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Nancy A Speck - One of the best experts on this subject based on the ideXlab platform.
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a mutation in the s switch region of the Runt Domain alters the dynamics of an allosteric network responsible for cbfβ regulation
Journal of Molecular Biology, 2006Co-Authors: Zhe Li, John H Bushweller, S M Lukasik, Jolanta Grembecka, Izabela Bielnicka, Nancy A SpeckAbstract:Abstract The Runt Domain is the DNA binding Domain of the core binding factor (CBF) Runx subunits. The CBFs are transcription factors that play critical roles in hematopoiesis, bone, and neuron development in mammals. A common non-DNA binding CBFβ subunit heterodimerizes with the Runt Domain of the Runx proteins and allosterically regulates its affinity for DNA. Previous NMR dynamics studies suggested a model whereby CBFβ allosterically regulates DNA binding by quenching conformational exchange in the Runt Domain, particularly in the S-switch region and the βE′-F loop. We sought to test this model, and to this end introduced all possible single amino acid substitutions into the S-switch region and the βE′-F loop, and screened for mutations that enhanced DNA-binding. We demonstrate that one Runt Domain mutant, R164N, binds both DNA and CBFβ with higher affinity, but it is less sensitive to allosteric regulation by CBFβ. Analysis of NMR relaxation data shows that the chemical exchange exhibited by the wild-type Runt Domain is largely quenched by the R164N substitution. These data support a model in which the dynamic behavior of a network of residues connecting the CBFβ and DNA binding sites on the Runt Domain plays a critical role in the mechanism of allosteric regulation. This study provides an important functional link between dynamic behavior and protein allosteric function, consistent with results on other allosterically regulated proteins.
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the cbfb smmhc oncoprotein inhibits binding of the runx1 Runt Domain to dna
Blood, 2005Co-Authors: John H Bushweller, Stephen M Lukasik, Nancy A SpeckAbstract:Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2, and RUNX3 encode for CBFα subunits, and CBFB encodes the CBFβ subunit. Homozygous disruption of either the Runx1 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil Domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11 gene. A knock-in of the CBFB-MYH11 allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1 and Cbfb knockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt Domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt Domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt Domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt Domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.
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Development of Small Molecule Inhibitor of the AML1-ETO Oncoprotein.
Blood, 2005Co-Authors: Jolanta Grembecka, Nancy A Speck, Yunpeng Zhou, Miki Newman, Kristin Graf, Mathew Cheney, Milton L. Brown, John H BushwellerAbstract:Core binding factor (CBF) is a heterodimeric transcription factor composed of RUNX1 (CBFa) and CBFb subunits which are essential for normal blood cell development. RUNX1 binds DNA and the affinity of this interaction is regulated by the binding of CBFb to the RUNX1 subunit. The RUNX1(AML1) gene is disrupted by a variety of chromosomal translocations, including t(8;21)(q22;q22), which produces the chimeric protein AML1-ETO associated with AML (M2 subtype). The AML1-ETO fusion protein is a dominant inhibitor of wildtype RUNX1-CBFb activity in vivo and functions as a transcriptional repressor of RUNX1-CBFb target genes, causing a blockage in normal hematopoietic development and predisposing for the development of leukemia. We have validated the importance of the RUNX1 Runt Domain - CBFb interface as a potential therapeutic target by assessing the effect of mutations introduced into the Runt Domain of AML1-ETO on AML1-ETO activity. Therefore, treatments targeting the AML1-ETO protein and blocking its interactions with CBFb are likely to be therapeutically beneficial. We initiated efforts to develop small molecule inhibitors of the RUNX1-CBFb interaction as possible therapeutics for the treatment of the associated leukemia. Based on the X-ray structures of the RUNX1 Runt Domain in complex with CBFb and on mutagenesis data, the interaction areas for the virtual screening searches were defined. The computer program LUDI was utilized for virtual screening using CAP (Chemicals Available for Purchase) database of 70,000 commercially available compounds with drug-like properties. The resultant hits were subjected to further scoring and visual inspection of the potential interactions with the RUNX1 Runt Domain to select compounds for experimental testing by FRET (Fluorescence Resonance Energy Transfer). Experimental screening resulted in identification of six initial lead compounds that inhibit the interaction of RUNX1 with CBFb. These compounds were further optimized using standard medicinal chemistry approaches to increase their affinity and determine the structure-activity relationship (SAR). This resulted in a number of compounds with low micromolar affinity which effectively block the heterodimerization of CBF. These compounds represent the first small molecule inhibitors targeting RUNX1 and inhibiting its interaction with CBFb. They represent a good starting point for the development of a therapeutically useful inhibitor. Several approaches are being explored to modify these compounds to achieve selectivity towards AML1-ETO versus wild type RUNX1.
