are part of a transcriptional regulatory circuit that counteracts the activity of the transcription factor Runx3 to maintain CD4 + T cell lineage integrity.

The hormone-sensing mammary epithelial cell (HS-MEC-expressing oestrogen receptor-alpha (ERα) and progesterone receptor (PGR)) is often represented as being terminally differentiated and lacking significant progenitor activity after puberty. Therefore while able to profoundly influence the proliferation and function of other MEC populations, HS-MECs are purported not to respond to sex hormone signals by engaging in significant cell proliferation during adulthood. This is a convenient and practical simplification that overshadows the sublime, and potentially critical, phenotypic plasticity found within the adult HS-MEC population. This concept is exemplified by the large proportion (~80 %) of human breast cancers expressing PGR and/or ERα, demonstrating that HS-MECs clearly proliferate in the context of breast cancer. Understanding how HS-MEC proliferation and differentiation is driven could be key to unraveling the mechanisms behind uncontrolled HS-MEC proliferation associated with ERα- and/or PGR-positive breast cancers. Herein we review evidence for the existence of a HS-MEC progenitor and the emerging plasticity of the HS-MEC population in general. This is followed by an analysis of hormones other than oestrogen and progesterone that are able to influence HS-MEC proliferation and differentiation: androgens, prolactin and transforming growth factor-beta1.

To explore the effects of Elemene, the essential oil of Curcuma wenyujin, on Bone morphogenetic protein/drosophila mothers against decapentaplegic proteins (BMP/SMADs) signal pathway in ankylosing spondylitis (AS) fibroblasts.Hip joint capsules were obtained from AS patients (n=10) receiving total hip replacement. Healthy hip joint capsules from patients with hip fracture (n=10) receiving surgery were included as a control. Primary fibroblast cell lines were established from these tissue samples. Fibroblasts were incubated with Elemene for 48 hours. The protein expression was detected by Western blot. The mRNA expression was detected by real-time fluorescent quantitative polymerase chain reaction (PCR).The results showed that the expression of proteins including SMAD1, pSMAD1, SMAD4 and Runt-related transcription factor 2 (RUNX2), and mRNA of RUNX2, which were over-expressed in AS fibroblasts were decreased in the AS fibroblasts cultured in medium with Elemene.Ele could have a hand in anti-osteogenic differentiation of AS fibroblasts by inhibiting the BMP/SMADs signal pathway and subsequently blocking expression of ossification marker genes RUNX2 that initiate the osteogenic differentiation.

The core binding factor (CBF) gene RUNX1 is a target of chromosomal translocations in leukemia, including t(8;21) in acute myeloid leukemia (AML). Normal CBF function is essential for activity of AML1-ETO, product of the t(8;21), and for survival of several leukemias lacking RUNX1 mutations. Using virtual screening and optimization, we developed Runt domain inhibitors which bind to the Runt domain and disrupt its interaction with CBFβ. On-target activity was demonstrated by the Runt domain inhibitors' ability to depress hematopoietic cell formation in zebrafish embryos, reduce growth and induce apoptosis of t(8;21) AML cell lines, and reduce progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. Runt domain inhibitors had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines. Our results confirmed that Runt domain inhibitors might prove efficacious in various AMLs and in T-ALL.

Among the particular immunomodulation properties of mesenchymal stem cells (MSCs), one relies on their capacity to regulatory T cell (Treg) induction from effector T cells. Stable expression of Foxp3 has a dominant role in suppressive phenotype and stability of induced regulatory T cells (iTregs). How MSCs induce stable Foxp3 expression in iTregs remains unknown. We previously showed MSCs could enhance demethylation of Treg-specific demethylated region (TSDR) in iTregs in cell-cell contact manner (unpublished data). Here, we evaluated the possible effect of MSCs on the mRNA expression of Runx complex genes (Runx1, Runx3, and CBFB) that perch on TSDR in iTregs and play the main role in suppressive properties of Tregs, a regulatory pathway that has not yet been explored by MSCs. Also, we investigated the mRNA expression of MBD2 that promotes TSDR demethylation in Tregs. We first showed that in vitro MSC-iTreg induction was associated with strong mRNA modifications of genes involved in Runx complex. We next injected high doses of MSCs in a murine model of C57BL/6 into Balb/C allogeneic skin transplantation to prolong allograft survival. When splenocytes of grafted mice were analyzed, we realized that the Foxp3 expression was increased at day 5 and 10 post-graft merely in MSC-treated mice. Furthermore, Foxp3 mRNA expression was associated with modified Runx complex mRNA expression comparable to what was shown in in vitro studies. Hence, our data identify a possible mechanism in which MSCs convert conventional T cells to iTreg through strong modifications of mRNA of genes that are involved in Runx complex of Foxp3.

