Runt Domain Of AML1 Research Articles

Selection of aptamers using β-1,3-glucan recognition protein tagged proteins and curdlan beads.

RNA aptamers are nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilise target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins, but also to the protein tags and matrix beads. In this study, we performed SELEX using β-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilise the AML1 Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Conjugation of two RNA aptamers improves binding affinity to AML1 Runt domain.

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.

Solution structure of a DNA mimicking motif of an RNA aptamer against transcription factor AML1 Runt domain

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.

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

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.

CBFβ is critical for AML1-ETO and TEL-AML1 activity

AbstractAML1-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.

Previously Unknown Interactions Between AML1 and MLL Provide Epigenetic Regulation of Gene Expression in Normal Hematopoiesis and in Leukemia

In all organisms, the fundamental process of transcriptional regulation requires transcription factors, which bind to DNA in response to extra-cellular signals and regulate transcription of target genes. In eukaryotes, this process also involves epigenetic regulation, which includes DNA and histone modifications. Hematopoiesis and leukemia are excellent model systems for studying the higher eukaryotic regulations of gene expression and for identifying important molecules involved in genetic and epigenetic transcriptional regulation. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcriptional factor AML1/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. We have previously shown that the regulation of PU.1 by AML1 is mediated by 3 AML1 binding sites in the PU.1 upstream regulatory element (URE), located −14 kb relative to the transcription start site in mice (Huang et al. Nat Genet. 2008). To understand whether AML1 plays a critical role in regulating the PU.1 locus at the chromatin level, we have utilized this PU.1 regulation system as a model to study the potential interplay between AML1/CBFβ and MLL. We found that MLL binds to the evolutionarily conserved Runt-domain of AML1, enhances the formation of the AML1/CBFβ heterodimer, and blocks the ubiquitin-proteasome mediated degradation of AML1. We also found that AML1/CBFβ is required for maintenance of the H3K4-me3 histone mark at the PU.1 upstream regulatory element (URE) and promoter region, and that MLL is required for AML1 to regulate PU.1 expression. In contrast, we found that several leukemia-associated MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-AF10, no longer stabilize AML1 but rather induce AML1 degradation and downregulate PU.1 expression. Taken together, our data indicate that MLL and some of its leukemia-associated fusion proteins physically and functionally interact with AML1. Their effects on target genes, particularly PU.1, may be critical for the normal regulation of hematopoiesis and for the aberrant regulation that underlies acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).

AML1-ETO interacts with Sp1 and antagonizes Sp1 transactivity through RUNT domain

AML1-ETO fusion protein is observed in approximately 12% of acute myeloid leukemia. In the present research, we found that AML1-ETO is able to inhibit Sp1 transactivity. We also found that this inhibition of Sp1 transactivity by AML1-ETO is achieved by interaction between Sp1 and RUNT domain of AML1. AML1b is able to abrogate the inhibition of AML1-ETO. Since Sp1 is involved in hematopoietic cell differentiation, we proposed that AML1-ETO promotes leukemogenesis by blocking cell differentiation through inhibition of Sp1 transactivity. Structured summary MINT- 6549474: AML1-ETO (genbank_protein_gi:AAB34820) physically interacts ( MI:0218) with Sp1 (uniprotkb: P08047) by anti bait coimmunoprecipitation ( MI:0006) MINT- 6549439: Sp1 (uniprotkb: P08047) physically interacts ( MI:0218) with AML1-ETO (uniprotkb: AAB34820) by anti tag coimmunoprecipitation ( MI:0007) MINT- 6549458: Sp1 (uniprotkb: P08047) physically interacts ( MI:0218) with AML1a (uniprotkb: Q01196-2) by anti tag coimmunoprecipitation ( MI:0007)

A Possible Mechanism for Synergy in Transcriptional Activation between AML1 and PU.1: Exclusion of the Co-Repressor CBFA2T3 (ETO-2, MTG16).

AML1 and PU.1, important regulators of hematopoietic differentiation, interact with each other and are known to synergize in transcriptional activation (Zhang et al, 1996). PU.1 and AML1 also interact with mSin3, a component of a co-repressor complex that can include N-CoR, HDAC and CBFA2T1 (ETO, MTG8) or CBFA2T3 (ETO2, MTG16). CBFA2T3 is highly expressed in hematopoietic cells and is a target of a chromosomal translocation found in acute myeloid leukemia (t(16;21)). In transfected 293T cells, we demonstrate that both AML1 and PU.1 co-immunoprecipitate with the conserved N-terminal TAFH domain of CBFA2T3 but not the C-terminal MYND domain. Although AML1 and PU.1 independently co-immunoprecipitate with CBFA2T3, when all three proteins are over-expressed in 293T cells, AML1 and PU.1 co-immunoprecipitate with each other while excluding CBFA2T3. CBFA2T3 interacts with the non-runt portion of AML1 (AML1 C-terminus) while PU.1 can interact with both the runt domain of AML1 and AML1 C-terminus. Presumably, the interaction between AML1 and PU.1 shields the CBFA2T3 binding sites on both proteins. Since this region includes the binding site for mSin3, other co-repressors may also be excluded from an AML1/PU.1 complex. This may be one basis for the co-operation between AML1 and PU.1 in transcriptional activation.

