t(8;21)(q22;q22) fusion proteins preferentially bind to duplicated AML1/RUNX1 DNA-binding sequences to differentially regulate gene expression

Okumura, Akiko; Peterson, Luke F.; Okumura, Fumihiro; Boyapati, Anita; Zhang, Dong‐Er

doi:10.1182/blood-2007-11-124735

Cited by 41 publications

(40 citation statements)

References 66 publications

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“…In addition, coexpression of the cofactor CBF␤, which enhances DNA-binding of RUNX1, 33 did not improve REtr-m5 binding to DNA (data not shown). Similar to the observations of others, 32 the monomeric form of RUNX1/ETO (RE-dNHR2) and a DNA-binding defective mutant of RUNX1/ETO (RE-L148D) failed to bind to the R3 oligonucleotide. Loss of RE-m5 DNA binding was also observed by EMSA analysis using a radioactive labeled RUNX1 highaffinity oligonucleotide ( Figure 3G).…”

Section: Hot Spots Of Runx1/eto Dimer-tetramer Transition 607supporting

confidence: 67%

“…32 Although the RUNX3 and PU.1 oligonucleotides used in our study did contain 2 RUNX1 consensus-binding sites, RE-m5 or REtr-m5 failed to bind to these oligonucleotides. One can anticipate that the antiparallel orientation of both ␣-helices in the RUNX1/ETO-m5 dimer does not favor the conformational change observed on DNA binding for RUNX1.…”

Section: Discussionmentioning

confidence: 90%

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confidence: 99%

“…In addition, monomeric forms of REtr (REtr-dNHR2) did not bind to the oligos ( Figure 3A-B), confirming the results of others who have shown that tetramerization of RUNX1/ETO is required for specific DNA binding. 32 Compared with the binding strength of the REtr tetrameric form, the dimeric and monomeric forms of REtr displayed a 40-fold reduction in binding affinity to the DNA oligos ( Figure 3C-D).We also introduced the m5 mutations into full-length RUNX1/ ETO and tested its DNA-binding capability as mentioned in the previous paragraph. Full-length RUNX1/ETO did bind specifically to the R3 oligonucleotide, whereas a mutant of R3 lacking the RUNX1-binding sites was not bound by RUNX1/ETO and DNA …”

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confidence: 99%

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Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Wichmann

Becker

Chen-Wichmann

et al. 2010

Blood

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RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimertetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the selfrenewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimertetramer interface, suitable for a molecular intervention in t(8;21) leukemias. IntroductionChromosomal translocations are frequent events during malignant cell transformation, particularly during leukemogenesis. 1 The translocation t(8;21), one of the most frequent chromosomal anomalies in acute myeloid leukemia (AML), involves the RUNX1 gene (also known as AML1, CBF␣2, or PEBP2␣B) on chromosome 21 and the ETO gene (also known as MTG8 or RUNX1T1) on chromosome 8. The ubiquitously expressed RUNX1 is a transcription factor and belongs to the key regulators of hematopoietic cell differentiation. 2 The fusion protein RUNX1/ETO contains the DNA-binding domain (Runt, RHD) of the RUNX1 transcription factor but lacks the C-terminal transactivation sequence that is replaced by almost the entire ETO protein. 2 forms of RUNX1/ETO coexist in AML-leukemia samples: the originally discovered full-length RUNX1/ETO and a splice variant called RUNX1/ETO9a, which lacks 178 amino acids at the C-terminus. Only RUNX1/ETO9a does not require cooperative events for inducing leukemia development in mice. 3,4 We and others have shown that RUNX1/ETO has a modular structure. Besides the Runt domain, RUNX1/ETO contains 4 functional domains, which are generally referred to as nervy homology region (NHR1 to NHR4). The NHR domains serve as docking interface for a variety of different proteins, including the E-protein HEB, 5,6 the apoptosis-related protein SON, 7 and nuclear corepressor proteins, such as N-CoR, SMRT, mSIN3A, and MTGR1, as well as histone deacetylases (HDACs). [8][9][10][11] In addition, the NHR2 domain mediates tetramer formation through hydrophobic and ionic/polar interactions. Two ␣-helices align in a head-to-tail fashion to form an antiparallel dimer. Two dimers subsequently are positioned on top of each other in a sandwich-like fashion. The total interaction area composing all contact points of the 4 ␣-helices is approximately 10 000 Å. 2 Substitution of 7 leucines withi...