The C-terminal multimerization domain is essential for leukemia development by CBFβ-SMMHC in a mouse knockin model

Background The Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein. Results To address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform. Conclusions Our results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production. Electronic supplementary material The online version of this article (10.1186/s12861-017-0156-y) contains supplementary material, which is available to authorized users.

A full understanding of RUNX gene function in different epithelial lineages has been thwarted by the lethal phenotypes observed when constitutively knocking out these mammalian genes. However temporal expression of the Runx genes throughout the different phases of mammary gland development is indicative of a functional role in this tissue. A few studies have emerged describing how these genes impact on the fate of mammary epithelial cells by regulating lineage differentiation and stem/progenitor cell potential, with implications for the transformed state. The importance of the RUNX/CBFβ core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFβ appearing in a comprehensive gene list of predicted breast cancer driver mutations. Nonetheless, the evidence to date shows that the RUNX genes can have dualistic outputs with respect to promoting or constraining breast cancer phenotypes, and that this may be aligned to individual subtypes of the clinical disease. We take this opportunity to review the current literature on RUNX and CBFβ in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

Runt-related (Runx) transcription factors play essential roles during development and adult tissue homeostasis and are responsible for several human diseases. They regulate a variety of biological mechanisms in numerous cell lineages. Recent years have seen significant progress in our understanding of the functions performed by Runx proteins in the developing and postnatal mammalian nervous system. In both central and peripheral nervous systems, Runx1 and Runx3 display remarkably specific expression in mostly non-overlapping groups of postmitotic neurons. In the central nervous system, Runx1 is involved in the development of selected motor neurons controlling neural circuits mediating vital functions such as chewing, swallowing, breathing, and locomotion. In the peripheral nervous system, Runx1 and Runx3 play essential roles during the development of sensory neurons involved in circuits mediating pain, itch, thermal sensation and sense of relative position. Runx1 and Runx3 orchestrate complex gene expression programs controlling neuronal subtype specification and axonal connectivity. Runx1 is also important in the olfactory system, where it regulates the progenitor-to-neuron transition in undifferentiated neural progenitor cells in the olfactory epithelium as well as the proliferation and developmental maturation of specific glial cells termed olfactory ensheathing cells. Moreover, upregulated Runx expression is associated with brain injury and disease. Increasing knowledge of the functions of Runx proteins in the developing and postnatal nervous system is therefore expected to improve our understanding of nervous system development, homeostasis and disease.

The RUNX family of transcription factors plays important roles in tissue-specific gene expression. Many of their functions depend on specific post-translational modifications (PTMs), and in this review, we describe how PTMs govern RUNX DNA binding, transcriptional activity, protein stability, cellular localization, and protein-protein interactions. We also report how these processes can be disrupted in disease settings. Finally, we describe how alterations of RUNX1, or the enzymes that catalyze its post-translational modifications, contribute to hematopoietic malignancies.

During hematopoiesis, a variety of cells are generated from stem cells through successive rounds of cell fate determination processes. Studies in the last two decades have demonstrated the involvement of Runx transcription factor family members in differentiation of multiple types of hematopoietic cells. Along with evolutionary conservation, the Runx family is considered to be one of the ancestral regulators of hematopoiesis. It is conceivable that the Runx family is involved in shaping the immune system, which is then comprised of innate and acquired lymphoid cells in vertebrates. In this chapter, we will first summarize roles of Runx proteins during the development of T- and B-lymphocytes, which appeared later during evolution and express antigen specific receptors as a result of DNA recombination processes. We also discuss the recent findings that have unraveled the functions of Runx during differentiation of innate lymphoid cells (ILCs).

The Runx family genes play important roles in development and cancer, largely via their regulation of tissue stem cell behavior. Their involvement in two organs, blood and skin, is well documented. This review summarizes currently known Runx functions in the stem cells of these tissues. The fundamental core mechanism(s) mediated by Runx proteins has been sought; however, it appears that there does not exist one single common machinery that governs both tissue stem cells. Instead, Runx family genes employ multiple spatiotemporal mechanisms in regulating individual tissue stem cell populations. Such specific Runx requirements have been unveiled by a series of cell type-, developmental stage- or age-specific gene targeting studies in mice. Observations from these experiments revealed that the regulation of stem cells by Runx family genes turned out to be far more complex than previously thought. For instance, although it has been reported that Runx1 is required for the endothelial-to-hematopoietic cell transition (EHT) but not thereafter, recent studies clearly demonstrated that Runx1 is also needed during the period subsequent to EHT, namely at perinatal stage. In addition, Runx1 ablation in the embryonic skin mesenchyme eventually leads to complete loss of hair follicle stem cells (HFSCs) in the adult epithelium, suggesting that Runx1 facilitates the specification of skin epithelial stem cells in a cell extrinsic manner. Further in-depth investigation into how Runx family genes are involved in stem cell regulation is warranted.

