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Giuseppina Nucifora

Publications and source records attributed to Giuseppina Nucifora.

At least 19 recordsLinked to original sources
PubMed2006

Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro.

Animals↗
PubMed2006

Normal and transforming functions of RUNX1: a perspective.

Converging studies from many investigators indicate that RUNX1 has a critical role in the correct maintenance of essential cellular functions during embryonic development and after birth. The discovery that this gene is also frequently mutated in human leukemia has increased the interest in the role that RUNX1 plays in both normal and transforming pathways. Here, we provide an overview of the many roles of RUNX1 in hematopoietic self-renewal and differentiation and summarize the information that is currently available on the many mechanisms of RUNX1 deregulation in human leukemia.

Animals↗
PubMed2006

Combinatorial action of RUNX1 and PU.1 in the regulation of hematopoiesis.

The hematopoietic stem cell (HSC) has the potential to differentiate into mature cells with distinct phenotypes and functions. As suggested in recent reports, this plasticity can expand to include nonhematopoietic lineages, and, indeed, the HSC may repopulate liver and muscle tissues, as well. Considering the flexibility in HSC differentiation, these processes are regulated by a relatively small number of factors, some of which are expressed in all lineages, whereas others are activated only in a specific cell type. Combined evidence from many studies suggests that alternative subsets of these factors work in a combinatorial manner to regulate specific promoters for the induction of a specific lineage. RUNX1 and PU.1 have a fundamental role in HSC differentiation in that multifactor complexes are assembled around these proteins leading to tissue-specific and synergistic gene activation. Here we describe the relationship of RUNX1 with PU.1 as a facet of the combinatorial relationships that determine hematopoietic lineage commitment.

Animals↗
PubMed2005

EVI1 and hematopoietic disorders: history and perspectives.

The ecotropic viral integration site 1 (EVI1) gene was identified almost 20 years ago as the integration site of an ecotropic retrovirus leading to murine myeloid leukemia. Since its identification, EVI1 has slowly been recognized as one of the most aggressive oncogenes associated with human leukemia. Despite the effort of many investigators, still very little is known about this gene. The mechanism by which EVI1 operates in the transformation of hematopoietic cells is not known, but it is clear that EVI1 upregulates cell proliferation, impairs cell differentiation, and induces cell transformation. In this review, we summarize the biochemical properties of EVI1 and the effects of EVI1 in biological models.

Amino Acid Sequence↗
PubMed2005

Corepressor CtBP1 interacts with and specifically inhibits CBP activity.

Transcription repression in eukaryotes is mediated by a wide variety of transcription factors that usually recruit corepressors and form corepressor complexes at the specific promoter sites. One of these corepressors is the C-terminal-binding protein (CtBP) which was first identified as a protein that binds to the C-terminal region of the adenovirus E1A protein. CtBP has a strong role in both development and oncogenesis. Till date, the mechanism of transcription repression by CtBP is unknown. Here, we report that CtBP physically interacts in vivo with HAT enzymes from different families. The vast majority of the HAT enzymes have a potential consensus site for CtBP binding within the bromodomain but we show that additional site(s) exists for CBP. The interaction between CtBP and CBP is functionally important and leads to impairment of histone H3 acetylation by CBP at specific lysine residues (Lys9, Lys14, and Lys18) in a dose-dependent and NADH-dependent manner. Based on these results, we propose that CtBP1 mediates repression by blocking histone acetylation by HAT complexes.

Alcohol Oxidoreductases↗
PubMed2005

The distal zinc finger domain of AML1/MDS1/EVI1 is an oligomerization domain involved in induction of hematopoietic differentiation defects in primary cells in vitro.

AML1/MDS1/EVI1 (AME) is a chimeric transcription factor produced by the (3;21)(q26;q22) translocation. This chromosomal translocation is associated with de novo and therapy-related acute myeloid leukemia and with the blast crisis of chronic myelogenous leukemia. AME is obtained by in-frame fusion of the AML1 and MDS1/EVI1 (ME) genes. The mechanisms by which AME induces a neoplastic transformation in bone marrow cells are unknown. AME interacts with the corepressors CtBP and HDAC1, and it was shown that AME is a repressor in contrast to the parent transcription factors AML1 and ME, which are transcription activators. Studies with murine bone marrow progenitors indicated that the introduction of a point mutation that destroys the CtBP-binding consensus impairs but does not abolish the disruption of cell differentiation and replication associated with AME expression, suggesting that additional events are required. Several chimeric proteins, such as AML1/ETO, BCR/ABL, and PML/RARa, are characterized by the presence of a self-interaction domain critical for transformation. We report that AME is also able to oligomerize and displays a complex pattern of self-interaction that involves at least three oligomerization regions, one of which is the distal zinc finger domain. Although the deletion of this short domain does not preclude the self-interaction of AME, it significantly reduces the differentiation defects caused in vitro by AME in primary murine bone marrow progenitors. The addition of a point mutation that inhibits CtBP binding completely abrogates the effects of AME on differentiation, suggesting that AME induces hematopoietic differentiation defects through at least two separate but cooperating pathways.