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biochemical and in vivo characterization of amino acid substitutions in the runx1 aml1 Runt Domain found in fpd aml aml m0 and cleidocranial dysplasia ccd patients
Blood, 2004Co-Authors: Christina J Matheny, Takeshi Corpora, John H Bushweller, Maren E Speck, Tinglei Gu, Nancy A SpeckAbstract:Runx1 and CBF β are the DNA-binding and non DNA-binding subunits of a core-binding factor that is required for hematopoiesis, and that is frequently mutated in leukemia. Runx2 is the DNA-binding subunit of a core-binding factor required for bone formation. Mono-allelic deletion, nonsense, frameshift, and missense mutations have been found in RUNX1 in familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML) and in myelodysplastic syndrome (MDS), and biallelic mutations in RUNX1 are found in 20% of AML M0 patients. Similar types of mono-allelic mutations have been found in RUNX2 in patients with cleidocranial dysplasia (CCD), an inherited skeletal syndrome. FPD/AML and CCD pedigrees have revealed varying degrees of disease severity depending on the nature of the specific mutation. Additionally, it has been observed that mutations involving amino acids in the DNA binding Runt Domain that directly contact DNA are associated primarily with Runx1 and hematopoietic disorders, while mutations predicted to disrupt CBF β binding or the Runt Domain structure are found only in Runx2 in CCD patients. We introduced 21 amino acid substitutions into the Runt Domain of Runx1 identified in FPD/AML, AML M0, and CCD patients, and quantified their effects on DNA binding, heterodimerization with CBFβ, and the Runt Domain structure using yeast one- and two-hybrid, quantitative electrophoretic mobility shift, heteronuclear single quantum correlation spectroscopy, and urea denaturation experiments. To address the impact on in vivo function, several of these point mutations were engineered into the endogenous Runx1 allele in mice. These five mutations include: R177X, R174Q, T149A, T161A, and L148F. R177X is found in FPD/AML patients and truncates Runx1 two amino acids before the C-terminal boundary of the Runt Domain. R174Q (found in FPD/AML and CCD) disrupts DNA binding 1000-fold, but does not disrupt CBFb binding or perturb the Runt Domain fold. T149A (found only in CCD) disrupts CBFβ binding 13-fold while T161A (not found in patients) disrupts CBFβ binding 40-fold. Both T149A and T161A slightly perturb the Runt Domain fold, but do not alter DNA binding affinity. L148F (found in CCD) also disrupts the Runt Domain fold, and decreases DNA binding. All animals heterozygous for these alleles are viable. Mice homozygous for R177X and R174Q die during gestation. Mice homozygous for the T149A and T161A mutations, on the other hand, are born at normal Mendelian frequencies, but 62% and 100%, respectively, die by or at three weeks of age from an undetermined cause. The effects of these mutations on hematopoietic progenitor and platelet numbers, both of which are affected in FPD/AML patients, will be presented. We conclude that mutations that affect CBFβ binding result in hypomorphic Runx1 alleles, while mutations involving DNA contacts result in more severe inactivation of Runx1 function. Thus FPD/AML, AML M0, and MDS require mutations that severely inactivate Runx1 function, while CCD can result from more subtle alterations in Runx2.
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Biochemical and In Vivo Characterization of Amino Acid Substitutions in the Runx1 (AML1) Runt Domain Found in FPD/AML, AML M0, and Cleidocranial Dysplasia (CCD) Patients.