The observation that the Runx genes act as targets for transcriptional activation by retroviral insertion identified a new family of dominant oncogenes. However, it is now clear that Runx genes are 'conditional' oncogenes whose over-expression is growth inhibitory unless accompanied by another event such as concomitant over-expression of MYC or loss of p53 function. Remarkably, while the oncogenic activities of either MYC or RUNX over-expression are suppressed while p53 is intact, the combination of both neutralises p53 tumour suppression in vivo by as yet unknown mechanisms. Moreover, there is emerging evidence that endogenous, basal RUNX activity is important to maintain the viability and proliferation of MYC-driven lymphoma cells. There is also growing evidence that the human RUNX genes play a similar conditional oncogenic role and are selected for over-expression in end-stage cancers of multiple types. Paradoxically, reduced RUNX activity can also predispose to cell immortalisation and transformation, particularly by mutant Ras. These apparently conflicting observations may be reconciled in a stage-specific model of RUNX involvement in cancer. A question that has yet to be fully addressed is the extent to which the three Runx genes are functionally redundant in cancer promotion and suppression.

Epithelial-mesenchymal transition (EMT) is an evolutionary conserved morphogenetic program necessary for the shaping of the body plan during development. It is guided precisely by growth factor signaling and a dedicated network of specialised transcription factors. These are supported by other transcription factor families serving auxiliary functions during EMT, beyond their general roles as effectors of major signaling pathways. EMT transiently induces in epithelial cells mesenchymal properties, such as the loss of cell-cell adhesion and a gain in cell motility. Together, these newly acquired properties enable their migration to distant sites where they eventually give rise to adult epithelia. However, it is now recognized that EMT contributes to the pathogenesis of several human diseases, notably in tissue fibrosis and cancer metastasis. The RUNX family of transcription factors are important players in cell fate determination during development, where their spatio-temporal expression often overlaps with the occurrence of EMT. Furthermore, the dysregulation of RUNX expression and functions are increasingly linked to the aberrant induction of EMT in cancer. The present chapter reviews the current knowledge of this emerging field and the common themes of RUNX involvement during EMT, with the intention of fostering future research.

All RUNX genes have been implicated in the development of solid tumors, but the role each RUNX gene plays in the different tumor types is complicated by multiple interactions with major signaling pathways and tumor heterogeneity. Moreover, for a given tissue type, the specific role of each RUNX protein is distinct at different stages of differentiation. A regulatory function for RUNX in tissue stem cells points sharply to a causal effect in tumorigenesis. Understanding how RUNX dysregulation in cancer impinges on normal biological processes is important for identifying the molecular mechanisms that lead to malignancy. It will also indicate whether restoration of proper RUNX function to redirect cell fate is a feasible treatment for cancer. With the recent advances in RUNX research, it is time to revisit the many mechanisms/pathways that RUNX engage to regulate cell fate and decide whether cells proliferate, differentiate or die.

RUNX1 and RUNX3 are the main RUNX genes expressed in B lymphocytes. Both are expressed throughout B-cell development and play key roles at certain key developmental transitions. The tumour-associated Epstein-Barr virus (EBV) has potent B-cell transforming ability and manipulates RUNX3 and RUNX1 transcription through novel mechanisms to control B cell growth. In contrast to resting mature B cells where RUNX1 expression is high, in EBV-infected cells RUNX1 levels are low and RUNX3 levels are high. Downregulation of RUNX1 in these cells results from cross-regulation by RUNX3 and serves to relieve RUNX1-mediated growth repression. RUNX3 is upregulated by the EBV transcription factor (TF) EBNA2 and represses RUNX1 transcription through RUNX sites in the RUNX1 P1 promoter. Recent analysis revealed that EBNA2 activates RUNX3 transcription through an 18 kb upstream super-enhancer in a manner dependent on the EBNA2 and Notch DNA-binding partner RBP-J. This super-enhancer also directs RUNX3 activation by two further RBP-J-associated EBV TFs, EBNA3B and 3C. Counter-intuitively, EBNA2 also hijacks RBP-J to target a super-enhancer region upstream of RUNX1 to maintain some RUNX1 expression in certain cell backgrounds, although the dual functioning EBNA3B and 3C proteins limit this activation. Interestingly, the B-cell genome binding sites of EBV TFs overlap extensively with RUNX3 binding sites and show enrichment for RUNX motifs. Therefore in addition to B-cell growth manipulation through the long-range control of RUNX transcription, EBV may also use RUNX proteins as co-factors to deregulate the transcription of many B cell genes during immortalisation.