Alcohol Oxidoreductases↗
PubMed2004

EVI1 abrogates interferon-alpha response by selectively blocking PML induction.

EVI1 is an oncogene frequently associated with chronic and acute myeloid leukemia. In hematopoietic cells, EVI1 impairs several pathways including proliferation, differentiation, and apoptosis. Interferon-alpha (IFN-alpha) is a powerful cytokine that controls the immune response and limits the expansion of several tissues including bone marrow. These properties contribute to the effectiveness of IFN-alpha in the treatment of many neoplastic disorders especially chronic myeloid leukemia. We report here that in murine hematopoietic progenitors the expression of EVI1 completely abrogates the antiproliferative and apoptotic effects of IFN-alpha. EVI1 does not repress the JAK/STAT signaling pathway or the activation of many IFN-responsive genes. On the contrary, EVI1 prolongs the phosphorylation of STAT1 and the activation of an IFN-dependent reporter gene. However, EVI1 specifically represses the IFN-dependent induction of the tumor suppressor PML and blocks the apoptotic pathways activated by PML. We show that the position of the ISRE, which is located within the first exon of PML, is critical to block PML induction by IFN-alpha. The relocation of the ISRE to a position upstream of the transcription start site is sufficient to re-establish the response to IFN in the presence of EVI1. Our data suggest that stabilized STAT1 phosphorylation and prolonged binding of the STAT1 complex to the first exon could impair PML transcription and inhibit the activation of PML-dependent apoptotic pathways resulting in loss of IFN response. These results point to a novel mechanism utilized by an oncogene to escape normal cell response to growth-controlling cytokines.

Animals↗
PubMed2004

A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic leukemia.

AML1 is among the most frequent targets of chromosomal rearrangements in human leukemias. We report here the molecular analysis of a t(4;21)(q28;q22) that has disrupted AML1 in a patient with de novo T-cell acute lymphoblastic leukemia. By using 3'-RACE analysis, we show that this rearrangement results in the fusion of a novel gene immediately downstream of exon 5 or exon 6 of AML1, indicating that the AML1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. The sequence of the novel gene, located at 4q28, does not have any significant homology with any of the known genes in the human GenBank DNA database. However, the first 118 bases are identical to a part of a human ovarian EST. Also, its high homology with mouse and rat sequences suggests that this sequence most probably represents a part of a novel gene, which we named FGA7 (Fused Gene 7 to AML1). Following the AML1 open reading frame, the FGA7 sequence encodes an unknown protein of 27 amino acids. We isolated three bacterial artificial chromosome (BAC) clones that contain the FGA7 sequence and confirmed the breakpoint of the gene on the patient's metaphase spreads by fluorescence in situ hybridization using these BACs as probes. RT-PCR and Northern blot analyses revealed that FGA7 is expressed in ovarian and skeletal muscle tissues. The predicted AML1-FGA7 chimeric proteins contained a limited number of residues fused to AML1 in a situation similar to that reported for the AML1-EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full-length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor activates.

Amino Acid Sequence↗
PubMed2004

Arsenic trioxide and thalidomide combination produces multi-lineage hematological responses in myelodysplastic syndromes patients, particularly in those with high pre-therapy EVI1 expression.

Twenty-eight myelodysplastic syndromes (MDS) patients were treated with arsenic trioxide (ATO) and thalidomide. Seven patients responded including one complete hematologic and cytogenetic response and one with regression in spleen size. Two trilineage responses were seen in patients with inv(3)(q21q26.2). Three of five patients who had high pre-therapy EVI1 levels showed unexpectedly good responses while two died early in the first cycle. In vitro studies using 32Dcl3 cells forced to express EVI1 confirmed increased sensitivity of these cells to ATO. Both low/high risk MDS may benefit significantly from therapy with ATO/thalidomide, and those with high pre-therapy EVI1 expression may be uniquely sensitive.

Aged↗
PubMed2004

Targeted therapies in myelodysplastic syndromes: ASH 2003 review.

