scholarly journals Multimerization via Its Myosin Domain Facilitates Nuclear Localization and Inhibition of Core Binding Factor (CBF) Activities by the CBFβ-Smooth Muscle Myosin Heavy Chain Myeloid Leukemia Oncoprotein

2002 ◽  
Vol 22 (23) ◽  
pp. 8278-8291 ◽  
Author(s):  
Tanawan Kummalue ◽  
Jianrong Lou ◽  
Alan D. Friedman
Keyword(s):  
Amino Acids ◽  
Smooth Muscle ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Heavy Chain ◽  
Myeloid Leukemia ◽  
Dna Binding Domain ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Muscle Myosin

ABSTRACT In CBFβ-SMMHC, core binding factor beta (CBFβ) is fused to the α-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBFβ-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBFβ-SMMHC(ΔACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the ΔACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G1 to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBFβ domain to 149 or 83 C-terminal CBFβ-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBFβ-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBFβ-SMMHC(83C) did not. The majority of CBFβ-SMMHC and CBFβ-SMMHC(149C) was detected in the nucleus, whereas the ΔACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBFβ-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBFβ-SMMHC(ΔACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBFβ-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G1 to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBFβ-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBFβ-SMMHC expression.

scholarly journals The Leukemic Protein Core Binding Factor β (CBFβ)–Smooth-Muscle Myosin Heavy Chain Sequesters CBFα2 into Cytoskeletal Filaments and Aggregates

1998 ◽  
Vol 18 (12) ◽  
pp. 7432-7443 ◽  
Author(s):  
Neeraj Adya ◽  
Terryl Stacy ◽  
Nancy A. Speck ◽  
Pu Paul Liu
Keyword(s):  
Smooth Muscle ◽  
Dna Binding ◽  
Mouse Model ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Chimeric Protein ◽  
Smooth Muscle Myosin ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Muscle Myosin

ABSTRACT The fusion gene CBFB-MYH11 is generated by the chromosome 16 inversion associated with acute myeloid leukemias. This gene encodes a chimeric protein involving the core binding factor β (CBFβ) and the smooth-muscle myosin heavy chain (SMMHC). Mouse model studies suggest that this chimeric protein CBFβ-SMMHC dominantly suppresses the function of CBF, a heterodimeric transcription factor composed of DNA binding subunits (CBFα1 to 3) and a non-DNA binding subunit (CBFβ). This dominant suppression results in the blockage of hematopoiesis in mice and presumably contributes to leukemogenesis. We used transient-transfection assays, in combination with immunofluorescence and green fluorescent protein-tagged proteins, to monitor subcellular localization of CBFβ-SMMHC, CBFβ, and CBFα2 (also known as AML1 or PEBP2αB). When expressed individually, CBFα2 was located in the nuclei of transfected cells, whereas CBFβ was distributed throughout the cell. On the other hand, CBFβ-SMMHC formed filament-like structures that colocalized with actin filaments. Upon cotransfection, CBFα2 was able to drive localization of CBFβ into the nucleus in a dose-dependent manner. In contrast, CBFα2 colocalized with CBFβ-SMMHC along the filaments instead of localizing to the nucleus. Deletion of the CBFα-interacting domain within CBFβ-SMMHC abolished this CBFα2 sequestration, whereas truncation of the C-terminal-end SMMHC domain led to nuclear localization of CBFβ-SMMHC when coexpressed with CBFα2. CBFα2 sequestration by CBFβ-SMMHC was further confirmed in vivo in a knock-in mouse model. These observations suggest that CBFβ-SMMHC plays a dominant negative role by sequestering CBFα2 into cytoskeletal filaments and aggregates, thereby disrupting CBFα2-mediated regulation of gene expression.

Core-binding factor β (CBFβ), but not CBFβ–smooth muscle myosin heavy chain, rescues definitive hematopoiesis in CBFβ-deficient embryonic stem cells

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2248-2256 ◽  
Author(s):  
Janelle D. Miller ◽  
Terryl Stacy ◽  
P. Paul Liu ◽  
Nancy A. Speck
Keyword(s):  
Smooth Muscle ◽  
Fusion Protein ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Es Cells ◽  
Embryonic Stem ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Muscle Myosin ◽  
Definitive Hematopoiesis

Abstract Core-binding factor β (CBFβ) is the non–DNA-binding subunit of the heterodimeric CBFs. Genes encoding CBFβ (CBFB),and one of the DNA-binding CBFα subunits, Runx1 (also known as CBFα2, AML1, and PEBP2αB), are required for normal hematopoiesis and are also frequent targets of chromosomal translocations in acute leukemias in humans. Homozygous disruption of either the Runx1or Cbfb gene in mice results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system, and severely impairs fetal liver hematopoiesis. Results of this study show that Cbfb-deficient mouse embryonic stem (ES) cells can differentiate into primitive erythroid colonies in vitro, but are impaired in their ability to produce definitive erythroid and myeloid colonies, mimicking the in vivo defect. Definitive hematopoiesis is restored by ectopic expression of full-length Cbfbtransgenes, as well as by a transgene encoding only the heterodimerization domain of CBFβ. In contrast, the CBFβ–smooth muscle myosin heavy chain (SMMHC) fusion protein generated by the inv(16) associated with acute myeloid leukemias (M4Eo) cannot rescue definitive hematopoiesis by Cbfb-deficient ES cells. Sequences responsible for the inability of CBFβ-SMMHC to rescue definitive hematopoiesis reside in the SMMHC portion of the fusion protein. Results also show that the CBFβ-SMMHC fusion protein transdominantly inhibits definitive hematopoiesis, but not to the same extent as homozygous loss of Runx1 orCbfb. CBFβ-SMMHC preferentially inhibits the differentiation of myeloid lineage cells, while increasing the number of blastlike cells in culture.

