The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation.

The AML-1/CBF beta transcription factor complex is targeted by both the t(8;21) and the inv(16) chromosomal alterations, which are frequently observed in acute myelogenous leukemia. AML-1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. The t(8;21) translocation fuses the first 177 amino acids of AML-1 to MTG8 (also known as ETO), generating a chimeric protein that retains the DNA-binding domain of AML-1. Analysis of endogenous AML-1 DNA-binding complexes suggested the presence of at least two AML-1 isoforms. Accordingly, we screened a human B-cell cDNA library and isolated a larger, potentially alternatively spliced, form of AML1, termed AML1B. AML-1B is a protein of 53 kDa that binds to a consensus AML-1-binding site and complexes with CBF beta. Subcellular fractionation experiments demonstrated that both AML-1 and AML-1/ETO are efficiently extracted from the nucleus under ionic conditions but that AML-1B is localized to a salt-resistant nuclear compartment. Analysis of the transcriptional activities of AML-1, AML-1B, and AML-1/ETO demonstrated that only AML-1B activates transcription from the T-cell receptor beta enhancer. Mixing experiments indicated that AML-1/ETO can efficiently block AML-1B-dependent transcriptional activation, suggesting that the t(8;21) translocation creates a dominant interfering protein.

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Four of the seven zinc fingers of the Evi-1 myeloid-transforming gene are required for sequence-specific binding to GA(C/T)AAGA(T/C)AAGATAA.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4291 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4291-4300 ◽

Cited By ~ 84

Author(s):

R Delwel ◽

T Funabiki ◽

B L Kreider ◽

K Morishita ◽

J N Ihle

Keyword(s):

Dna Binding ◽

Specific Binding ◽

Zinc Fingers ◽

Consensus Sequence ◽

Myelogenous Leukemia ◽

Chromosome Band ◽

Aberrant Expression ◽

Relative Specificity ◽

Acute Myelogenous ◽

Binding Sequence

Expression of the Evi-1 gene is activated in murine myeloid leukemias by retroviral insertions and in human acute myelogenous leukemia by translocations and inversions involving chromosome band 3q26 where the gene resides. Aberrant expression of the Evi-1 gene has been shown to interfere with myeloid differentiation, which is proposed to be the basis for its role in leukemias. The Evi-1 gene encodes a 145-kDa DNA-binding protein containing two domains of seven and three Cys2-His2 zinc fingers. Previous studies identified a portion of the consensus DNA-binding sequence for the first domain of zinc fingers. The experiments presented here extend these studies and demonstrate that the first domain recognizes a consensus of 15 nucleotides consisting of GA(C/T)AAGA(T/C)AAGATAA. The first three fingers of the first domain do not detectably bind DNA but contribute to the binding by conferring a relative specificity for GACAA verses GATAA in the first position. The first three fingers also contribute to optimal binding of the 15-nucleotide consensus sequence.

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Heterogeneity in CBF beta/MYH11 fusion messages encoded by the inv(16)(p13q22) and the t(16;16)(p13;q22) in acute myelogenous leukemia

Blood ◽

10.1182/blood.v85.12.3695.bloodjournal85123695 ◽

1995 ◽

Vol 85 (12) ◽

pp. 3695-3703 ◽

Cited By ~ 75

Author(s):

SA Shurtleff ◽

S Meyers ◽

SW Hiebert ◽

SC Raimondi ◽

DR Head ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Acute Myelogenous Leukemia ◽