The myelodysplastic syndromes (MDS) continue to pose conceptual and practical conundrums because of their heterogeneity and therapeutic challenges. They are not restricted to the presence of clonal cells that are prone to excessive proliferation and premature apoptosis. In MDS the bone marrow microenvironment also is abnormal and exhibits an excess of proinflammatory cytokines, especially tumor necrosis factor (TNF), neoangiogenesis, and poorly defined immune defects. Thalidomide, a drug with anti-TNF, antiangiogenic, and immunomodulatory activities, and other agents with anti-TNF effects, such as pentoxifylline, etanercept, and infliximab, have produced hematologic improvement in 20% to 40% of patients. These agents may provide effective therapy for a subset of lower-risk MDS patients, even if the drugs target the bone marrow microenvironment predominantly. However, in higher-risk MDS patients, especially those with more than 10% blasts, it is important to eliminate abnormal cell clones; drugs used for this purpose have included arsenic trioxide, topotecan, the farnesyl transferase inhibitor tipifarnib, and demethylating agents, such as 5-azacytidine and decitabine. To increase the therapeutic index, a combination strategy may be preferable for higher-risk MDS patients, in whom the seed (clone) and the soil (bone marrow microenvironment) must be targeted simultaneously. The challenge is to recognize the subset that is likely to respond to a given drug so that patients can be preselected for therapy.

Animals↗
PubMed2004

EVI1 induces myelodysplastic syndrome in mice.

Myelodysplasia is a hematological disease in which genomic abnormalities accumulate in a hematopoietic stem cell leading to severe pancytopenia, multilineage differentiation impairment, and bone marrow (BM) apoptosis. Mortality in the disease results from pancytopenia or transformation to acute myeloid leukemia. There are frequent cytogenetic abnormalities, including deletions of chromosomes 5, 7, or both. Recurring chromosomal translocations in myelodysplasia are rare, but the most frequent are the t(3;3)(q21;q26) and the inv(3)(q21q26), which lead to the inappropriate activation of the EVI1 gene located at 3q26. To better understand the role of EVI1 in this disease, we have generated a murine model of EVI1-positive myelodysplasia by BM infection and transplantation. We find that EVI1 induces a fatal disease of several stages that is characterized by severe pancytopenia. The disease does not progress to acute myeloid leukemia. Comparison of in vitro and in vivo results suggests that EVI1 acts at two levels. The immediate effects of EVI1 are hyperproliferation of BM cells and downregulation of EpoR and c-Mpl, which are important for terminal erythroid differentiation and platelet formation. These defects are not fatal, and the mice survive for about 10 months with compensated hematopoiesis. Over this time, compensation fails, and the mice succumb to fatal peripheral cytopenia.

Animals↗
PubMed2003

EVI1 promotes cell proliferation by interacting with BRG1 and blocking the repression of BRG1 on E2F1 activity.

EVI1 is a complex protein required for embryogenesis and inappropriately expressed in many types of human myeloid leukemia. Earlier we showed that the forced expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and increased proliferation. In this report, we show that EVI1 physically interacts with BRG1 and its functional homolog BRM in mammalian cells. We found that the C terminus of EVI1 interacts strongly with BRG1 and that the central and C-terminal regions of BRG1 are involved in EVI1-BRG1 interaction. Using reporter gene assays, we demonstrate that EVI1 activates the E2F1 promoter in NIH3T3 cells but not in BRG1-negative SW13 cells. Ectopic expression of BRG1 is able to repress the E2F1 promoter in vector-transfected SW13 cells but not in EVI1-transfected SW13 cells. Finally, we show that EVI1 up-regulates cell proliferation in BRG1-positive 32Dcl3 cells but not in BRG1-negative SW13 cells. Taken together, these data support the hypothesis that the interaction with BRG1 is important for up-regulation of cell-growth by EVI1.

Animals↗
PubMed2003

SUV39H1 interacts with AML1 and abrogates AML1 transactivity. AML1 is methylated in vivo.

Acute myeloid leukemia 1 (AML1) belongs to a family of DNA-binding proteins highly conserved through evolution. AML1 regulates the expression of several hematopoietic genes and is essential for murine fetal liver hematopoiesis. We report here that the histone methyltransferase SUV39H1, a mammalian ortholog of the Drosophila melanogaster SU(VAR) 3-9, forms complex with AML1. SUV39H1 methylates lysine 9 of the histone protein H3 leading to the formation of the high-affinity binding site on chromatin for proteins of the heterochromatin protein 1 family (HP1). The interaction of AML1 with SUV39H1 requires the N-terminus of AML1 where the Runt domain is located. Binding of AML1 to SUV39H1 abrogates the transactivating and DNA-binding properties of AML1 and dissociates the net-like nuclear structure of AML1. It has been reported that AML1 is capable of interaction with histone acetyl transferases (CBP, p300, and MOZ) and with component of the histone deacetylase complex (Sin3), and that the interaction with these coregulators affects the strength of AML1 in promoter regulation. Our data suggest that other enzymes are also involved in gene regulation by AML1 activity by modulating the affinity of AML1 for DNA.