During the past two decades, Runt domain transcription factors (RUNX1, 2, and 3) have been investigated in regard to their function, structural elements, genetic variants, and roles in normal development and pathological conditions. The Runt family proteins are evolutionarily conserved from Drosophila to mammals, emphasizing their physiological importance. A hypoxic microenvironment caused by insufficient blood supply is frequently observed in developing organs, growing tumors, and tissues that become ischemic due to impairment or blockage of blood vessels. During embryonic development and tumor growth, hypoxia triggers a stress response that overcomes low-oxygen conditions by increasing erythropoiesis and angiogenesis and triggering metabolic changes. This review briefly introduces hypoxic conditions and cellular responses, as well as angiogenesis and its related signaling pathways, and then describes our current knowledge on the functions and molecular mechanisms of Runx family proteins in hypoxic responses, especially in angiogenesis.

The core binding factor (CBF) transcription factor is somewhat unique in that it is composed of a DNA binding RUNX subunit (RUNX1, 2, or 3) and a non-DNA binding CBFβ subunit, which modulates RUNX protein activity by modulating the auto-inhibition of the RUNX subunits. Since the discovery of this fascinating transcription factor more than 20 years ago, there has been a robust effort to characterize the structure as well as the biochemical properties of CBF. More recently, these efforts have also extended to the fusion proteins that arise from the subunits of CBF in leukemia. This chapter highlights the work of numerous labs which has provided a detailed understanding of the structure and function of this transcription factor and its fusion proteins.

The observation that the Runx genes act as targets for transcriptional activation by retroviral insertion identified a new family of dominant oncogenes. However, it is now clear that Runx genes are 'conditional' oncogenes whose over-expression is growth inhibitory unless accompanied by another event such as concomitant over-expression of MYC or loss of p53 function. Remarkably, while the oncogenic activities of either MYC or RUNX over-expression are suppressed while p53 is intact, the combination of both neutralises p53 tumour suppression in vivo by as yet unknown mechanisms. Moreover, there is emerging evidence that endogenous, basal RUNX activity is important to maintain the viability and proliferation of MYC-driven lymphoma cells. There is also growing evidence that the human RUNX genes play a similar conditional oncogenic role and are selected for over-expression in end-stage cancers of multiple types. Paradoxically, reduced RUNX activity can also predispose to cell immortalisation and transformation, particularly by mutant Ras. These apparently conflicting observations may be reconciled in a stage-specific model of RUNX involvement in cancer. A question that has yet to be fully addressed is the extent to which the three Runx genes are functionally redundant in cancer promotion and suppression.

The Runt-domain (RD) transcription factors (RUNX genes) are an important family of transcriptional mediators that interact with a variety of proteins including the Hippo pathway effector proteins, YAP and TAZ. In this chapter we focus on two examples of RUNX-TAZ/YAP interactions that have particular significance in human cancer. Specifically, recent evidence has found that RUNX2 cooperates with TAZ to promote epithelial to mesenchymal transition mediated by the soluble N-terminal ectodomain of E-Cadherin, sE-Cad. Contrastingly, in gastric cancer, RUNX3 acts as a tumor suppressor via inhibition of the YAP-TEAD complex and disruption of downstream YAP-mediated gene transcription, and the oncogenic phenotype. The reports highlighted in this chapter add to the growing repertoire of instances of Hippo pathway crosstalk that have been identified in cancer. Elucidation of these increasingly complex interactions may help to identify novel strategies to target Hippo pathway dysregulation in human cancer.

In order to achieve a persistent infection, viruses must overcome the host immune system. Host restriction factors dominantly block virus transmission, but are subject to down regulation by viral accessory proteins. HIV encodes several accessory factors that overcome different cellular restriction factors. For example, the HIV-1 protein Vif down regulates the human APOBEC3 family of restriction factors by targeting them for proteolysis by the ubiquitin-proteasome pathway. Recently, this function was shown to require the transcription cofactor CBFβ, which acts as a template to assist in Vif folding and allow for assembly of an APOBEC3-targeting E3 ligase complex. In uninfected cells, CBFβ is an essential binding partner of RUNX transcription factors. By binding CBFβ, Vif has also been shown to perturb transcription of genes regulated by the RUNX proteins, including restrictive APOBEC3 family members. Here we review how the link between CBFβ and Vif supports transcriptional and post-transcriptional repression of innate immunity. The ability of a single viral protein to coopt multiple host pathways is an economical strategy for a pathogen with limited protein coding capacity to achieve a productive infection.

