RUNX1::ETO and CBFß::MYH11 converge on aberrant activation of BCAT1 to confer a therapeutic vulnerability in core-binding factor-acute myeloid leukaemia.

Effectively targeting transcription factors in therapeutic interventions remains challenging, especially in core-binding factor-acute myeloid leukaemia (CBF-AML) characterized by RUNX1::ETO and CBFß::MYH11 fusions. However, recent studies have drawn attention towards aberrant amino acid metabolisms as actionable therapeutic targets. Here, by integrating the expression profile and genetic makeup in AML cohort, we found higher BCAT1 expression in CBF-AML patients compared with other subtypes. Metabolic profiling revealed that high BCAT1 expression led to reprogrammed branch amino acid metabolism in CBF-AML and was associated with sphingolipid pathway relating to the fitness of leukaemia cells, supported by transcriptomic profiling. Mechanistically, we demonstrated in cell lines and primary patient samples that BCAT1 was directly activated by RUNX1::ETO and CBFß::MYH11 fusion proteins similarly in a RUNX1-dependent manner through rewiring chromatin conformation at the BCAT1 gene locus. Furthermore, BCAT1 inhibition resulted in blunted cell cycle, enhanced apoptosis and myeloid differentiation of CBF-AML cells in vitro, and alleviated leukaemia burden and prolonged survival in vivo. Importantly, pharmacological inhibition of BCAT1 using the specific inhibitor Gabapentin demonstrated therapeutic effects, as evidenced by delayed leukaemia progression and improved survival in vivo. In conclusion, our study uncovers BCAT1 as a genetic vulnerability and a promising targeted therapeutic opportunity for CBF-AML.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.

A novel HIV-1 inhibitor that blocks viral replication and rescues APOBEC3s by interrupting vif/CBFß interaction.

HIV remains a health challenge worldwide, partly because of the continued development of resistance to drugs. Therefore, it is urgent to find new HIV inhibitors and targets. Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3) are important host restriction factors that inhibit HIV-1 replication by their cytidine deaminase activity. HIV-1 viral infectivity factor (Vif) promotes proteasomal degradation of APOBEC3 proteins by recruiting the E3 ubiquitin ligase complex, in which core-binding factor ß (CBFß) is a necessary molecular chaperone. Interrupting the interaction between Vif and CBFß can release APOBEC3 proteins to inhibit HIV-1 replication and may be useful for developing new drug targets for HIV-1. In this study, we identified a potent small molecule inhibitor CBFß/Vif-3 (CV-3) of HIV-1 replication by employing structure-based virtual screening using the crystal structure of Vif and CBFß (PDB: 4N9F) and validated CV-3's antiviral activity. We found that CV-3 specifically inhibited HIV-1 replication (IC50 = 8.16 µm; 50% cytotoxic concentration >100 µm) in nonpermissive lymphocytes. Furthermore, CV-3 treatment rescued APOBEC3 family members (human APOBEC3G (hA3G), hA3C, and hA3F) in the presence of Vif and enabled hA3G packaging into HIV-1 virions, which resulted in Gly-to-Ala hypermutations in viral genomes. Finally, we used FRET to demonstrate that CV-3 inhibited the interaction between Vif and CBFß by simultaneously forming hydrogen bonds with residues Gln-67, Ile-102, and Arg-131 of CBFß. These findings demonstrate that CV-3 can effectively inhibit HIV-1 by blocking the interaction between Vif and CBFß and that this interaction can serve as a new target for developing HIV-1 inhibitors.

Hypoxia-inducible factor 2α is a novel inhibitor of chondrocyte maturation.

Hypoxic environment is essential for chondrocyte maturation and longitudinal bone growth. Although hypoxia-inducible factor 1 alpha (Hif-1α) has been known as a key player for chondrocyte survival and function, the function of Hif-2α in cartilage is mechanistically and clinically relevant but remains unknown. Here we demonstrated that Hif-2α was a novel inhibitor of chondrocyte maturation through downregulation of Runx2 stability. Mechanistically, Hif-2α binding to Runx2 inhibited chondrocyte maturation by Runx2 degradation through disrupting Runx2/Cbfß complex formation. The Hif-2α-mediated-Runx2 degradation could be rescued by Cbfß transfection due to the increase of Runx2/Cbfß complex formation. Consistently, mesenchymal cells derived from Hif-2α heterozygous mice were more rapidly differentiated into hypertrophic chondrocytes than those of wild-type mice in a micromass culture system. Collectively, these findings demonstrate that Hif-2α is a novel inhibitor for chondrocyte maturation by disrupting Runx2/Cbfß complex formation and consequential regulatory activity.

