NPM-RAR Enhances NFκB Transcriptional Activity

Abstract Abstract 5117 Acute Promyelocytic Leukemia (APL) is characterized by recurrent translocations of 17q21, which result in expression of fusion proteins of the retinoic acid receptor alpha (RARα). We have been studying the t(5;17)(q35;q21) variant of APL, which fuses the N-terminal 117 amino acids of nucleophosmin (NPM) to the C-terminus of RARα. NPM-RAR is capable of binding to retinoic acid response elements (RAREs), and we have previously shown that NPM-RAR can recruit both corepressor and coactivator complexes to retinoic acid target promoters, to modulate transcription in a ligand-dependent fashion, though with a different sensitivity to ligand than RARα. The N-terminal NPM-sequence of NPM-RAR contains a dimerization domain capable of mediating not only homodimerization of the fusion protein, but also heterodimerization with wild-type NPM, to potentially modulate NPM functionality. NPM serves as a molecular chaperone, and binds to a diverse set of proteins. Amongst these are the p50 and p65 subunits of NFκB. NPM has been shown to serve as a coactivator for the NFκB transcriptional complex. We hypothesized that NPM-RAR might interact with NPM to modulate NFκB dependent transcription. We focused on NFκB regulation of the Superoxide Dismutase 2 (MnSOD, SOD2) promoter, since NPM regulation of NFκB mediated transcription has been most extensively studied in this model system. We found that NPM-RAR enhances both basal and TNFα-induced transcription of an NFκB-reporter construct (containing a minimal MnSOD promoter/enhancer) in HepG2 and Hela cells. This effect was not dependent upon translocation of NFκB to the nucleus, indicating that the positive effect on NFκB was mediated downstream of the canonical activation pathway of NFκB. The MnSOD promoter does not contain a consensus RARE, and this effect was not seen in cells transfected with RARα, suggesting that this effect is not mediated through the DNA binding motifs of the NPM-RAR C-terminal RARα sequences. NPM-RAR does not directly interact with NFκB; preliminary experiments using siRNA to down-regulate NPM indicate that NPM-RAR enhancement of MnSOD transcription is dependent upon the presence of wild-type NPM. This leads us to propose a model whereby NPM-RAR binds to the NPM/NFκB transcriptional complex through interaction with NPM, to enhance NFκB mediated transcription. Disclosures: No relevant conflicts of interest to declare.

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Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

Applied and Environmental Microbiology ◽

10.1128/aem.00791-07 ◽

2007 ◽

Vol 73 (18) ◽

pp. 5711-5715 ◽

Cited By ~ 75

Author(s):

Sung Kuk Lee ◽

Howard H. Chou ◽

Brian F. Pfleger ◽

Jack D. Newman ◽

Yasuo Yoshikuni ◽

...

Keyword(s):

Directed Evolution ◽

Cross Talk ◽

Biological Systems ◽

Wild Type ◽

Dimerization Domain ◽

Expression Levels ◽

Inducible Promoters ◽

C Terminus ◽

Increased Sensitivity ◽

Better Than

ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-β-d-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

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tau4/tau c/AF-2 of the thyroid hormone receptor relieves silencing of the retinoic acid receptor silencer core independent of both tau4 activation function and full dissociation of corepressors.

Molecular and Cellular Biology ◽

10.1128/mcb.17.8.4259 ◽

1997 ◽

Vol 17 (8) ◽

pp. 4259-4271 ◽

Cited By ~ 15

Author(s):

A Baniahmad ◽

D Thormeyer ◽

R Renkawitz

Keyword(s):

Amino Acids ◽

Retinoic Acid ◽

Thyroid Hormone ◽

Transcriptional Activation ◽

Hormone Receptors ◽

Thyroid Hormone Receptor ◽

Retinoic Acid Receptor ◽

Activation Function ◽

Acid Receptor ◽

C Terminus

Members of the thyroid hormone (TR)-retinoic acid receptor (RAR) subfamily of nuclear hormone receptors silence gene expression in the absence of hormone. Addition of cognate ligands leads to dissociation of corepressors, association of coactivators, and transcriptional activation. Here, we used the hRAR alpha silencer core, which encompasses the ligand binding domain, including receptor regions D and E of RAR alpha without the activation function called tau4/tau c/AF-2 and without the F region, to analyze the mechanisms by which transcriptional silencing is relieved. Although the RAR silencer core is able to bind ligand, it acts as a constitutive transcriptional silencer. We have fused various small activation domains to the C terminus of the silencer core and analyzed hormone-dependent changes in receptor function. We show that nine amino acids derived from the hTRbeta are sufficient to transform the RAR silencer core into a hormone-dependent activator. Lengthening the linker between the silencer core and these nine amino acids is not critical for mediating ligand-induced relief of silencing and activation. In addition, we show that a transactivation function at the C terminus is not required for relief of silencing by the hormone, but it is required for transcriptional activation. Furthermore, we created functional silencer fusions which lose their repressive function upon addition of hormone, although the corepressors SMRT and N-CoR remain attached to the receptor.