The RUNX family of transcription factors plays important roles in tissue-specific gene expression. Many of their functions depend on specific post-translational modifications (PTMs), and in this review, we describe how PTMs govern RUNX DNA binding, transcriptional activity, protein stability, cellular localization, and protein-protein interactions. We also report how these processes can be disrupted in disease settings. Finally, we describe how alterations of RUNX1, or the enzymes that catalyze its post-translational modifications, contribute to hematopoietic malignancies.

RUNX1 and RUNX3 are the main RUNX genes expressed in B lymphocytes. Both are expressed throughout B-cell development and play key roles at certain key developmental transitions. The tumour-associated Epstein-Barr virus (EBV) has potent B-cell transforming ability and manipulates RUNX3 and RUNX1 transcription through novel mechanisms to control B cell growth. In contrast to resting mature B cells where RUNX1 expression is high, in EBV-infected cells RUNX1 levels are low and RUNX3 levels are high. Downregulation of RUNX1 in these cells results from cross-regulation by RUNX3 and serves to relieve RUNX1-mediated growth repression. RUNX3 is upregulated by the EBV transcription factor (TF) EBNA2 and represses RUNX1 transcription through RUNX sites in the RUNX1 P1 promoter. Recent analysis revealed that EBNA2 activates RUNX3 transcription through an 18 kb upstream super-enhancer in a manner dependent on the EBNA2 and Notch DNA-binding partner RBP-J. This super-enhancer also directs RUNX3 activation by two further RBP-J-associated EBV TFs, EBNA3B and 3C. Counter-intuitively, EBNA2 also hijacks RBP-J to target a super-enhancer region upstream of RUNX1 to maintain some RUNX1 expression in certain cell backgrounds, although the dual functioning EBNA3B and 3C proteins limit this activation. Interestingly, the B-cell genome binding sites of EBV TFs overlap extensively with RUNX3 binding sites and show enrichment for RUNX motifs. Therefore in addition to B-cell growth manipulation through the long-range control of RUNX transcription, EBV may also use RUNX proteins as co-factors to deregulate the transcription of many B cell genes during immortalisation.

Core Binding Factor β (CBFβ) is complexed with the RUNX family of transcription factors in the nucleus to support activation or repression of genes related to bone (RUNX2), hematopoiesis (RUNX1) and gastrointestinal (RUNX3) development. Furthermore, RUNX proteins contribute to the onset and progression of different types of cancer. Although CBFβ localizes to cytoskeletal architecture, its biological role in the cytoplasmic compartment remains to be established. Additionally, the function and localization of CBFβ during the cell cycle are important questions relevant to its biological role. Here we show that CBFβ dynamically distributes in different stages of cell division and importantly is present during telophase at the midbody, a temporal structure important for successful cytokinesis. A functional role for CBFβ localization at the midbody is supported by striking defects in cytokinesis that include polyploidy and abscission failure following siRNA-mediated downregulation of endogenous CBFβ or overexpression of the inv(16) fusion protein CBFβ-SMMHC. Our results suggest that CBFβ retention in the midbody during cytokinesis reflects a novel function that contributes to epigenetic control.

Runx2 is a major transcription factor that induces osteoblast differentiation by bone morphogenetic proteins (BMPs). Conversely, YY1 is a transcription factor that inhibits BMP2-induced cell differentiation. Until now, there has been no understanding of how osteoblast differentiation by Runx2 and YY1 is regulated. In this study we focused on the relationship between Runx2 and YY1. We confirmed that alkaline phosphatase staining is repressed by YY1. Runx2 interacted with YY1 through Runt and the C-terminus domain of Runx2. YY1 markedly repressed the Runx2-mediated enhancement of transcriptional activity on the osteocalcin and alkaline phosphatase promoters. Knockdown of YY1 enhanced BMP2- and Runx2-induced osteoblast differentiation. YY1 decreased Runx2 DNA binding affinity. The results indicate that YY1 represses osteoblast differentiation by an interaction with Runx2 and inhibits the transcriptional activity of Runx2.

Runx transcription factors contribute to hematopoiesis and are frequently implicated in hematologic malignancies. All three Runx isoforms are expressed at the earliest stages of hematopoiesis; however, their function in hematopoietic stem cells (HSCs) is not fully elucidated. Here, we show that Runx factors are essential in HSCs by driving the expression of the hematopoietic transcription factor PU.1. Mechanistically, by using a knockin mouse model in which all three Runx binding sites in the -14kb enhancer of PU.1 are disrupted, we observed failure to form chromosomal interactions between the PU.1 enhancer and its proximal promoter. Consequently, decreased PU.1 levels resulted in diminished long-term HSC function through HSC exhaustion, which could be rescued by reintroducing a PU.1 transgene. Similarly, in a mouse model of AML/ETO9a leukemia, disrupting the Runx binding sites resulted in decreased PU.1 levels. Leukemia onset was delayed, and limiting dilution transplantation experiments demonstrated functional loss of leukemia-initiating cells. This is surprising, because low PU.1 levels have been considered a hallmark of AML/ETO leukemia, as indicated in mouse models and as shown here in samples from leukemic patients. Our data demonstrate that Runx-dependent PU.1 chromatin interaction and transcription of PU.1 are essential for both normal and leukemia stem cells.

