scholarly journals Auto-Inhibition of Ets-1 Is Counteracted by DNA Binding Cooperativity with Core-Binding Factor α2

2000 ◽  
Vol 20 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Tamara L. Goetz ◽  
Ting-Lei Gu ◽  
Nancy A. Speck ◽  
Barbara J. Graves
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Transcriptional Control ◽  
Potential Role ◽  
Control Mechanism ◽  
Synergistic Activity ◽  
Core Binding Factor ◽  
Partner Protein ◽  
Binding Factor

ABSTRACT Auto-inhibition is a common transcriptional control mechanism that is well characterized in the regulatory transcription factor Ets-1. Autoinhibition of Ets-1 DNA binding works through an inhibitory module that exists in two conformations. DNA binding requires a change in the inhibitory module from the packed to disrupted conformation. This structural switch provides a mechanism to tightly regulate Ets-1 DNA binding. We report that the Ets-1 partner protein core-binding factor α2 (CBFα2; also known as AML1 or PEBP2) stimulates Ets-1 DNA binding and counteracts auto-inhibition. Support for this conclusion came from three observations. First, the level of cooperative DNA binding (10-fold) was similar to the level of repression by auto-inhibition (10- to 20-fold). Next, a region necessary for cooperative DNA binding mapped to the inhibitory module. Third, an Ets-1 mutant with a constitutively disrupted inhibitory module did not bind DNA cooperatively with CBFα2. Furthermore, two additional lines of evidence indicated that CBFα2 affects the structural switch by direct interactions with Ets-1. First, the retention of cooperative DNA binding on nicked duplexes eliminated a potential role of through-DNA effects. Second, cooperative DNA binding was observed on composite sites with altered spacing or reversed orientation. We suggest that only protein interactions can accommodate this observed flexibility. These findings provide a mechanism by which CBF relieves the auto-inhibition of Ets-1 and illustrates one strategy for the synergistic activity of regulatory transcription factors.

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.

The role of the KRSIK motif of human angiogenin in heparin and DNA binding

RSC Advances ◽  
2016 ◽  
Vol 6 (86) ◽  
pp. 82644-82647 ◽  
Author(s):  
Kwon Joo Yeo ◽  
Jun-Goo Jee ◽  
Jin-Wan Park ◽  
Yu-Jin Lee ◽  
Kyoung-Seok Ryu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Potential Role ◽  
Interaction Site ◽  
Insight Into

The 50KRSIK54 motif is the main interaction site of hAng for heparin and DNA binding, providing an insight into the potential role of the motif for the internalization and DNA binding of hAng, which is essential for the regulation of angiogenesis.

The Multimerization Domain of Cbfß-SMMHC Is Required for Leukemogenesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3666-3666 ◽  
Author(s):  
Ling Zhao ◽  
Lemlem Alemu ◽  
Jun Cheng ◽  
Tao Zhen ◽  
Alan D. Friedman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Fetal Liver ◽  
Core Binding Factor ◽  
Binding Factor ◽  
Definitive Hematopoiesis