Regulation of RUNX1 dosage is crucial for efficient blood formation from hemogenic endothelium.

During ontogeny, hematopoietic stem and progenitor cells arise from hemogenic endothelium through an endothelial-to-hematopoietic transition that is strictly dependent on the transcription factor RUNX1. Although it is well established that RUNX1 is essential for the onset of hematopoiesis, little is known about the role of RUNX1 dosage specifically in hemogenic endothelium and during the endothelial-to-hematopoietic transition. Here, we used the mouse embryonic stem cell differentiation system to determine if and how RUNX1 dosage affects hemogenic endothelium differentiation. The use of inducible Runx1 expression combined with alterations in the expression of the RUNX1 co-factor CBFß allowed us to evaluate a wide range of RUNX1 levels. We demonstrate that low RUNX1 levels are sufficient and necessary to initiate an effective endothelial-to-hematopoietic transition. Subsequently, RUNX1 is also required to complete the endothelial-to-hematopoietic transition and to generate functional hematopoietic precursors. In contrast, elevated levels of RUNX1 are able to drive an accelerated endothelial-to-hematopoietic transition, but the resulting cells are unable to generate mature hematopoietic cells. Together, our results suggest that RUNX1 dosage plays a pivotal role in hemogenic endothelium maturation and the establishment of the hematopoietic system.

Inhibition of Vif-Mediated Degradation of APOBEC3G through Competitive Binding of Core-Binding Factor Beta.

Vif counteracts the host restriction factor APOBEC3G (A3G) and other APOBEC3s by preventing the incorporation of A3G into progeny virions. We previously identified Vif mutants with a dominant-negative (D/N) phenotype that interfered with the function of wild-type Vif, inhibited the degradation of A3G, and reduced the infectivity of viral particles by increased packaging of A3G. However, the mechanism of interference remained unclear, in particular since all D/N Vif mutants were unable to bind Cul5 and some mutants additionally failed to bind A3G, ruling out competitive binding to A3G or the E3 ubiquitin ligase complex as the sole mechanism. The goal of the current study was to revisit the mechanism of D/N interference by Vif mutants and analyze the possible involvement of core binding factor beta (CBFß) in this process. We found a clear correlation of D/N properties of Vif mutants with their ability to engage CBFß. Only mutants that retained the ability to bind CBFß exhibited the D/N phenotype. Competition studies revealed that D/N Vif mutants directly interfered with the association of CBFß and wild-type Vif. Furthermore, overexpression of CBFß counteracted the interference of D/N Vif mutants with A3G degradation by wild-type Vif. Finally, overexpression of Runx1 mimicked the effect of D/N Vif mutants and inhibited the degradation of A3G by wild-type Vif. Taken together, we identified CBFß as the key player involved in D/N interference by Vif.IMPORTANCE Of all the accessory proteins encoded by HIV-1 and other primate lentiviruses, Vif has arguably the strongest potential as a target for antiviral therapy. This conclusion is based on the observation that replication of HIV-1 in vivo is critically dependent on Vif. Thus, inhibiting the function of Vif via small-molecule inhibitors or other approaches has significant therapeutic potential. We previously identified dominant-negative (D/N) Vif variants whose expression interferes with the function of virus-encoded wild-type Vif. We now show that D/N interference involves competitive binding of D/N Vif variants to the transcriptional cofactor core binding factor beta (CBFß), which is expressed in cells in limiting quantities. Overexpression of CBFß neutralized the D/N phenotype of Vif. In contrast, overexpression of Runx1, a cellular binding partner of CBFß, phenocopied the D/N Vif phenotype by sequestering endogenous CBFß. Thus, our results provide proof of principle that D/N Vif variants could have therapeutic potential.

Enhanced osteogenic differentiation of human mesenchymal stem cells by direct delivery of Cbfß protein.