Myelogenous Leukemia ◽

Pcr Analysis ◽

Chain Gene ◽

Genomic Locus ◽

Acute Myelogenous

Inv(16)(p13q22) is one of the most frequent chromosomal rearrangements found in acute myelogenous leukemia (AML), representing approximately 16% of documented karyotypic abnormalities. The inv(16) breakpoints have been cloned and shown to involve the non-DNA binding component of the AML-1 transcription factor complex termed core binding factor beta gene (CBF beta) on 16q and the smooth muscle myosin heavy chain gene (MYH11) on 16p. In this study, we analyzed 37 cases of inv(16)-containing AML and 4 cases with t(16;16)(p13;q22) for expression of the CBF beta/MYH11 chimeric message by reverse transcription-polymerase chain reaction (PCR) analysis. CBF beta/MYH11 chimeric messages were detected in 33 of 37 cases with the inv(16) and in the 4 t(16;16)-containing cases. Sequence analysis of PCR products showed extensive breakpoint heterogeneity in both CBF beta and MYH11. In addition to the previously described breakpoint in CBF beta at nucleotide (nt) 495 (amino acid 165), we have identified a second novel fusion point at nt 399 (amino acid 133) in 7% of the cases. Similarly, a unique breakpoint site was identified in MYH11 at nt 1098, as well as at three previously characterized sites at nts 994, 1201, and 1921. Of the 4 PCR-negative cases, 2 of 3 tested lacked CBF beta rearrangements by Southern blot analysis, suggesting the possible involvement of a different genomic locus in some cases with cytogenetic evidence of inv(16). To assess whether the portions of CBF-beta contained within the CBF beta/MYH11 chimeric products retain the ability to interact with their heterodimeric DNA-binding partner AML-1, we performed in vitro DNA- binding analysis. Recombinant CBF-beta polypeptides consisting of the N-terminal 165 amino acids retained their ability to interact with AML-1, whereas mutants containing only the N-terminal 133 amino acids interacted with AML-1 less efficiently. These data suggest that different CBF beta/MYH11 products may vary subtly in their biologic activities.

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Human Cdc5, a regulator of mitotic entry, can act as a site-specific DNA binding protein

Journal of Cell Science ◽

10.1242/jcs.113.24.4523 ◽

2000 ◽

Vol 113 (24) ◽

pp. 4523-4531

Author(s):

X.H. Lei ◽

X. Shen ◽

X.Q. Xu ◽

H.S. Bernstein

Keyword(s):

Dna Binding ◽

Binding Site ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Family Members ◽

Site Specific ◽

Coordinated Expression ◽

Preferential Binding ◽

Dna Protein Interaction ◽

A Site

G(2)/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved. We recently identified human Cdc5, a positive regulator of G(2)/M in mammalian cells. We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G(2)/M through transcriptional activation. Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members. In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding. We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus. We show that this DNA-protein interaction is capable of activating transcription. We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5. Several of these contained sequences similar to the binding site. We demonstrate that these bind human Cdc5 with similar specificity and affinity. These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome. This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.

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Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.954-962.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 954-962

Author(s):

C V Dang ◽

J Barrett ◽

M Villa-Garcia ◽

L M Resar ◽

G J Kato ◽

...

Keyword(s):

Dna Binding ◽

Reporter Gene ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Leucine Zipper ◽

Chimeric Protein ◽

Dna Binding Domain ◽

Cytoplasmic Protein ◽

Activation Domain ◽

In Cells

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

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CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.4051 ◽

1997 ◽

Vol 17 (7) ◽

pp. 4051-4058 ◽

Cited By ~ 144

Author(s):

V S Shapiro ◽

K E Truitt ◽

J B Imboden ◽

A Weiss

Keyword(s):

Transcriptional Activation ◽

Interleukin 2 ◽

Cell Receptor ◽

Unique Element ◽

Composite Element ◽

Shift Analysis ◽

Nf Kappab ◽

Responsive Element ◽

A Site ◽

Acetate Activation

Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-kappaB family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.

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High frequency of clonal immunoglobulin or T cell receptor gene rearrangements in acute myelogenous leukemia expressing terminal deoxyribonucleotidyltransferase.