Abstract Among acute myeloid leukemia (AML) with cytogenetic abnormalities, core binding factor (CBF) leukemia acounts for 20-30% of adult AML, and 20-30% of pediatric AML. The chromosome 16 inversion (inv(16)), which results in a fusion gene CBFB -MYH11 and an encoded chimeric protein CBFβ-SMMHC (core binding factor β - smooth muscle myosin heavy chain), is observed primarily in AML subtype M4Eo. Using Cbfb-MYH11 knock-in mouse models we previously demonstrated that CBFβ-SMMHC needs its C terminal domains for leukemogenesis (Kamikubo et al, Blood 121:638, 2013). In this study we generated a new CBFB-MYH11 knock-in mouse model to determine the role of the multimerization domain at the C terminus of CBFβ-SMMHC for hematopoietic defects and leukemogenesis. Previous studies have shown that the C-terminal 29-residue assembly competent domain (ACD) is essential for multimerization of SMMHC. Within ACD, clustered point mutations in helices D and E specifically disrupts multimerization of CBFβ-SMMHC without interfering with the repression function of CBFβ-SMMHC (Zhang et al., Oncogene 25:7289, 2006). Therefore, we generated knock-in mice expressing CBFβ-SMMHC with mutated helices D and E (mDE) to study the role of the multimerization domain in vivo. Heterozygous embryos (Cbfb+/mDE) were viable and showed no defects in fetal liver definitive hematopoiesis, while homozygous embryos (CbfbmDE/mDE) showed complete blockage of definitive hematopoiesis, hemorrhage in the central nervous system and midgestation lethality, similar to the phenotype in Cbfb+/MYH11 mice and the Cbfb or Runx1 null mice. This phenotype is also similar to that in the homozygous knockin embryos expressing C-terminally-deleted CBFβ-SMMHC (Kamikubo et al, Blood 121:638, 2013). The fetal liver of E12.5 CbfbmDE/mDE embryos gave no colonies while the fetal liver of Cbfb+/mDE mice generated similar number of colonies as the WT controls. We further looked at the peripheral blood of E10.5 CbfbmDE/mDE embryos and found that the primitive hematopoiesis was not affected, while E10.5 Cbfb+/MYH11 embryos showed a developmental delay at this stage. Analysis of peripheral blood showed decreased B cell population in young adult Cbfb+/mDE mice, while the myeloid compartment was unchanged. In aged mice (>12 months), however, there was an increase of immature myeloid cells in the peripheral blood. Importantly, there was no leukemia development in the Cbfb+/mDE mice one year after ENU treatment (to induce cooperating mutations), while Cbfb+/MYH11 micedied of leukemia within 2 months of ENU treatment. Notably bone marrow cells in the Cbfb+/mDE and Cbfb+/MYH11 mice expressed their respective fusion proteins at similar levels. Overall our data suggest that the C terminal multimerization domain is required for the defects in primitive and definitive hematopoiesis caused by CBFβ-SMMHC, and the domain is essential for leukemogenesis by CBFβ-SMMHC. Further mechanistic studies of this domain may lead to new drug targets for treating inv(16) leukemia. For this purpose we have performed gene expression profiling with microarray and RNA-seq technologies, comparing gene expression changes in adult bone marrow c-Kit+ cells as well as embryonic primitive blood cells from Cbfb+/mDE and Cbfb+/MYH11 mice. Preliminary analysis indicates that the gene expression profile of the hematopoietic cells from the Cbfb+/mDE mice was much similar to that of Cbfb+/+ than Cbfb+/MYH11 mice. Validation and pathway analysis of those differentially expressed genes are ongoing and the results will be presented at the annual meeting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Potential role of lncRNA cyp2c91–protein interactions on diseases of the immune system

2015 ◽  
Vol 6 ◽  
Author(s):  
Prashanth Suravajhala ◽  
Lisette J. A. Kogelman ◽  
Gianluca Mazzoni ◽  
Haja N. Kadarmideen
Keyword(s):  
Immune System ◽  
Protein Interactions ◽  
Potential Role

scholarly journals FimW Is a Negative Regulator Affecting Type 1 Fimbrial Expression in Salmonella enterica Serovar Typhimurium

2001 ◽  
Vol 183 (2) ◽  
pp. 435-442 ◽  
Author(s):  
Juliette K. Tinker ◽  
Lisa S. Hancox ◽  
Steven Clegg
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Parental Strain ◽  
Salmonella Enterica ◽  
Specific Binding ◽  
Negative Regulator ◽  
E Coli ◽  
Serovar Typhimurium