Multimerization and Corepression Mediated by the CBFβ-SMMHC Assembly Competence Domain Are Partially Separable and Corepression Is Required to Inhibit Core Binding Factor Activities.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1972-1972
Author(s):  
Linsheng Zhang ◽  
Scott W. Hiebert ◽  
Alan D. Friedman
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Dna Binding ◽  
Fusion Protein ◽  
Nuclear Localization ◽  
Outer Surface ◽  
Gene Activation ◽  
Salt Bridges ◽  
Core Binding Factor ◽  
Binding Factor

Core Binding Factor (CBF) is a family of heterodimeric transcription factors consisting of a CBFα subunit (AML1/RUNX1, AML2, or AML3) and CBFβ. The CBFα subunits bind DNA and regulate transcription. CBFβ increases the DNA affinity of the CBFα subunits. CBF induces lineage-specific genes and also accelerates the cell cycle via gene activation. CBFβ-SMMHC, encoded by the inv(16) or t(16;16) chromosomes in 8% of acute myeloid leukemia (AML) cases, is a fusion protein containing amino acids 1–165 of the 182 residue CBFβ and the α-helical rod domain of smooth muscle myosin heavy chain (SMMHC). The SMMHC domain mediates multimerization dependent upon a 28 residue, C-terminal Assembly Competence Domain (ACD). CBFβ-SMMHC potentially inhibits CBF by sequestering CBFα subunits in multimeric complexes in the nucleus. The CBFβ domain of CBFβ-SMMHC retains the ability to interact with AML1, and the resulting heterodimer can bind DNA. In addition, the SMMHC domain interacts directly with corepressors, including mSin3A, allowing AML1:CBFβ-SMMHC to directly repress CBF-regulated genes. Repression is obviated, however, upon deletion of the ACD. We have now carried out a mutagenic analysis of the ACD, in an effort to separate its contributions to multimerization and repression. We designated the 4 α-helices of the ACD as A, B, C, and D, and the subseqent helix as E. Each helix consists of 7 residues, abcdefg, with a and d hydrophobic and mediating dimerization, e and g often forming salt-bridges to stabilize this dimer, and b, c, and f available to mediate multimerization and corepressor binding. We mutated the bcf residues as a cluster within helix A, B, C, D, or E and also generated additional combinations: BC, CD, DE, BCD, ABCD, and ABCDE, the latter carrying alterations in 15 residues on the outer surface of the dimeric coiled-coil. Mutants DE, BCD, ABCD, and ABCDE were defective for multimerization, inhibition of CBF DNA-binding, nuclear localization, and inhibition of Ba/F3 proliferation. Even when directed to the nucleus using a nuclear localization signal, DE and ABCD did not inhibit CBF DNA-binding or CBF-mediated cell cycle progression. In addition, mutants B, C, or D did not inhibit Ba/F3 proliferation, and mutants A or E only mildly reduced proliferation. As each of these single helix mutants assembled into multimers, multimerization and sequestration of CBFα subunits is not sufficient for CBF inhibition. Mutation of helix C or E also prevented transrepression by a Gal4 DNA-binding domain-SMMHC fusion protein, in NIH 3T3 cells (other mutants remain to be evaluated in this assay). Of note, mutants C and E effectively coimmunoprecipitated with mSin3A from Ba/F3 extracts, suggesting that additional corepressors participate in suppression of CBF activities in these cells. In summary, we have identified CBFβ-SMMHC variants which multimerize and are defective for repression of CBF-regulated genes, but we have yet to identify mutants which cannot multimerize but still inhibit CBF. This may reflect overlap of the corepressor-binding site with residues required for multimerization. Nevertheless, we have identified amino acids on the outer surface of the ACD critical for inhibition of CBF. Drugs targeting the BCD or DE cluster of bcf residues may simultaneously prevent CBFβ-SMMHC multimerization and corepressor interaction and assist in the therapy of AMLs expressing this oncoprotein.

scholarly journals The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

2003 ◽  
Vol 23 (2) ◽  
pp. 607-619 ◽  
Author(s):  
Kristie L. Durst ◽  
Bart Lutterbach ◽  
Tanawan Kummalue ◽  
Alan D. Friedman ◽  
Scott W. Hiebert
Keyword(s):  
Amino Acids ◽  
Smooth Muscle ◽  
Fusion Protein ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transcriptional Repression ◽  
Coiled Coil ◽  
Smooth Muscle Myosin ◽  
Muscle Myosin ◽  
Repression Domain

ABSTRACT Inversion(16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor β to the coiled-coil region of a smooth muscle myosin heavy chain (CBFβ/SMMHC). CBFβ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBFβ/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBFβ/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBFβ/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor.

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 ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 464-464
Author(s):  
Christina J. Matheny ◽  
Takeshi Corpora ◽  
Maren E. Speck ◽  
Ting-Lei Gu ◽  
John H. Bushweller ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Domain Structure ◽  
Cleidocranial Dysplasia ◽  
Amino Acid Substitutions ◽  
Runt Domain ◽  
Core Binding Factor ◽  
Binding Factor

Abstract 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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