Core binding factor ß (Cbfß) is a non-DNA binding cofactor of Runx2 that potentiates DNA binding. Previously, it has been reported that Cbfß plays an essential role in osteogenic differentiation and skeletal development by inhibition adipogenesis. Here, we delivered the recombinant Cbfß protein into human mesenchymal stem cells (MSCs) and triggered osteogenic lineage commitment. The efficient delivery of Cbfß was achieved by fusing 30Kc19 protein, which is a cell-penetrating protein derived from the silkworm. After the production of the recombinant Cbfß-30Kc19 protein in the Escherichia coli expression system, and confirmation of its intracellular delivery, MSCs were treated with the Cbfß-30Kc19 once or twice up to 300 µg/ml. By investigating the upregulation of osteoblast-specific genes and phenotypical changes, such as calcium mineralization, we demonstrated that Cbfß-30Kc19 efficiently induced osteogenic differentiation in MSCs. At the same time, Cbfß-30Kc19 suppressed adipocyte formation and downregulated the expression of adipocyte-specific genes. Our results demonstrate that the intracellularly delivered Cbfß-30Kc19 enhances osteogenesis in MSCs, whereas it suppresses adipogenesis by altering the transcriptional regulatory network involved in osteoblast-adipocyte lineage commitment. Cbfß-30Kc19 holds great potential for the treatment of bone-related diseases, such as osteoporosis, by allowing transcriptional regulation in MSCs, and overcoming the limitations of current therapies.

Cbfß governs osteoblast-adipocyte lineage commitment through enhancing ß-catenin signaling and suppressing adipogenesis gene expression.

The mechanism underlying how transcription factors regulate mesenchymal stem cell lineage commitment remains unclear. To determine the role of core-binding factor subunit beta (Cbfß) in osteoblast lineage commitment, we generated three mouse models by deleting Cbfß at different osteoblast lineage stages. We demonstrated that the Cbfßf/fPrx1-Cre, Cbfßf/fCol2α1-Cre, and Cbfßf/fOsx-Cre mice exhibited severe osteoporosis with substantial accumulation of marrow adipocytes resembling aged bone from enhanced adipogenesis, indicating that mesenchymal stem cells and osteoblasts can be programed and reprogramed, respectively, into adipocytes. Consistently, Cbfß-deficient calvarial cells and bone marrow mesenchymal stem cells displayed strong adipogenic potential, with 5- to ∼70-fold increased adipocyte gene expression, which can be rescued by Cbfß overexpression. Canonical Wnt signaling was impeded in the Cbfß-deficient cells, with ∼80% decrease of Wnt10b expression. Accordingly, ChIP and luciferase assays demonstrated that Cbfß/RUNX2 binds to Wnt10b promoter driving Wnt10b expression. Furthermore, Wnt3a suppressed adipogenesis but did not rescue osteoblastogenesis in Cbfß-deficient cells. Notably, mixing culture of Cbfß-deficient with normal cells demonstrates that Cbfß functions not only through WNT paracrine pathway but also through endogenous signaling. Further analysis shows that Cbfß/RUNX2 inhibits c/ebpα expression at transcriptional level. Our results show that, besides its osteogenic role, Cbfß governs osteoblast-adipocyte lineage commitment both cell nonautonomously through enhancing ß-catenin signaling and cell autonomously through suppressing adipogenesis gene expression to maintain osteoblast lineage commitment, indicating Cbfß may be a therapeutic target for osteoporosis.

Vif-CBFß interaction is essential for Vif-induced cell cycle arrest.

Interaction between HIV-1 Vif and host factor CBFß leads to the assembly of the Vif-Cul5-EloB/C ubiquitin ligase (E3 complex). By inducing the formation of E3 complex, Vif depletes host APOBEC3 restriction factors and promotes HIV-1 infection. In addition, Vif is known to arrest host cells at G2/M phase (G2 arrest), benefiting HIV-1 replication and contributing to the depletion of CD4+ T cells. However, whether CBFß is also involved in Vif-induced cell cycle arrest remains unclear. In the present study, we report that CBFß is an essential factor for Vif-induced G2 arrest. Reducing endogenous CBFß expression significantly compromised Vif's potency in cell cycle regulation. In addition, tests with CBFß and Vif mutants indicated that Vif-CBFß interaction is crucial for Vif to induce G2 arrest. Furthermore, suppressors against Vif-hijacked E3 complex or proteasome-mediated proteolysis also abolished Vif's ability to cause G2 arrest. In general, our data indicated that Vif induces G2 arrest through depletion of a yet-unknown cellular factor, where the involvement of CBFß is essential. On the other hand, our data also suggested that, antiviral drugs targeting the Vif-CBFß interaction have the potential to abolish Vif's ability to cause APOBEC3 degradation as well as G2 arrest in host cells, thus reducing both HIV-1 replication and Vif-induced CD4+ T-cell depletion.

Classifying AML patients with inv(16) into high-risk and low-risk relapsed patients based on peritransplantation minimal residual disease determined by CBFß/MYH11 gene expression.