3T3 Cells↗
PubMed2003

P/CAF and GCN5 acetylate the AML1/MDS1/EVI1 fusion oncoprotein.

Lysine acetyltransferases modulate the activity of many genes by modifying the lysine residues of both core histones and transcription-related factors. These modifications are tightly controlled in the cell because they are involved in vital processes such as cell cycle progression, differentiation, and apoptosis. Therefore, any deregulation of acetylation/deacetylation equilibrium or inappropriate modifications could lead to different diseases. Since previous studies have shown that some oncoproteins also undergo this modification, acetylation could be involved in the processes of cell transformation and oncogenesis. Here, we report that AML1/MDS1/EVI1 (AME), a repressor produced by the t(3;21) associated with human leukemia, physically interacts with the acetyltransferases P/CAF and GCN5. Our data suggest that AME has at least two binding sites for these acetyltransferases, one of which is in the Runt domain. Both P/CAF and GCN5 efficiently acetylate AME in vivo in the central region. AME acetylation has no effect on its interaction with the co-repressor CtBP1. Finally, we demonstrate that the co-expression of AME and either P/CAF or GCN5 abrogates the repression of an AML1-dependent reporter gene.

Acetylation↗
PubMed2003

Small ubiquitin-like modifier conjugation regulates nuclear export of TEL, a putative tumor suppressor.

Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Ras-dependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the "pointed" domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.

3T3 Cells↗
PubMed2003

Association of 3q21q26 syndrome with different RPN1/EVI1 fusion transcripts.

BACKGROUND AND OBJECTIVES: Patients with acute myeloblastic leukemia (AML) with features of myelodysplastic syndrome and abnormalities of megakaryocytopoiesis often have cytogenetic aberrations of 3q21 and 3q26 bands involving the paracentric inversion [inv(3) (q21q26)] or a reciprocal translocation [t(3;3) (q21;q26)]. These abnormalities frequently cause inappropriate expression of the EVI1 gene located at 3q26. Other genes that have been implicated at the rearrangement breakpoint are GR6 and RPN1 (both on 3q21). The aim of this study was to investigate the expression of the EVI1 fusion genes in AML patients with 3q21q26 syndrome. DESIGN AND METHODS: We used reverse transcription polymerase chain reaction to evaluate the expression of EVI1 and GR6, and particularly of the fusion genes RPN1-EVI1 and GR6-EVI1 in 9 AML patients with either inv(3)(q21q26) (7 cases) or t(3;3)(q21;q26) (2 cases). RESULTS: EVI1 and GR6 were always expressed, as was RPN1-EVI1; GR6-EVI1 was absent. In 8/9 patients, the part of EVI1 retained in RPN1-DEVI1 contained blocks B and C of the PR domain commonly found in the MDS1-EVI1 gene. In the remaining patient [with inv(3) (q21q26)], only block C was retained: we named this variant fusion gene RPN1-DEVI1. This patient lacked the micromegakaryocytopoiesis frequently found in 3q21q26 syndrome. INTERPRETATION AND CONCLUSIONS: These findings support the hypothesis that EVI1 activation plays a dominant role in the pathogenesis of the 3q21q26 syndrome. EVI1 expression might occur either as a consequence of rearrangements leading to the formation of different fusion transcripts, such as RPN1-EVI1 and RPN1-DEVI1 or following disruption of the PR activation domain of the MDS1-EVI1 gene.

Adult↗
PubMed2002

The leukemia-associated transcription repressor AML1/MDS1/EVI1 requires CtBP to induce abnormal growth and differentiation of murine hematopoietic cells.

The leukemia-associated fusion gene AML1/MDS1/EVI1 (AME) encodes a chimeric transcription factor that results from the (3;21)(q26;q22) translocation. This translocation is observed in patients with therapy-related myelodysplastic syndrome (MDS), with chronic myelogenous leukemia during the blast crisis (CML-BC), and with de novo or therapy-related acute myeloid leukemia (AML). AME is obtained by in-frame fusion of the AML1 and MDS1/EVI1 genes. We have previously shown that AME is a transcriptional repressor that induces leukemia in mice. In order to elucidate the role of AME in leukemic transformation, we investigated the interaction of AME with the transcription co-regulator CtBP1 and with members of the histone deacetylase (HDAC) family. In this report, we show that AME physically interacts in vivo with CtBP1 and HDAC1 and that these co-repressors require distinct regions of AME for interaction. By using reporter gene assays, we demonstrate that AME represses gene transcription by CtBP1-dependent and CtBP1-independent mechanisms. Finally, we show that the interaction between AME and CtBP1 is biologically important and is necessary for growth upregulation and abnormal differentiation of the murine hematopoietic precursor cell line 32Dc13 and of murine bone marrow progenitors.

Acute Disease↗