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.

Patients with compromised bone conditions still suffer the problem of deficient osseointegration during dental implant treatment. Developing mesenchymal stem cell (MSC) sheet functionalized titanium implant with proper inductive cue to promote osteogenesis and angiogenesis coupling shall be a good solution. In the present study, the antimiR-138 delivered MSC sheet is used to functionalize the Ti implant. The cell sheet can well integrate with the Ti implant to form the MSC sheet-implant complex (MSIC). The antimiR-138 delivered MSIC shows greatly improved osteogenesis and angiogenesis coupling both in vitro and in vivo. In vitro, the antimiR-138 delivered MSIC significantly promotes the expression of endogenous osteogenesis and angiogenesis related genes and proteins, alkaline phosphatase activity, extracellular matrix mineralization and collagen secretion compared to the antimiR-control and the nothing delivered control. The in vivo ectopic implantation assay uncovers the robust vascularized bone formation of the antimiR-138 delivered MSIC. The antimiR-138 delivered MSIC with promoted osteogenesis and angiogenesis coupling is anticipated to lead to rigid osseointegration in the compromised bone conditions.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

Objective The role of the Notch pathway has already been identified as a crucial regulator of bone development. However, the Notch signaling pathway has gone largely unexplored during osseointegration. This study aims to investigate the role of Notch signaling on osteogenic differentiation of rat derived bone marrow mesenchymal stem cells (BMSCs) on sandblasted, large-grit, acid-etched (SLA) treated Ti disks. Methods The involved target genes in Notch pathways were identified by in vitro microarray and bioinformatics analyses with or without osteogenic induction. Adhesion, proliferation, and osteogenic related assay were subsequently conducted with target gene shRNA treatment. Results We found that 11 genes in the Notch signaling pathway were differentially expressed after osteogenic induction on SLA-treated Ti disks, which included up-regulated genes ( Notch2, Dll1, Dll3, Ncstn, Ncor2, and Hes5) and down-regulated genes (Notch3, Lfng, Mfng, Jag2 and Maml2) . With Notch3 shRNA treatment, the adhesion and proliferation of BMSCs on SLA-treated Ti disks were inhibited. Moreover, the expression levels of alkaline phosphatase (ALP), osteocalcin (OCN), calcium deposition, BMP2 and Runx2 increased significantly compared with that observed in control groups, suggesting that the function of Notch3 was inhibitory in the osteogenic differentiation of BMSCs on SLA-treated titanium. Conclusions Inhibition Notch3 can enhance osteogenic differentiation of BMSCs on SLA-treated Ti disks, which potentially provides a gene target for improving osseointegration.

Aims To evaluate the short term effect of zoledronic acid on bone remodeling in the streptozotocin induced diabetes rats. Materials and Methods Diabetes was induced by an injection of streptozotocin (60 mg/kg). The rats were treated with zoledronic acid (0.1 mg/kg) at the onset of diabetes (Z-I group) and 2 weeks later (Z-II group). Rats were sacrificed at the 1, 2, 3, 4 and 5 weeks after the onset of diabetes. Real–time PCR and western blot were performed to detect the expression of the following osteogenic gene mRNAs and their proteins: bone morphogenetic proteins 2 (BMP2), Runx2, Osterix and Noggin. The bone mineral density (BMD) and the mechanical resistance test was measured. Results BMP2, Runx2 and Osterix mRNA and protein expression in group D had regulated down, while Noggin expression increased. Z-I treatment could reverse the results. However group Z-II showed only a transient reversing effect. On the 5th week in group D, the BMD decreased, the bone trabecular distance increased, while the trabecular thickness and bone trabecular volume were reduced, the biomechanics index decreased significantly. Zoledronic acid treatment restored these alterations. Conclusions Zoledronic acid administered in the early stage of the diabetes could prevent the osteopenia. The underlying mechanisms might be that zoledronic acid treatment reversed the effect of diabetes on the expression of osteoblast-regulating transcription factors: BMP2, Runx2 and Osterix.