Ninety acute myeloid leukemia (AML) patients with inv(16) were monitored CBFß/MYH11 transcript around allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 23 patients received HLA-matched sibling donor transplantation (MSDT) and 67 patients received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were analyzed in this study. Patients were divided into four groups based on CBFß/MYH11 expression prior to transplantation (pre-MRD): with negative (group 1)/positive (group 2) pre-MRD before MSDT; with negative (group 3)/positive (group 4) pre-MRD before haplo-HSCT. The results showed that patients in group 2 had the highest cumulative incidence of relapse (2-year CIR, 40.7%), the lowest leukemia-free survival (2-year LFS, 50.8%), and overall survival (2-year OS, 62.5%). The other three groups of patients had comparable outcomes. The patients were also classified into the other three groups according to CBFß/MYH11 value of + 1 month after transplantation: group 5: pre- and post-transplant MRD were both negative; group 6: the value of post-transplant MRD was lower than 0.2%; group 7: the value of post-transplant MRD was higher than 0.2%. Group 7 had the highest CIR and the lowest LFS. These results indicated that AML patients with inv(16) were able to be separated into high-risk and low-risk relapse groups based on peritransplant MRD determined by RQ-PCR-based CBFß/MYH11. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT.

Transcriptional activation of CBFß by CDK11p110 is necessary to promote osteosarcoma cell proliferation.

BACKGROUND: Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. METHODS: In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFß) were further accessed in osteosarcoma. RESULTS: We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFß expression in osteosarcoma cells. The CBFß promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFß is a direct transcriptional target of CDK11. High expression of CBFß is associated with poor outcome in osteosarcoma patients. Expression of CBFß contributes to the proliferation and metastatic behavior of osteosarcoma cells. CONCLUSIONS: These data establish CBFß as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFß pathway may be a promising therapeutic strategy for osteosarcoma treatment.

Genetic compensation of RUNX family transcription factors in leukemia.

Runt (Runt domain)-related transcription factor 1 (RUNX1) is a transcription factor belonging to the core-binding factor (CBF) family. It is considered to be a master regulator of hematopoiesis and has been regarded as a tumor suppressor because it is essential for definitive hematopoiesis in vertebrates. It is one of the most frequent target genes of chromosomal translocation in leukemia, and germ line mutation of RUNX1 causes familial platelet disorder with associated myeloid malignancies. Somatic cell mutations and chromosomal abnormalities, including those of RUNX1, are observed in myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, and chronic myelomonocytic leukemia at a high frequency. In addition, recent studies reported by us and other groups suggested that WT RUNX1 is needed for survival and proliferation of certain types of leukemia. In this review, we describe the significance and paradoxical requirement of RUNX1 tumor suppressor in hematological malignancies based on recent findings such as "Genetic compensation of RUNX family transcription factors in leukemia," "RUNX1 inhibition-induced inhibitory effects on leukemia cells through p53 activation" and our novel promising theory "Cluster regulation of RUNX (CROX)" through the RUNX gene switch method using pyrrole-imidazole polyamides as a new technique that could contribute to the next generation of leukemia treatment strategies.

RUNX transcription factors at the interface of stem cells and cancer.

The RUNX1 transcription factor is a critical regulator of normal haematopoiesis and its functional disruption by point mutations, deletions or translocations is a major causative factor leading to leukaemia. In the majority of cases, genetic changes in RUNX1 are linked to loss of function classifying it broadly as a tumour suppressor. Despite this, several recent studies have reported the need for a certain level of active RUNX1 for the maintenance and propagation of acute myeloid leukaemia and acute lymphoblastic leukaemia cells, suggesting an oncosupportive role of RUNX1. Furthermore, in solid cancers, RUNX1 is overexpressed compared with normal tissue, and RUNX factors have recently been discovered to promote growth of skin, oral, breast and ovarian tumour cells, amongst others. RUNX factors have key roles in stem cell fate regulation during homeostasis and regeneration of many tissues. Cancer cells appear to have corrupted these stem cell-associated functions of RUNX factors to promote oncogenesis. Here, we discuss current knowledge on the role of RUNX genes in stem cells and as oncosupportive factors in haematological malignancies and epithelial cancers.

Charged Amino Acid-rich Leucine Zipper-1 (Crlz-1) as a Target of Wnt Signaling Pathway Controls Pre-B Cell Proliferation by Affecting Runx/CBFß-targeted VpreB and λ5 Genes.