Journal of Experimental Medicine ◽

10.1084/jem.165.6.1703 ◽

1987 ◽

Vol 165 (6) ◽

pp. 1703-1712 ◽

Cited By ~ 64

Author(s):

S V Seremetis ◽

P G Pelicci ◽

A Tabilio ◽

A Ubriaco ◽

F Grignani ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Receptor Gene ◽

Cell Receptor ◽

Myelogenous Leukemia ◽

Gene Rearrangements ◽

Lymphoid Lineage ◽

Acute Myelogenous ◽

T Cell Receptor Gene ◽

Cell Receptor Gene

Ig and T cell receptor rearrangements have been used as irreversible markers of lineage and clonality in the study of B- and T-lymphoid populations. We have addressed the issue of lymphoid lineage specificity of these rearrangements by analyzing a panel of 25 TdT- acute myelogenous leukemias, 13 TdT+ AMLs, and 4 TdT+ undifferentiated leukemias. We report that while gene rearrangements represent extremely rare events in classical TdT- AML (less than 8%), rearrangements of either the Ig or T beta locus or both were detectable in the majority of the TdT+ AMLs (greater than 60%), and rearrangements of both loci were detectable in all of the TdT+ undifferentiated leukemias. These data demonstrate a significant association between TdT expression and Ig or T beta gene rearrangements even outside the lymphoid lineage, further supporting a role for TdT in Ig and T cell receptor gene assembly. These data also indicate that a coordinated program of lymphoid gene expression involving TdT-CD7-expression and Ig/T beta rearrangements can be activated before myeloid commitment. Whether the activation of this program represents a normal, albeit rare, event in early myelopoiesis or a transformation-related event present only in leukemic cells remains to be determined.

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NUP98 Is Fused to PMX1 Homeobox Gene in Human Acute Myelogenous Leukemia With Chromosome Translocation t(1;11)(q23;p15)

Blood ◽

10.1182/blood.v94.2.741 ◽

1999 ◽

Vol 94 (2) ◽

pp. 741-747 ◽

Cited By ~ 96

Author(s):

Takuro Nakamura ◽

Yukari Yamazaki ◽

Yoshiaki Hatano ◽

Ikuo Miura

Keyword(s):

Transcriptional Activation ◽

Acute Myelogenous Leukemia ◽

Constitutive Expression ◽

Homeobox Gene ◽

Fusion Transcript ◽

Chromosome Translocation ◽

Myelogenous Leukemia ◽

Homeodomain Protein ◽

Transcriptional Activation Domain ◽

Acute Myelogenous

Abstract The nucleoporin gene NUP98 was found fused to theHOXA9, HOXD13, or DDX10 genes in human acute myelogenous leukemia (AML) with chromosome translocations t(7;11)(p15;p15), t(2;11)(q35;p15), or inv(11)(p15;q22), respectively. We report here the fusion between the NUP98 gene and another homeobox gene PMX1 in a case of human AML with a t(1;11)(q23;p15) translocation. The chimeric NUP98-PMX1transcript was detected; however, there was no reciprocalPMX1-NUP98 fusion transcript. Like the NUP98-HOXA9fusion, NUP98 and PMX1 were fused in frame and the N-terminal GLFG-rich docking region of the NUP98 and the PMX1 homeodomain were conserved in the NUP98-PMX1 fusion, suggesting that PMX1 homeodomain expression is upregulated and that the fusion protein may act as an oncogenic transcription factor. The fusion to NUP98 results in the addition of the strong transcriptional activation domain located in the N-terminal region of NUP98 to PMX1. These findings suggest that constitutive expression and alteration of the transcriptional activity of the PMX1 homeodomain protein may be critical for myeloid leukemogenesis.

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The t(12;21) translocation converts AML-1B from an activator to a repressor of transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.16.4.1349 ◽

1996 ◽

Vol 16 (4) ◽

pp. 1349-1355 ◽

Cited By ~ 195

Author(s):

S W Hiebert ◽

W Sun ◽

J N Davis ◽

T Golub ◽

S Shurtleff ◽

...