Blood, 2004Co-Authors: Christina J Matheny, Takeshi Corpora, John H Bushweller, Maren E Speck, Tinglei Gu, Nancy A SpeckAbstract:Runx1 and CBF β are the DNA-binding and non DNA-binding subunits of a core-binding factor that is required for hematopoiesis, and that is frequently mutated in leukemia. Runx2 is the DNA-binding subunit of a core-binding factor required for bone formation. Mono-allelic deletion, nonsense, frameshift, and missense mutations have been found in RUNX1 in familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML) and in myelodysplastic syndrome (MDS), and biallelic mutations in RUNX1 are found in 20% of AML M0 patients. Similar types of mono-allelic mutations have been found in RUNX2 in patients with cleidocranial dysplasia (CCD), an inherited skeletal syndrome. FPD/AML and CCD pedigrees have revealed varying degrees of disease severity depending on the nature of the specific mutation. Additionally, it has been observed that mutations involving amino acids in the DNA binding Runt Domain that directly contact DNA are associated primarily with Runx1 and hematopoietic disorders, while mutations predicted to disrupt CBF β binding or the Runt Domain structure are found only in Runx2 in CCD patients. We introduced 21 amino acid substitutions into the Runt Domain of Runx1 identified in FPD/AML, AML M0, and CCD patients, and quantified their effects on DNA binding, heterodimerization with CBFβ, and the Runt Domain structure using yeast one- and two-hybrid, quantitative electrophoretic mobility shift, heteronuclear single quantum correlation spectroscopy, and urea denaturation experiments. To address the impact on in vivo function, several of these point mutations were engineered into the endogenous Runx1 allele in mice. These five mutations include: R177X, R174Q, T149A, T161A, and L148F. R177X is found in FPD/AML patients and truncates Runx1 two amino acids before the C-terminal boundary of the Runt Domain. R174Q (found in FPD/AML and CCD) disrupts DNA binding 1000-fold, but does not disrupt CBFb binding or perturb the Runt Domain fold. T149A (found only in CCD) disrupts CBFβ binding 13-fold while T161A (not found in patients) disrupts CBFβ binding 40-fold. Both T149A and T161A slightly perturb the Runt Domain fold, but do not alter DNA binding affinity. L148F (found in CCD) also disrupts the Runt Domain fold, and decreases DNA binding. All animals heterozygous for these alleles are viable. Mice homozygous for R177X and R174Q die during gestation. Mice homozygous for the T149A and T161A mutations, on the other hand, are born at normal Mendelian frequencies, but 62% and 100%, respectively, die by or at three weeks of age from an undetermined cause. The effects of these mutations on hematopoietic progenitor and platelet numbers, both of which are affected in FPD/AML patients, will be presented. We conclude that mutations that affect CBFβ binding result in hypomorphic Runx1 alleles, while mutations involving DNA contacts result in more severe inactivation of Runx1 function. Thus FPD/AML, AML M0, and MDS require mutations that severely inactivate Runx1 function, while CCD can result from more subtle alterations in Runx2.
John H Bushweller - One of the best experts on this subject based on the ideXlab platform.
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CBFβ is critical for AML1-ETO and TEL-AML1 activity
Blood, 2009Co-Authors: Liya Roudaia, Matthew D Cheney, Ekaterina Manuylova, Wei Chen, Michelle Morrow, Sangho Park, Prabhjot Kaur, Owen Williams, John H BushwellerAbstract:AML1-ETO and TEL-AML1 are chimeric proteins resulting from the t(8;21)(q22;q22) in acute myeloid leukemia, and the t(12;21)(p13;q22) in pre-B-cell leukemia, respectively. The Runt Domain of AML1 in both proteins mediates DNA binding and heterodimerization with the core binding factor β (CBFβ) subunit. To determine whether CBFβ is required for AML1-ETO and TEL-AML1 activity, we introduced amino acid substitutions into the Runt Domain that disrupt heterodimerization with CBFβ but not DNA binding. We show that CBFβ contributes to AML1-ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and is indispensable for its cooperativity with the activated receptor tyrosine kinase TEL-PDGFβR in generating acute myeloid leukemia in mice. Similarly, CBFβ is essential for TEL-AML1's ability to promote self-renewal of B cell precursors in vitro. These studies validate the Runt Domain/CBFβ interaction as a therapeutic target in core binding factor leukemias.
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a mutation in the s switch region of the Runt Domain alters the dynamics of an allosteric network responsible for cbfβ regulation
Journal of Molecular Biology, 2006Co-Authors: Zhe Li, John H Bushweller, S M Lukasik, Jolanta Grembecka, Izabela Bielnicka, Nancy A SpeckAbstract:Abstract The Runt Domain is the DNA binding Domain of the core binding factor (CBF) Runx subunits. The CBFs are transcription factors that play critical roles in hematopoiesis, bone, and neuron development in mammals. A common non-DNA binding CBFβ subunit heterodimerizes with the Runt Domain of the Runx proteins and allosterically regulates its affinity for DNA. Previous NMR dynamics studies suggested a model whereby CBFβ allosterically regulates DNA binding by quenching conformational exchange in the Runt Domain, particularly in the S-switch region and the βE′-F loop. We sought to test this model, and to this end introduced all possible single amino acid substitutions into the S-switch region and the βE′-F loop, and screened for mutations that enhanced DNA-binding. We demonstrate that one Runt Domain mutant, R164N, binds both DNA and CBFβ with higher affinity, but it is less sensitive to allosteric regulation by CBFβ. Analysis of NMR relaxation data shows that the chemical exchange exhibited by the wild-type Runt Domain is largely quenched by the R164N substitution. These data support a model in which the dynamic behavior of a network of residues connecting the CBFβ and DNA binding sites on the Runt Domain plays a critical role in the mechanism of allosteric regulation. This study provides an important functional link between dynamic behavior and protein allosteric function, consistent with results on other allosterically regulated proteins.