The transcription factor PLZF [promyelocytic leukemia zinc finger, encoded by zinc finger BTB domain containing 16 (Zbtb16)] is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T-helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylation-based ChIP-seq and microarray analysis of both natural killer T (NKT) cells and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. First, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T-helper–specific transcription factor genes that in turn control T-helper–specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the multilayered architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program.

Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell subsets: conventional DC1 (cDC1), cDC2 and plasmacytoid DCs (pDC), none of which have been identified in horses. Therefore, the objective of this study was to identify and characterize DC subsets in equine peripheral blood, emphasizing on pDC. Surface marker analysis allowed distinction of putative DC subsets, according to their differential expression of CADM-1 and MHC class II. Equine pDC were found to be Flt3(+) CD4(low) CD13(-) CD14(-) CD172a(-) CADM-1(-) MHCII(low). The weak expression of CD4 on equine pDC contrasts with findings in several other mammals. Furthermore, pDC purified by fluorescence-activated cell sorting were found to be the only cell subset able to produce large amounts of IFN-α upon TLR9-agonist stimulation. The pDC identity was confirmed by demonstrating high-levels of PLAC8, RUNX2 and TCF4 expression, showing pDC-restricted expression in other mammals.

Significance The Runt-related transcription factors (RUNX) are critical regulators of development. Mutation or dysregulation of RUNX genes have been associated with diverse cancer types. Phosphorylation of the strictly conserved threonine 173 (T173) residue within the Runt domain of RUNX3 disrupts DNA binding activity, which facilitates the recruitment of RUNX proteins to mitotic structures. Moreover, RUNX3 deficiency is associated with delayed mitotic entry. The tight conservation of T173 and its flanking residues from unicellular organism to human, and the fact that mitosis is indispensable to all metazoans, strongly indicate that T173 phosphorylation is fundamental to the role of RUNX in these divergent organisms. Cancer-associated mutation T173I further corroborates the critical role of RUNX phosphorylation in mitosis.

A TCRβ enhancer, known as the Eβ enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of Eβ during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the Eβ function. A single mutation at one of the two Runx binding motifs within the Eβ severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCRβ expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated Eβ activity, demonstrating that Runx complex binding is essential to initiate Eβ activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCRβ expression, whereas Eβ itself is continuously required for TCRβ expression. These findings imply that Runx complexes are essential for Eβ activation at the initiation phase, but are not necessary for maintaining Eβ activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for Eβ activity are differentially regulated, depending on the developmental stage and cellular activation status.

In B cells infected by the cancer-associated Epstein-Barr virus (EBV), RUNX3 and RUNX1 transcription is manipulated to control cell growth. The EBV-encoded EBNA2 transcription factor (TF) activates RUNX3 transcription leading to RUNX3-mediated repression of the RUNX1 promoter and the relief of RUNX1-directed growth repression. We show that EBNA2 activates RUNX3 through a specific element within a −97 kb super-enhancer in a manner dependent on the expression of the Notch DNA-binding partner RBP-J. We also reveal that the EBV TFs EBNA3B and EBNA3C contribute to RUNX3 activation in EBV-infected cells by targeting the same element. Uncovering a counter-regulatory feed-forward step, we demonstrate EBNA2 activation of a RUNX1 super-enhancer (−139 to −250 kb) that results in low-level RUNX1 expression in cells refractory to RUNX1-mediated growth inhibition. EBNA2 activation of the RUNX1 super-enhancer is also dependent on RBP-J. Consistent with the context-dependent roles of EBNA3B and EBNA3C as activators or repressors, we find that these proteins negatively regulate the RUNX1 super-enhancer, curbing EBNA2 activation. Taken together our results reveal cell-type-specific exploitation of RUNX gene super-enhancers by multiple EBV TFs via the Notch pathway to fine tune RUNX3 and RUNX1 expression and manipulate B-cell growth.

Interleukin (IL)-7 is a cytokine essential for T lymphocyte development and homeostasis. However, little is known about the roles of IL-7 receptor α-chain (IL-7Rα) in late stages of T-cell development. To address this question, we established IL-7Rα–floxed mice and crossed them with CD4-Cre transgenic mice. Resultant IL-7R conditional knockout (IL-7RcKO) mice exhibited marked reduction in CD8 single positive (SP) T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells in thymus. The proportion and proliferation of both mature CD4SP and CD8SP thymocytes were decreased without affecting Runx expression. In addition, expression of the glucocorticoid-induced TNF receptor was reduced in CD4SP and CD8SP thymocytes, and expression of CD5 was decreased in CD8SP thymocytes. IL-7RcKO mice also showed impaired Treg and NKT cell proliferation and inhibition of NKT cell maturation. Bcl-2 expression was reduced in CD4SP and CD8SP thymocytes but not in Tregs and NKT cells, and introduction of a Bcl-2 transgene rescued frequency and CD5 expression of CD8SP thymocytes. Furthermore, IL-7RcKO mice exhibited greatly increased numbers of B cells and, to a lesser extent, γδ T and dendritic cells in thymus. Overall, this study demonstrates that IL-7Rα differentially controls development and maturation of thymocyte subpopulations in late developmental stages and suggests that IL-7R expression on αβ T cells suppresses development of other cell lineages in thymus.