Keyword(s):

T Cell ◽

Fusion Protein ◽

B Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Related Factor ◽

Core Motif ◽

T Cell Receptor Beta ◽

Beta Enhancer ◽

Receptor Beta

The t(12;21) translocation is present in up to 30% of childhood B-cell acute lymphoblastic and fuses a potential dimerization motif from the ets-related factor TEL to the N terminus of AML1. The t(12;21) translocation encodes a 93-kDa fusion protein that localizes to a high-salt- and detergent-resistant nuclear compartment. This protein binds the enhancer core motif, TGTGGT, and interacts with the AML-1-binding protein, core-binding factor beta. Although TEL/AML-1B retains the C-terminal domain of AML-1B that is required for transactivation of the T-cell receptor beta enhancer, it fails to activate transcription but rather inhibits the basal activity of this enhancer. TEL/AML-1B efficiently interferes with AML-1B dependent transactivation of the T-cell receptor beta enhancer, and coexpression of wild-type TEL does not reverse this inhibition. The N-terminal TEL helix-loop-helix domain is essential for TEL/AML-1B-mediated repression. Thus, the t(12;21) fusion protein dominantly interferes with AML-1B-dependent transcription, suggesting that the inhibition of expression of AML-1 genes is critical for B-cell leukemogenesis.

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Indistinguishable nuclear factor binding to functional core sites of the T-cell receptor delta and murine leukemia virus enhancers.

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4817 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4817-4823 ◽

Cited By ~ 59

Author(s):

J M Redondo ◽

J L Pfohl ◽

C Hernandez-Munain ◽

S Wang ◽

N A Speck ◽

...

Keyword(s):

T Cell ◽

Transcriptional Activation ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Cell Receptor ◽

Cell Tropism ◽

Core Motif ◽

Virus Core ◽

Core Site ◽

Murine Leukemia

We have previously shown that the delta E3 site is an essential element for transcriptional activation by the human T-cell receptor (TCR) delta enhancer and identified two factors, NF-delta E3A and NF-delta E3C, that bound to overlapping core (TGTGGTTT) and E-box motifs within delta E3. In this study, we show that protein binding to the core motif is necessary but not sufficient for transcriptional activation by the delta E3 element. In contrast, protein binding to the E-box motif does not contribute significantly to enhancer activity. A similar core motif present within the enhancers of T-cell-tropic murine retroviruses has been shown to contribute to transcriptional activity of the viral long terminal repeat in T lymphocytes and to viral T-cell tropism. We therefore determined the relationship between the nuclear factors that bind to the TCR delta and Moloney murine leukemia virus core motifs. On the basis of electrophoretic mobility shift binding and competition studies, biochemical analysis of affinity-labeled DNA-binding proteins, and the binding of a purified core binding factor, the proteins that bound to the TCR delta core site were indistinguishable from those that bound to the murine leukemia virus core site. These data argue that DNA-binding proteins that interact with the core site of murine leukemia virus long terminal repeats and contribute to viral T-cell tropism also play an essential role in the T-cell-specific expression of cellular genes.

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Fusion AML1 transcript in a radiation-associated leukemia results in a truncated inhibitory AML1 protein

Blood ◽

10.1182/blood.v97.7.2168 ◽

2001 ◽

Vol 97 (7) ◽

pp. 2168-2170 ◽

Cited By ~ 23

Author(s):

Robert Hromas ◽

Tracey Busse ◽

Audra Carroll ◽

David Mack ◽

Rinah Shopnick ◽

...

Keyword(s):

Transcriptional Activation ◽

Fusion Transcript ◽

Myelogenous Leukemia ◽

Chain Reaction ◽

Hematopoietic Development ◽

Transcriptional Activation Domain ◽

Protein Functions ◽

Acute Myelogenous ◽

Polymerase Chain

Abstract AML1 is a transcription factor that is essential for normal hematopoietic development. It is the most frequent target for translocations in acute leukemia. Recently, fluorescence in situ hybridization was used to identify a novel syndrome of radiation-associated secondary acute myelogenous leukemia that had AML1 translocations. Using polymerase chain reaction, the AML1 fusion transcript was isolated from the patient who had a t(19;21) radiation-associated leukemia. The AML1gene is fused out of frame to chromosome 19 sequences, resulting in a truncated AML protein bearing the DNA binding domain but not the transcriptional activation domain. This fusion AML1 protein functions as an inhibitor of the normal AML1 protein.

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