ABSTRACT Type 1 fimbriae are proteinaceous surface appendages that carry adhesins specific for mannosylated glycoproteins. These fimbriae are found on most members of the family Enterobacteriaceae and are known to facilitate binding to a variety of eukaryotic cells, including those found on the mucosal surfaces of the alimentary tract. We have shown that the regulation of type 1 fimbrial expression in Salmonella enterica serovar Typhimurium is controlled, in part, by the products of four genes found within the fimgene cluster: fimZ, fimY, fimW, andfimU. To better understand the specific role of FimW in fimbrial expression, a mutation was constructed in this gene by the insertion of a kanamycin resistance DNA cassette into the chromosome. The resulting fimW mutation was characterized by mannose-sensitive hemagglutination and agglutination with fimbria-specific antiserum. Assays suggested that this mutant was more strongly fimbriate than the parental strain, exhibiting a four- to eightfold increase in fimbrial production. The fimWmutation was introduced into a second strain of Salmonella enterica serovar Typhimurium, and this mutant was also found to be strongly fimbriate compared to the parental strain. Consistent with the role of this protein as a negative regulator, fimA-lacZexpression in serovar Typhimurium, as well as in Escherichia coli, was increased twofold in the absence of functional FimW. Primer extension analysis determined that fimWtranscription is initiated from its own promoter 31 bp upstream of the translation start site. Analysis using a fimW-lacZ reporter indicated that fimW expression in serovar Typhimurium was increased under conditions that select for poorly fimbriate bacteria and low fimA expression. FimW also appears to act as an autoregulator, since expression from the fimW-lacZ reporter was increased in a fimW mutant. FimW was partially purified by fusion with the E. coli maltose-binding protein. Use of this FimW protein extract, as well as others, in DNA-binding assays was unable to identify a specific binding site for FimW in thefimA, fimZ, fimY, orfimW promoter regions. To analyze protein-protein interactions, FimW was expressed in a LexA-based two-hybrid system inE. coli. A significant interaction between FimW and the DNA-binding activator protein, FimZ, was detected using this system. These results indicate that FimW is a negative regulator of serovar Typhimurium type 1 fimbrial expression and may function by interfering with FimZ-mediated activation of fimA expression.

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.

The Role of C-KIT Mutations in the Leukemogenesis of Core-Binding Factor Acute Myeloid Leukemia: A Comparative Analysis on Paired Diagnosis and Relapse Samples.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2307-2307
Author(s):  
Der-Cherng Liang ◽  
Lee-Yung Shih ◽  
Chein-Fuang Huang ◽  
Ya-Tzu Chang ◽  
Huei-Ying Li ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Comparative Analysis ◽  
Crucial Role ◽  
Myeloid Leukemia ◽  
Tandem Repeats ◽  
Core Binding Factor ◽  
Kit Mutation ◽  
Binding Factor ◽  
Acute Myeloid

Abstract C-KIT is a member of the type III receptor tyrosine kinase family and plays a crucial role in normal hematopoiesis and acute myeloid leukemia (AML). C-KIT mutations have been described in core-binding factor (CBF) AML at initial diagnosis. The role of C-KIT mutations in the relapse of CBF AML is not clear. In the present study, we analyzed C-KIT mutations on paired diagnosis and relapse samples in CBF AML. Among 1014 adults and 162 children with AML, CBF AML was detected in 11.4% of adults and 25.3% of children. Mutational analysis of C-KIT was performed by direct sequencing for all cDNA PCR products amplified with 5 overlapping primer pairs, which cover the whole coding sequences of C-KIT gene from exon 1 through exon 21. In AML with t(8;21)/AML1-ETO, 33.0 % (29/88) of adults and 44.4 % (12/27) of children had C-KIT mutations. In AML with inv(16)/CBFβ-MYH11, 22.2 % (6/27) of adults and 38.5 %(5/13) of children had C-KIT mutations. Taken together, C-KIT mutations were present in 30.4 % (35/115) of adults and 42.5 % (17/40) of children with CBF AML. Forty-two patients with CBF AML relapsed. Twenty-two(18 adults and 4 children) of the 23 patients with CBF AML and C-KIT(+) at diagnosis had relapse samples available for comparative analysis. All the 22 patients relapsed with C-KIT mutations, 21 of them showed the identical C-KIT mutation patterns as those at diagnosis. Of the 20 relapsed patients with t(8;21)/AML1-ETO and C-KIT(+), 3 had mutations in exon 8: T417_D419delinsY, Y418_D419delinsA, and [Y418N;Y418_D419insFF], respectively; one had mutation in exon 9: I478V; another one had mutation in exon 11: [D572_P573insL; E561_D572dup]; 14 had mutations in exon 17: 5 D816V, 3 N822K, 3 D816Y, and one each with D816H, D820G, and D820Y; the remaining one patient relapsed twice, the patterns of C-KIT mutations changed but remained in exon 17: D816A at diagnosis, D816V at the first relapse, and N822K at the second relapse. Genotyping analysis with 15 loci of short tandem repeats at 13 different chromosomes showed identity for the diagnosis and the two relapse samples. Of the 2 adults with inv(16)/CBFβ-MYH11 and C-KIT(+) who relapsed, both had mutations in exon 17: N822K and D816Y, respectively. C-KIT mutations were absent in all of the 35 complete remission samples examined. In those with CBF AML and C-KIT(−) at diagnosis, 19 patients including 16 adults and 3 children relapsed; C-KIT mutations were not present in all the relapse samples except one who acquired D816H mutation. The present study showed that all patients with de novo CBF AML harboring C-KIT mutations at diagnosis retained the mutations at relapse, indicating that C-KIT mutations play a crucial role in the leukemogenesis in a substantial proportion of patients with CBF AML.