This study reports the in vitro and in vivo biological activities of recombinant human bone morphogenetic protein 2 (rhBMP-2) released from the core-shell structure of a nanofibrous barrier membrane as a sustained delivery model for bone regeneration. RhBMP-2 incorporating poly(ethylene glycol) was used as the core, and poly(caprolactone) was used as the shell surrounding the core. To determine its release profiles, the release solution was collected and the amount of rhBMP-2 was measured by ELlSA at different time points. In vitro rhBMP-2, released from the delivery system over at least 24 days, reached a stable rate of 500 pg per day and guided bone marrow mesenchymal stem cells (BMMSCs) to express osteogenic genes. The distribution and proliferation of BMMSCs in the nanofibrous barrier membrane was measured by laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM). The biological activity of rhBMP-2 was tested in BMMSC/membrane culture in vitro and in a rabbit calvarium defect model in vivo. Osteogenic genes osteonectin (ON) and core binding factor-α1 (Cbf-α1) expression of the BMMSCs cultured on the BMP-2-PEG/PCL membrane were significantly higher than those of cells on the PEG/PCL membrane at the late time points using real-time PCR (p < 0.05). The membranes containing the rhBMP-2 group exhibited the fastest and most bone formation compared to others in rabbit cranial defect models (p < 0.05). This study revealed that rhBMP-2 could be incorporated into a core-shell electrospun membrane, and preserve sustained release capability and biological activity.

Human immunodeficiency virus type 1 (HIV-1) requires the cellular transcription factor core binding factor subunit β (CBFβ) to stabilize its viral infectivity factor (Vif) protein and neutralize the APOBEC3 restriction factors. CBFβ normally heterodimerizes with the RUNX family of transcription factors, enhancing their stability and DNA-binding affinity. To test the hypothesis that Vif may act as a RUNX mimic to bind CBFβ, we generated a series of CBFβ mutants at the RUNX/CBFβ interface and tested their ability to stabilize Vif and impact transcription at a RUNX-dependent promoter. While several CBFβ amino acid substitutions disrupted promoter activity, none of these impacted the ability of CBFβ to stabilize Vif or enhance degradation of APOBEC3G. A mutagenesis screen of CBFβ surface residues identified a single amino acid change, F68D, that disrupted Vif binding and its ability to degrade APOBEC3G. This mutant still bound RUNX and stimulated RUNX-dependent transcription. These separation-of-function mutants demonstrate that HIV-1 Vif and the RUNX transcription factors interact with cellular CBFβ on genetically distinct surfaces.

The three RUNX family members are lineage specific master regulators, which also have important, context-dependent roles in carcinogenesis as either tumor suppressors or oncogenes. Here we review evidence for such roles in breast cancer (BCa). RUNX1, the predominant RUNX family member in breast epithelial cells, has a tumor suppressor role reflected by many somatic mutations found in primary tumor biopsies. The classical tumor suppressor gene RUNX3 does not consist of such a mutation hot spot, but it too seems to inhibit BCa; it is often inactivated in human BCa tumors and its haploinsufficiency in mice leads to spontaneous BCa development. The tumor suppressor activities of RUNX1 and RUNX3 are mediated in part by antagonism of estrogen signaling, a feature recently attributed to RUNX2 as well. Paradoxically, however RUNX2, a master osteoblast regulator, has been implicated in various aspects of metastasis in general and bone metastasis in particular. Reciprocating the anti-estrogenic tumor suppressor activity of RUNX proteins, inhibition of RUNX2 by estrogens may help explain their context-dependent anti-metastatic roles. Such roles are reserved to non-osseous metastasis, because ERα is associated with increased, not decreased skeletal dissemination of BCa cells. Finally, based on diverse expression patterns in BCa subtypes, the successful use of future RUNX-based therapies will most likely require careful patient selection.

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.

Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 up-regulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5′ of MYC to MYC . These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.

Summary T 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., Graphical Abstract Highlights ► Association of Cd4 and Cd8 genes enables coordinate gene regulation ► Cd4 and Cd8 associate with one another in CD8-expressing cells ► RUNX transcription factor mediates association of Cd4 and Cd8 genes ► Targeted deletions within Cd4 modulate expression and nuclear location of Cd8