The proliferation of pre-B cells is known to further increase the clonal diversity of B cells at the stage of pre-B cells by allowing the same rearranged heavy chains to combine with differently rearranged light chains in a subsequent developmental stage. Crlz-1 (charged amino acid-rich leucine zipper-1) was found to control this proliferation of pre-B cells by working as a Wnt (wingless-related mouse mammary tumor virus integration site) target gene in these cells. Mechanistically, Crlz-1 protein functioned by mobilizing cytoplasmic CBFß (core binding factor ß) into the nucleus to allow Runx (runt-related transcription factor)/CBFß heterodimerization. Runx/CBFß then turned on its target genes such as EBF (early B cell factor), VpreB, and λ5 and thereby pre-B cell receptor signaling, leading to the expression of cyclins D2 and D3 Actually, the proliferative function of Crlz-1 was demonstrated by not only Crlz-1 or ß-catenin knockdown but also Crlz-1 overexpression. Furthermore, the mechanistic view that the proliferative function of Crlz-1 is caused by relaying Wnt/ß-catenin to pre-B cell receptor signaling pathways through the regulation of Runx/CBFß heterodimerization was also verified by employing niclosamide, XAV939, and LiCl as Wnt inhibitors and activator, respectively.

Identification of a tripartite interaction between the N-terminus of HIV-1 Vif and CBFß that is critical for Vif function.

BACKGROUND: HIV-1 Vif interacts with the cellular core-binding factor ß (CBFß) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation. Previous studies have identified some amino acids important for Vif-CBFß interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFß, Cul5, EloB, and EloC was resolved. However, a comprehensive analysis of Vif-CBFß interactions that are important for Vif function has not been performed. RESULTS: Here, we carried out double-alanine scanning mutagenesis of the first 60 amino acids of Vif and determined their effects on interaction with CBFß and their ability to induce A3G degradation as well as rescue HIV-1 replication in the presence of A3G. We found that multiple Vif residues are involved in the extensive N-terminal Vif-CBFß interaction and that the 5WQVMIVW11 region of Vif is the major determinant. A minimum of three alanine substitutions are required to completely abrogate the Vif-CBFß interaction and Vif's ability to rescue HIV-1 infectivity in the presence of A3G. Mutational analysis of CBFß revealed that F68 and I55 residues are important and participate in a tripartite hydrophobic interaction with W5 of Vif to maintain a stable and functional Vif-CBFß complex. We also determined that CBFß amino acids 73WQGEQR78, which are not resolved in the structure of the pentameric complex, are not involved in interaction with HIV-1 Vif. CONCLUSIONS: Our results provide detailed insight into the Vif-CBFß interactions that are critical for Vif function and may contribute to the rational design of HIV-1 inhibitors that block Vif-mediated degradation of A3 proteins.

Structure and Biophysics of CBFß/RUNX and Its Translocation Products.

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 CBFß 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.

Runx Genes in Breast Cancer and the Mammary Lineage.

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/CBFß core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFß 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 CBFß in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

Covalent Modifications of RUNX Proteins: Structure Affects Function.

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.

Multiple lysines combined in HIV-1 Vif determines the responsiveness to CBF-ß.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type-1 (HIV-1) is critical for HIV-1 infectivity. CBF-ß is required for HIV-1 Vif function, as it increases the steady-state level of the HIV-1 Vif protein to promote host restriction factor APOBEC3 degradation. However, the precise mechanism by which CBF-ß promotes HIV-1 Vif levels remains unclear. In the present study, we provided evidences that CBF-ß promoted steady-state levels of HIV-1 Vif by inhibiting the degradation of HIV-1 Vif through the proteasome pathway. Our results reveal a new mechanism by which a cellular protein supports viral infectivity by inhibiting viral protein degradation.

Molecular characterization of cbfß gene and identification of new transcription variants: implications for function.

The CBFß gene encodes a transcription factor that, in combination with CBFα (also called Runx, runt-related transcription factor) regulates expression of several target genes. CBFß interacts with all Runx family members, such as RUNX2, a regulator of bone-related gene transcription that contains a conserved DNA-binding domain. CBFß stimulates DNA binding of the Runt domain, and is essential for most of the known functions of RUNX2. A comparative analysis of the zebrafish cbfß gene and protein, and of its orthologous identified homologous proteins in different species indicates a highly conserved function. We cloned eleven zebrafish cbfß gene transcripts, one resulting in the known Cbfß protein (with 187 aa), and three additional variants resulting from skipping exon 5a (resulting in a protein with 174 aa) or exon 5b (resulting in a protein with 201 aa), both observed for the first time in zebrafish, and a completely novel isoform containing both exon 5a and 5b (resulting in a protein with 188 aa). Functional analysis of these isoforms provides insight into their role in regulating gene transcription. From the other variants two are premature termination Cbfß forms, while the others show in-frame exon-skipping causing changes in the Cbfß domain that may affect its function.