Identification of Proteinase 3 (PR3) Gene Overexpresison and Protein Delocalization in Core Binding Factor Leukemias as a Mechanism Leading to Increased Chemosensitivity.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 204-204
Author(s):  
Daniela Cilloni ◽  
Ilaria Defilippi ◽  
Sonia Carturan ◽  
Chiara Maffè ◽  
Marisa Pautasso ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Mean Value ◽  
Binding Activity ◽  
Proteinase 3 ◽  
Loss Of Function ◽  
Core Binding Factor ◽  
Secondary Aml ◽  
Dna Binding Activity ◽  
Binding Factor

Abstract Proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells. When abnormally expressed it confers factor-independent growth to hematopoietic cells. The aim of this study was to investigate the role of PR3 gene in leukemic haematopoiesis. We analyzed the expression levels of PR3 by RQ-PCR in 113 BM samples collected from AML patients at diagnosis. The FAB distribution was as follows: M0=5, M1=12, M2=38, M3=12, M4=37, M5=5, M6=4. 19 patients were characterized by t(8;21) and 16 by inv(16). PR3 expression level was also analyzed in 57 BM and 42 PB samples from 88 MDS patients (44 RA, 32 RAEB and 12 secondary-AML) and in 15 BM and 40 PB samples from healthy volunteers. PR3 protein was analyzed by western blot (WB) and its localization determined by immunofluorescence assay using specific antibodies. The transcription factor C/EBPα, which negatively regulates PR3 expression was studied in parallel at the RNA and protein level by RQ-PCR and WB. The DNA binding activity of C/EBPα was investigated by EMSA assay. Gain and loss of function experiments were performed by transfecting COS and 293T cell lines with a plasmid containing the full length PR3 sequence and HL60, Me-1, and Kasumi cell lines with specific shRNA. We found that PR3 is significantly overexpressed in AML samples. The median value of 2−Δ ΔCt is 740, (range 15-5043). Interestingly, patients affected by Core Binding Factor leukemias showed significantly higher PR3 values compared to patients with normal karyotypes (NK) (p<0,0002 for t(8;21), p<0,001 for inv16) and lower C/EPBα levels. EMSA assay demonstrated the absence of C/EBPα DNA binding activity in CBF AML cells but not in NK AML. In addition, PR3 overexpression was detected in 60% of RA patients (mean value: 10, range 3–268), and in all the cases of RAEB (mean value 201: range:128–803) and secondary AML (mean value 589, range 207–7131). WB demonstrated the correlation between the mRNA and protein amount. Interestingly, immunofluorescence demonstrated the de-localization of the protein within the nucleous in CBF AML but it is completely cytoplasmatic in leukemic cells with normal karyotype and in MDS. Transfection experiments with PR3 plasmid demonstrated that PR3 overexpression results into a significantly increased proliferation and reduced apoptosis. By contrast transfection with shRNA triggers apoptosis and cell growth inhibition. In addition, WB demonstrated that nuclear PR3 is able to cleavage the p65subunit of NF-kB into a p56 isoform which lacks any transcriptional activity as confirmed by EMSA. In conclusion, PR3 gene expression and protein are significantly increased in AML and MDS, particularly in CBF leukemias in which the protein is not only increased but also completely delocalized within the nucleous. PR3 overepression My be due to a significant downmodulation of C/EBPα. Ectopic expression of PR3 induces increased proliferation and apoptosis arrest. The abnormal nuclear localization of PR3 in CBF leukemias results into the loss of function of NF-kB thus representing one mechanism of chemo sensitivity in this group of patients.

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