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the cbfb smmhc oncoprotein inhibits binding of the runx1 Runt Domain to dna
Blood, 2005Co-Authors: John H Bushweller, Stephen M Lukasik, Nancy A SpeckAbstract:Core-binding factors (CBFs) are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFα) subunit and a CBFβ subunit. Cbfβ allosterically increases the affinity of Runx1 for DNA ~2.5 fold. CBF subunits are encoded by four genes in mammals. RUNX1 (AML1), RUNX2, and RUNX3 encode for CBFα subunits, and CBFB encodes the CBFβ subunit. Homozygous disruption of either the Runx1 or the Cbfb genes in mice results in essentially identical phenotypes: midgestation embryonic lethality accompanied by extensive hemorrhaging and a profound block at the fetal liver stage of hematopoiesis. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFβ is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22). These translocations result in the production of novel fusion proteins containing most of the CBFβ protein fused to the C-terminal coiled-coil Domain from smooth muscle myosin heavy chain (SMMHC) encoded by the MYH11 gene. A knock-in of the CBFB-MYH11 allele in mice resulted in embryonic lethality with a profound block in hematopoietic development, the same phenotype observed for the Runx1 and Cbfb knockouts. We recently demonstrated that the CBFβ-SMMHC fusion protein binds to the DNA binding Runt Domain from Runx1 with both higher affinity and altered stoichiometry relative to native CBFβ. We also provided NMR-based evidence for multiple sites of contact between Runx1 and CBFβ-SMMHC, proving the role of the SMMHC sequence in creating this altered affinity. Here we demonstrate that CBFβ-SMMHC inhibits DNA binding of the Runx1 Runt Domain by ~6-fold for the CD4 dual-site silencer element. Cross-saturation NMR mapping on the Runt Domain in complex with CBFβ-SMMHC reveals that the SMMHC portion of the oncoprotein makes contacts with β-strands 1 and 2 in the Runt Domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFβ-SMMHC. These results also clearly suggest that targeting of the CBFβ-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.
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Development of Small Molecule Inhibitor of the AML1-ETO Oncoprotein.
Blood, 2005Co-Authors: Jolanta Grembecka, Nancy A Speck, Yunpeng Zhou, Miki Newman, Kristin Graf, Mathew Cheney, Milton L. Brown, John H BushwellerAbstract:Core binding factor (CBF) is a heterodimeric transcription factor composed of RUNX1 (CBFa) and CBFb subunits which are essential for normal blood cell development. RUNX1 binds DNA and the affinity of this interaction is regulated by the binding of CBFb to the RUNX1 subunit. The RUNX1(AML1) gene is disrupted by a variety of chromosomal translocations, including t(8;21)(q22;q22), which produces the chimeric protein AML1-ETO associated with AML (M2 subtype). The AML1-ETO fusion protein is a dominant inhibitor of wildtype RUNX1-CBFb activity in vivo and functions as a transcriptional repressor of RUNX1-CBFb target genes, causing a blockage in normal hematopoietic development and predisposing for the development of leukemia. We have validated the importance of the RUNX1 Runt Domain - CBFb interface as a potential therapeutic target by assessing the effect of mutations introduced into the Runt Domain of AML1-ETO on AML1-ETO activity. Therefore, treatments targeting the AML1-ETO protein and blocking its interactions with CBFb are likely to be therapeutically beneficial. We initiated efforts to develop small molecule inhibitors of the RUNX1-CBFb interaction as possible therapeutics for the treatment of the associated leukemia. Based on the X-ray structures of the RUNX1 Runt Domain in complex with CBFb and on mutagenesis data, the interaction areas for the virtual screening searches were defined. The computer program LUDI was utilized for virtual screening using CAP (Chemicals Available for Purchase) database of 70,000 commercially available compounds with drug-like properties. The resultant hits were subjected to further scoring and visual inspection of the potential interactions with the RUNX1 Runt Domain to select compounds for experimental testing by FRET (Fluorescence Resonance Energy Transfer). Experimental screening resulted in identification of six initial lead compounds that inhibit the interaction of RUNX1 with CBFb. These compounds were further optimized using standard medicinal chemistry approaches to increase their affinity and determine the structure-activity relationship (SAR). This resulted in a number of compounds with low micromolar affinity which effectively block the heterodimerization of CBF. These compounds represent the first small molecule inhibitors targeting RUNX1 and inhibiting its interaction with CBFb. They represent a good starting point for the development of a therapeutically useful inhibitor. Several approaches are being explored to modify these compounds to achieve selectivity towards AML1-ETO versus wild type RUNX1.