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Section: Hot Spots Of Runx1/eto Dimer-tetramer Transition 607supporting

confidence: 67%

Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Wichmann

Becker

Chen-Wichmann

et al. 2010

Blood

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“…26 Coimmunoprecipitation (Co-IP) assays in Kasumi-1 and AML1/ETO-induced U937-A/E/9/14/18 cells were performed strictly according to the manufacturer's protocol (Active Motif). Glutathione S-transferase (GST) fusion proteins were generated according to the GST Gene Fusion System Handbook (GE Healthcare, Piscataway, NJ).…”

Section: Chromatin Immunoprecipitation Sequencingmentioning

confidence: 99%

Genome-wide studies identify a novel interplay between AML1 and AML1/ETO in t(8;21) acute myeloid leukemia

Wang

et al. 2016

Blood

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Key Points• Wild-type AML1 and AML1/ ETO form a complex on chromatin via binding to adjacent different motifs and interacting through the runt homology domain.• The relative binding signals of AML1/ETO and AML1 and AP-1 recruitment determine whether AML1/ETO activates or represses its targets.The AML1/ETO fusion protein is essential to the development of t(8;21) acute myeloid leukemia (AML) and is well recognized for its dominant-negative effect on the coexisting wild-type protein AML1. However, the genome-wide interplay between AML1/ETO and wild-type AML1 remains elusive in the leukemogenesis of t(8;21) AML. Through chromatin immunoprecipitation sequencing and computational analysis, followed by a series of experimental validations, we report here that wild-type AML1 is able to orchestrate the expression of AML1/ETO targets regardless of being activated or repressed; this is achieved via forming a complex with AML1/ETO and via recruiting the cofactor AP-1 on chromatin. On chromatin occupancy, AML1/ETO and wild-type AML1 largely overlap and preferentially bind to adjacent and distinct short and long AML1 motifs on the colocalized regions, respectively. On physical interaction, AML1/ETO can form a complex with wild-type AML1 on chromatin, and the runt homology domain of both proteins are responsible for their interactions. More importantly, the relative binding signals of AML1 and AML1/ETO on chromatin determine which genes are repressed or activated by AML1/ETO. Further analysis of coregulators indicates that AML1/ETO transactivates gene expression through recruiting AP-1 to the AML1/ETO-AML1 complex. These findings enrich our knowledge of understanding the significance of the interplay between the wild-type protein and the oncogenic fusion protein in the development of leukemia. (Blood. 2016;127(2):233-242) IntroductionMany oncogenic fusion proteins generated by chromosomal translocations play a causal role in the development of leukemia. The oncogenic lesion almost exclusively occurs in a single allele within an individual leukemia, whereas the wild-type protein produced from the nontranslocated allele generally still exists.1,2 The coexistence of the wild-type protein with the oncogenic fusion protein raises the question about their significance in leukemogenesis. To address this question, acute myeloid leukemia (AML) with the t(8;21) translocation and resultant AML1/ETO fusion gene is an ideal model, particularly through understanding the interplay between the AML1/ETO fusion protein and the AML1 wild-type protein.AML1 (also known as RUNX1 or core-binding factor a [CBFa]), a critical regulator of normal hematopoiesis, 3 is widely expressed in multiple hematopoietic lineages and regulates the expression of a variety of hematopoietic genes through recognizing the motif TGTGGT.4 AML1 is frequently involved in chromosomal abnormalities in AML, [5][6][7] among which AML1/ETO is the most common fusion protein resulted from the t(8;21) translocation. 7 Structurally, AML1/ETO consists of the N-terminal portion o...

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RUNX1 repression‐independent mechanisms of leukemogenesis by fusion genes CBFB–MYH11 and AML1–ETO (RUNX1–RUNX1T1)

Hyde

Liu

2010

J of Cellular Biochemistry

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SUMMARY The Core Binding Factor (CBF) acute myeloid leukemias (AMLs) are a prognostically distinct subgroup that includes patients with the inv(16) and t(8:21) chromosomal rearrangements. Both of these rearrangements result in the formation of fusion proteins, CBFB-MYH11 and AML1-ETO respectively, that involve members of the CBF family of transcription factors. It has been proposed that both of these fusion proteins function primarily by dominantly repressing normal CBF transcription. However, recent reports have indicted that additional, CBF-repression independent activities may be equally important during leukemogenesis. This article will focus on these recent advances.

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t(8;21)(q22;q22) fusion proteins preferentially bind to duplicated AML1/RUNX1 DNA-binding sequences to differentially regulate gene expression

Cited by 41 publications

References 66 publications

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Genome-wide studies identify a novel interplay between AML1 and AML1/ETO in t(8;21) acute myeloid leukemia

RUNX1 repression‐independent mechanisms of leukemogenesis by fusion genes CBFB–MYH11 and AML1–ETO (RUNX1–RUNX1T1)

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