Mutations of the AML1 gene in acute myeloid leukemia of FAB types M0 and M7.

The AML1 gene encodes a transcription factor that, together with its heterodimeric partner CBFB, regulates a number of target genes that are essential for normal hemopoiesis. In acute myeloid leukemia (AML), AML1 is disrupted not only by chromosomal translocations but also by mutations in the runt domain, which binds both DNA and CBFB. Acquired mutations have been described predominantly in the AML FAB type M0. To date, most patients appear to have biallelic disease, suggesting a complete lack of normal AML1 function. Inherited loss of function mutations thought to lead to haploinsufficiency also have been described in patients who have a familial disorder with predisposition to AML (FPD/AML), indicating the role of AML1 in megakaryopoiesis. Using single-strand conformation polymorphism analysis, we studied the AML1 runt domain in 41 patients with M0 AML and identified potentially pathologic mutations in five (12%). Biallelic disease could be confirmed in only one patient, using loss of heterozygosity studies. At least three of the mutations would lead to truncated proteins similar to those reported in FPD/AML, suggesting that haploinsufficiency plays a role in the pathogenesis of this minimally differentiated type of leukemia. The incidence of acquired mutations in AML patients with acute megakaryoblastic leukemia (FAB type M7) was the same as that reported in other non-M0 patients, with only one mutation detected in 20 (5%) patients studied.

Mutational Analyses of the AML1 Gene in Patients with Myelodysplastic Syndrome

The AML1 gene is the most frequent target of translocations associated with human leukemias. We recently found somatic point mutations of the AML1 gene, V105ter and R139G, in two cases of myelodysplastic syndrome (MDS). Both mutations are present in the region encoding the Runt domain of AML1, and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with PEBP2 β /CBF β. On the other hand, the R139G mutant acts as a dominant negative inhibitor through competing with wild-type AML1 for interaction with PEBP2 β /CBF β. In this review, we summarize mutational changes of the AML1 gene in hematological malignancies, especially in MDS and discuss the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis

AbstractThe AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase–polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor β (PEBP2β/CBFβ). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2β/CBFβ. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

Mutations of the AML1 gene in myelodysplastic syndrome and their functional implications in leukemogenesis

The AML1 gene encodes a DNA-binding protein that contains the runt domain and is the most frequent target of translocations associated with human leukemias. Here, point mutations of the AML1 gene, V105ter (single-letter amino acid code) and R139G, (single-letter amino acid codes) were identified in 2 cases of myelodysplastic syndrome (MDS) by means of the reverse transcriptase-polymerase chain reaction single-strand conformation polymorphism method. Both mutations are present in the region encoding the runt domain of AML1 and cause loss of the DNA-binding ability of the resultant products. Of these mutants, V105ter has also lost the ability to heterodimerize with polyomavirus enhancer binding protein 2/core binding factor beta (PEBP2beta/CBFbeta). On the other hand, the R139G mutant acts as a dominant negative inhibitor by competing with wild-type AML1 for interaction with PEBP2beta/CBFbeta. This study is the first report that describes mutations of AML1 in patients with MDS and the mechanism whereby the mutant acts as a dominant negative inhibitor of wild-type AML1.

Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFbeta.

Mutations in the genes encoding the interacting proteins AML1 and CBFbeta are the most common genetic abnormalities in acute leukaemia, and congenital mutations in the related AML3 gene are associated with disorders of osteogenesis. Furthermore, the interaction of AML1 with CBFbeta is essential for haematopoiesis. We report the 2.6 A resolution crystal structure of the complex between the AML1 Runt domain and CBFbeta, which represents a paradigm for the mode of interaction of this highly conserved family of transcription factors. The structure demonstrates that point mutations associated with cleidocranial dysplasia map to the conserved heterodimer interface, suggesting a role for CBFbeta in osteogenesis, and reveals a potential protein interaction platform composed of conserved negatively charged residues on the surface of CBFbeta.

Solution properties of the free and DNA-bound Runt domain of AML1.

The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.