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biochemical and in vivo characterization of amino acid substitutions in the runx1 aml1 Runt Domain found in fpd aml aml m0 and cleidocranial dysplasia ccd patients
Blood, 2004Co-Authors: Christina J Matheny, Takeshi Corpora, John H Bushweller, Maren E Speck, Tinglei Gu, Nancy A SpeckAbstract:Runx1 and CBF β are the DNA-binding and non DNA-binding subunits of a core-binding factor that is required for hematopoiesis, and that is frequently mutated in leukemia. Runx2 is the DNA-binding subunit of a core-binding factor required for bone formation. Mono-allelic deletion, nonsense, frameshift, and missense mutations have been found in RUNX1 in familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML) and in myelodysplastic syndrome (MDS), and biallelic mutations in RUNX1 are found in 20% of AML M0 patients. Similar types of mono-allelic mutations have been found in RUNX2 in patients with cleidocranial dysplasia (CCD), an inherited skeletal syndrome. FPD/AML and CCD pedigrees have revealed varying degrees of disease severity depending on the nature of the specific mutation. Additionally, it has been observed that mutations involving amino acids in the DNA binding Runt Domain that directly contact DNA are associated primarily with Runx1 and hematopoietic disorders, while mutations predicted to disrupt CBF β binding or the Runt Domain structure are found only in Runx2 in CCD patients. We introduced 21 amino acid substitutions into the Runt Domain of Runx1 identified in FPD/AML, AML M0, and CCD patients, and quantified their effects on DNA binding, heterodimerization with CBFβ, and the Runt Domain structure using yeast one- and two-hybrid, quantitative electrophoretic mobility shift, heteronuclear single quantum correlation spectroscopy, and urea denaturation experiments. To address the impact on in vivo function, several of these point mutations were engineered into the endogenous Runx1 allele in mice. These five mutations include: R177X, R174Q, T149A, T161A, and L148F. R177X is found in FPD/AML patients and truncates Runx1 two amino acids before the C-terminal boundary of the Runt Domain. R174Q (found in FPD/AML and CCD) disrupts DNA binding 1000-fold, but does not disrupt CBFb binding or perturb the Runt Domain fold. T149A (found only in CCD) disrupts CBFβ binding 13-fold while T161A (not found in patients) disrupts CBFβ binding 40-fold. Both T149A and T161A slightly perturb the Runt Domain fold, but do not alter DNA binding affinity. L148F (found in CCD) also disrupts the Runt Domain fold, and decreases DNA binding. All animals heterozygous for these alleles are viable. Mice homozygous for R177X and R174Q die during gestation. Mice homozygous for the T149A and T161A mutations, on the other hand, are born at normal Mendelian frequencies, but 62% and 100%, respectively, die by or at three weeks of age from an undetermined cause. The effects of these mutations on hematopoietic progenitor and platelet numbers, both of which are affected in FPD/AML patients, will be presented. We conclude that mutations that affect CBFβ binding result in hypomorphic Runx1 alleles, while mutations involving DNA contacts result in more severe inactivation of Runx1 function. Thus FPD/AML, AML M0, and MDS require mutations that severely inactivate Runx1 function, while CCD can result from more subtle alterations in Runx2.
Katsuya Shigesada - One of the best experts on this subject based on the ideXlab platform.
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Concurrent transcriptional deregulation of AML1/RUNX1 and GATA factors by the AML1-TRPS1 chimeric gene in t(8;21)(q24;q22) acute myeloid leukemia.
Blood, 2007Co-Authors: Norio Asou, Katsuya Shigesada, Masatoshi Yanagida, Liqun Huang, Masayuki Yamamoto, Hiroaki Mitsuya, Motomi OsatoAbstract:The Runt Domain transcription factor AML1/RUNX1 is essential for the generation of hematopoietic stem cells and is the most frequent target of chromosomal translocations associated with leukemia. Here, we present a new AML1 translocation found in a patient with acute myeloid leukemia M4 with t(8;21)(q24;q22) at the time of relapse. This translocation generated an in-frame chimeric gene consisting of the N-terminal portion of AML1, retaining the Runt Domain, fused to the entire length of TRPS1 on the C-terminus. TRPS1 encodes a putative multitype zinc finger (ZF) protein containing 9 C2H2 type ZFs and 1 GATA type ZF. AML1-TRPS1 stimulated proliferation of hematopoietic colony-forming cells and repressed the transcriptional activity of AML1 and GATA-1 by 2 different mechanisms: competition at their cognate DNA-binding sites and physical sequestrations of AML1 and GATA-1, suggesting that simultaneous deregulation of AML1 and GATA factors constitutes a basis for leukemogenesis.
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Molecular basis for a dominant inactivation of RUNX1/AML1 by the leukemogenic inversion 16 chimera.
Blood, 2003Co-Authors: Gang Huang, Katsuya Shigesada, Motomi OsatoAbstract:The Runt Domain transcription factor, PEBP2/CBF, is a heterodimer composed of 2 subunits. The DNA-binding α subunit, or RUNX protein, interacts with a partner PEBP2β/CBFβ through the evolutionarily conserved Runt Domain. Each of the genes encoding RUNX1 and PEBP2β/CBFβ is frequently involved in acute myeloid leukemia. The chimeric protein, CBFβ(PEBP2β)/SMMHC, is generated as a result of inversion of chromosome 16 in such a way to retain the heterodimerization Domain of PEBP2β at the amino-terminal side fused to the C-terminal coiled-coil region of smooth muscle myosin heavy chain (SMMHC). Here we show that, in the chimeric protein, the second heterodimerization Domain is created by the fusion junction, enabling the chimeric protein to interact with RUNX1 at far greater affinity than PEBP2β and inactivate the RUNX1/AML1 function. To explain why and how heterozygous CBFB/MYH11 can inactivate homozygous RUNX1 near to completion, we propose a new model for this chimeric protein that consists of a Y-shaped dimer with unpaired N-terminal halves followed by a coiled-coil for the C-terminal region. (Blood. 2004;103:3200-3207)
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Functional analysis of RUNX2 mutations in cleidocranial dysplasia: novel insights into genotype-phenotype correlations.
Blood Cells Molecules and Diseases, 2003Co-Authors: Taketoshi Yoshida, Motomi Osato, Hirokazu Kanegane, Masatoshi Yanagida, Toshio Miyawaki, Katsuya ShigesadaAbstract:Abstract Cleidocranial dysplasia (CCD) is an inherited autosomal-dominant skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor, RUNX2. We have performed mutational analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2 : 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations alongside one polymorphism. The missense mutations were all clustered within the Runt Domain and their protein products showed neither DNA binding nor transactivation. On the other hand, some mutant RUNX2 had the Runt Domain intact and remained partially competent for transactivation. Coincidentally, one important phenotype of CCD, the short stature, was significantly milder in the patients with the intact Runt Domain than those without. Furthermore, a remarkable correlation was found between the short stature and the number of supernumerary teeth. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanelles, was invariably observed regardless of the degree of short stature or supernumerary teeth. Overall, these results suggest that CCD could result from a much smaller loss in the RUNX2 function than envisioned on the basis of the conventional haploinsufficiency model. This makes an interesting contrast to the case of familial and sporadic leukemias mediated by RUNX1 mutations, in which mutants acting in a dominant negative manner have been suggested to confer a higher propensity to develop leukemia.
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functional analysis of runx2 mutations in japanese patients with cleidocranial dysplasia demonstrates novel genotype phenotype correlations
American Journal of Human Genetics, 2002Co-Authors: Taketoshi Yoshida, Motomi Osato, Hirokazu Kanegane, Masatoshi Yanagida, Toshio Miyawaki, Katsuya ShigesadaAbstract:Cleidocranial dysplasia (CCD) is an autosomal dominant heritable skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor RUNX2. We have performed mutational analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2: 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations, in addition to 1 polymorphism. The missense mutations were all clustered within the Runt Domain, and their protein products were severely impaired in DNA binding and transactivation. In contrast, two RUNX2 mutants had the Runt Domain intact and remained partially competent for transactivation. One criterion of CCD, short stature, was much milder in the patients with the intact Runt Domain than in those without. Furthermore, a significant correlation was found between short stature and the number of supernumerary teeth. On the one hand, these genotype-phenotype correlations highlight a general, quantitative dependency, by skeleto-dental developments, on the gene dosage of RUNX2, which has hitherto been obscured by extreme clinical diversities of CCD; this gene-dosage effect is presumed to manifest on small reductions in the total RUNX2 activity, by approximately one-fourth of the normal level at minimum. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanelles, was invariably observed in all patients, including those with normal height. Thus, the cleidocranial bone formation, as mediated by intramembranous ossification, may require a higher level of RUNX2 than does skeletogenesis (mediated by endochondral ossification), as well as odontogenesis (involving still different complex processes). Overall, these results suggest that CCD could result from much smaller losses in the RUNX2 function than has been envisioned on the basis of the conventional haploinsufficiency model.
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mechanisms of transcriptional regulation by Runt Domain proteins
Seminars in Cell & Developmental Biology, 2000Co-Authors: John Wheeler, Katsuya Shigesada, Peter J GergenAbstract:Abstract Runt Domain proteins have vital roles in regulating transcription in developmental pathways extending from sex determination and segmentation in fruit fly embryos to the development of blood and bone in mammals. Many of the insights into the mechanisms by which these proteins act to regulate transcription originate either from studies on the Drosophila Runt gene, the founding member of this family, or from work on the mammalian PEBP2/CBF transcription factor. Genetic experiments in the Drosophila system reveal that Runt functions both to activate and to repress transcription of different downstream target genes and indicate that different mechanisms are used in the regulation of different specific downstream target genes. These studies have also identified other nuclear factors that work with Runt in some of these pathways. Studies in mammalian systems have provided additional evidence for the complexity of transcriptional regulation by Runt Domain proteins and have identified other transcription factors that cooperate with Runt Domain proteins to regulate the activity of different specific cis-regulatory enhancers. The emerging view from studies in both systems is that these proteins act as context-dependent regulators of transcription, activating or repressing gene expression dependent upon the constitution of a particular promoter/enhancer in a particular cell type. These results have yielded new insights into the molecular mechanisms that control animal development and provide a framework for investigating fundamental issues in eukaryotic transcriptional regulation.
Tomoko Kozu - One of the best experts on this subject based on the ideXlab platform.
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characterisation of an aptamer against the Runt Domain of aml1 runx1 by nmr and mutational analyses
FEBS Open Bio, 2018Co-Authors: Kenta Takada, Yusuke Nomura, Ryo Amano, Takashi Nagata, Yoichiro Tanaka, Masato Katahira, Tomoko Kozu, Yoshikazu Nakamura, Shigeru Sugiyama, Traiichi SakamotoAbstract:: Since the invention of systematic evolution of ligands by exponential enrichment, many short oligonucleotides (or aptamers) have been reported that can bind to a wide range of target molecules with high affinity and specificity. Previously, we reported an RNA aptamer that shows high affinity to the Runt Domain (RD) of the AML1 protein, a transcription factor with roles in haematopoiesis and immune function. From kinetic and thermodynamic studies, it was suggested that the aptamer recognises a large surface area of the RD, using numerous weak interactions. In this study, we identified the secondary structure by nuclear magnetic resonance spectroscopy and performed a mutational study to reveal the residue critical for binding to the RD. It was suggested that the large contact area was formed by a DNA-mimicking motif and a multibranched loop, which confers the high affinity and specificity of binding.
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conjugation of two rna aptamers improves binding affinity to aml1 Runt Domain
Journal of Biochemistry, 2017Co-Authors: Yusuke Nomura, Kaori Yamazaki, Ryo Amano, Kenta Takada, Takashi Nagata, Naohiro Kobayashi, Yoichiro Tanaka, Junichi Fukunaga, Masato Katahira, Tomoko KozuAbstract:: To develop a high-affinity aptamer against AML1 Runt Domain, two aptamers were conjugated based on their structural information. The newly designed aptamer Apt14 was generated by the conjugation of two RNA aptamers (Apt1 and Apt4) obtained by SELEX against AML1 Runt Domain, resulting in improvement in its binding performance. The residues of AML1 Runt Domain in contact with Apt14 were predicted in silico and confirmed by mutation and NMR analyses. It was suggested that the conjugated internal loop renders additional contacts and is responsible for the enhancement in the binding affinity. Conjugation of two aptamers that bind to different sites of the target protein is a facile and robust strategy to develop an aptamer with higher performance.
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solution structure of a dna mimicking motif of an rna aptamer against transcription factor aml1 Runt Domain
Journal of Biochemistry, 2013Co-Authors: Yusuke Nomura, Yoichiro Tanaka, Junichi Fukunaga, Kazuya Fujiwara, Manabu Chiba, Hiroaki Iibuchi, Taku Tanaka, Yoshikazu Nakamura, Gota Kawai, Tomoko KozuAbstract:: AML1/RUNX1 is an essential transcription factor involved in the differentiation of hematopoietic cells. AML1 binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt Domain. In a previous study, we obtained RNA aptamers against the AML1 Runt Domain by systematic evolution of ligands by exponential enrichment and revealed that RNA aptamers exhibit higher affinity for the Runt Domain than that for RDE and possess the 5'-GCGMGNN-3' and 5'-N'N'CCAC-3' conserved motif (M: A or C; N and N' form Watson-Crick base pairs) that is important for Runt Domain binding. In this study, to understand the structural basis of recognition of the Runt Domain by the aptamer motif, the solution structure of a 22-mer RNA was determined using nuclear magnetic resonance. The motif contains the AH(+)-C mismatch and base triple and adopts an unusual backbone structure. Structural analysis of the aptamer motif indicated that the aptamer binds to the Runt Domain by mimicking the RDE sequence and structure. Our data should enhance the understanding of the structural basis of DNA mimicry by RNA molecules.
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the Runt Domain of aml1 runx1 binds a sequence conserved rna motif that mimics a dna element
RNA, 2013Co-Authors: Junichi Fukunaga, Yusuke Nomura, Ryo Amano, Yoichiro Tanaka, Taku Tanaka, Yoshikazu Nakamura, Gota Kawai, Taiichi Sakamoto, Tomoko KozuAbstract:AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt Domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt Domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt Domain. Mutational analyses of the Runt Domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt Domain in vitro. These results demonstrated that the Runt Domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.
Gary S Stein - One of the best experts on this subject based on the ideXlab platform.
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the tissue specific nuclear matrix protein nmp 2 is a member of the aml cbf pebp2 Runt Domain transcription factor family interactions with the osteocalcin gene promoter
Biochemistry, 1995Co-Authors: Harold L Merriman, S W Hiebert, Andre J Van Wijnen, Joseph P Bidwell, Jane B Lian, Janet L Stein, Gary S SteinAbstract:: The nuclear matrix protein, NMP-2, was originally identified as an osteoblast-specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt -441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML-1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to the sequence TGCTGGT via its DNA-binding Runt Domain. Electrophoretic mobility shift assays reveal that a component of NMP-2 is a member of the AML/PEBP2/Runt Domain transcription factor family based on cross-competition with AML-1 consensus oligonucleotide. Limited immunoreactivity of NMP-2 with a polyclonal N-terminal AML-1 antibody and inability of the AML-1 partner protein CBF-beta to form complexes with NMP-2 indicate that NMP-2 is not identical to AML-1 but represents a variant AML/PEBP2/Runt Domain protein. Western and Northern blots reveal the presence of multiple AML-related proteins and AML-1 transcripts in several osseous cell lines. Furthermore, our results indicate that AML family members may selectively partition between nuclear matrix and nonmatrix compartments. Because proteins that contain a Runt Domain are implicated in tissue-specific transcriptional regulation, our results support the concept that the nuclear matrix mediates osteoblast-specific expression of the osteocalcin gene.
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The Tissue-Specific Nuclear Matrix Protein, NMP-2, Is a Member of the AML/ CBF/PEBP2/Runt Domain Transcription Factor Family: Interactions with the Osteocalcin Gene Promoter
Biochemistry, 1995Co-Authors: Harold L Merriman, S W Hiebert, Andre J Van Wijnen, Joseph P Bidwell, Jane B Lian, Janet L Stein, Gary S SteinAbstract:: The nuclear matrix protein, NMP-2, was originally identified as an osteoblast-specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt -441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML-1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to the sequence TGCTGGT via its DNA-binding Runt Domain. Electrophoretic mobility shift assays reveal that a component of NMP-2 is a member of the AML/PEBP2/Runt Domain transcription factor family based on cross-competition with AML-1 consensus oligonucleotide. Limited immunoreactivity of NMP-2 with a polyclonal N-terminal AML-1 antibody and inability of the AML-1 partner protein CBF-beta to form complexes with NMP-2 indicate that NMP-2 is not identical to AML-1 but represents a variant AML/PEBP2/Runt Domain protein. Western and Northern blots reveal the presence of multiple AML-related proteins and AML-1 transcripts in several osseous cell lines. Furthermore, our results indicate that AML family members may selectively partition between nuclear matrix and nonmatrix compartments. Because proteins that contain a Runt Domain are implicated in tissue-specific transcriptional regulation, our results support the concept that the nuclear matrix mediates osteoblast-specific expression of the osteocalcin gene.