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Roles of retinoic acid receptors in early embryonic morphogenesis and hindbrain patterning

Development ◽

10.1242/dev.128.11.2031 ◽

2001 ◽

Vol 128 (11) ◽

pp. 2031-2038 ◽

Cited By ~ 4

Author(s):

Olivia Wendling ◽

Norbert B. Ghyselinck ◽

Pierre Chambon ◽

Manuel Mark

Keyword(s):

Retinoic Acid ◽

Retinoic Acid Receptor ◽

Axial Rotation ◽

Retinoic Acid Receptors ◽

Retinoic Acid Signaling ◽

Wild Type ◽

Pharyngeal Arches ◽

Embryonic Morphogenesis ◽

Null Mutants

Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) α and RARγ (Aα/Aγ mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that we previously observed during the fetal period. We show that embryonic day (E)9.5 Aα/Aγ embryos display severe malformations, similar to those already described in retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds; and in the closure of the primitive gut. The hindbrain of E8.5 Aα/Aγ embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RARα and RARβ (Aα/Aβmutants), in which we have previously shown that the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an Aα/Aβ-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an Aα/Aγ-like phenotype. Altogether, our data suggest that RARα and/or RARγ transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RARβ is subsequently involved in setting up the caudal boundary of this territory.

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The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties

Blood ◽

10.1182/blood.v95.8.2683.008k29_2683_2690 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2683-2690 ◽

Cited By ~ 2

Author(s):

Robert L. Redner ◽

J. Don Chen ◽

Elizabeth A. Rush ◽

Hui Li ◽

Sheri L. Pollock

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Transcriptional Activation ◽

Rna Splicing ◽

Fusion Proteins ◽

Retinoic Acid Receptor ◽

Promyelocytic Leukemia ◽

Dominant Negative ◽

Wild Type ◽

Retinoic Acid Response Element

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.

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A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

Molecular and Cellular Biology ◽

10.1128/mcb.14.12.8399 ◽

1994 ◽

Vol 14 (12) ◽

pp. 8399-8407 ◽

Cited By ~ 109

Author(s):

J Flach ◽

M Bossie ◽

J Vogel ◽

A Corbett ◽

T Jinks ◽

...

Keyword(s):

Nuclear Protein ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Binding Protein ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Binding Motifs ◽

C Terminus ◽

N Terminus ◽

Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

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Intercellular crosstalk regulating ARRB2/RARRES1 is involved in transition from fibrosis to cancer

10.1101/2021.09.08.458161 ◽

2021 ◽

Author(s):

Robert Schierwagen ◽

Peter Dietrich ◽

Judith Heinzen ◽

Sabine Klein ◽

Frank E. Uschner ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Retinoic Acid ◽

Liver Tumors ◽

Retinoic Acid Receptor ◽

Cancer Development ◽

Wild Type ◽

Chronic Liver Injury ◽

Deficient Mice

AbstractProgressive fibrogenesis in chronic liver injury is often associated with cancer development. Beta-arrestin-2 (ARRB2) is a regulator of the profibrotic Angiotensin II type 1 receptor (AGTR1). The role of ARRB2 in liver fibrosis and in the transition from fibrosis to cancer is not fully understood and was investigated in this study.This study demonstrates that upregulation of the retinoic acid receptor responder 1 (RARRES1) in HSC mediated by ARRB2 leads to fibrosis. This process is driven by exosomal ARRB2 transfer to HSC, major fibrosis contributors, from injured hepatocytes, which highly express ARRB2. By contrast, downregulation of RARRES1 in hepatocytes induces malignant transformation and hepatocellular carcinoma (HCC) development. Consequently, Arrb2-deficient mice show higher number and size of liver tumors than wild-type mice in a hepatocellular carcinoma model with fibrosis. The identified relationship between ARRB2 and RARRES1 was observed in at least two species, including human cells and tissues in fibrosis and HCC and has a predictive value for survival in cancer patients. This study describes the discovery of a novel molecular pathway mediating the transition from fibrosis to cancer offering potential diagnostics and therapeutics.

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Four SR Proteins Play Opposing Roles in the Regulated Biosynthesis of Tissue Factor in Human Monocytic Cells.

Blood ◽

10.1182/blood.v114.22.2128.2128 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2128-2128

Author(s):

Sajiv Chandradas ◽

Gintaras Deikus ◽

Jonathan G. Tardos ◽

David H Bechhofer ◽

Vladimir Y Bogdanov

Keyword(s):

Complex Formation ◽

Tissue Factor ◽

Binding Sites ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Sr Proteins ◽

Human Monocytes ◽

Wild Type ◽

Binding Motifs ◽

Rna Probes

Abstract Abstract 2128 Poster Board II-105 Background. Circulating Tissue Factor (TF) is a major contributor to the etiology of thrombotic disorders. Blood monocytes are the primary source of circulating TF, which they express in two forms: full length TF (flTF), a transmembrane surface protein, and alternatively spliced TF (asTF), a secreted soluble protein. The presence or absence of the internal cassette exon 5 in the TF mRNA determines whether the encoded protein is flTF or asTF, respectively. While the procoagulant potential of flTF vastly exceeds that of asTF when assessed using conventional static assays, asTF exhibits unique angiogenic properties distinct from flTF. Investigation of the molecular mechanisms governing TF exon 5 processing is of much interest as it may afford novel approaches to modulate monocyte-mediated blood thrombogenicity as well as monocyte-induced angiogenesis. Using a splicing reporter system developed to study the processing of TF pre-mRNA, we previously determined that SR proteins ASF/SF2 and SRp55 are vital for TF exon 5 inclusion in human monocytes (Tardos et al, J Thromb Haemost 6:877-884, 2008). In the course of more recent experiments, we found that in contrast to ASF/SF2 and SRp55, two other SR proteins – SC35 and SRp40 – appear to promote exclusion of this variable exon: weakening of the binding motifs for SC35 (sites 33 and 81) and SRp40 (site 44), whose positions in exon 5 overlap with the binding sites for ASF/SF2 and SRp55, results in the decrease of the splicing event unique to asTF. The competition of these four SR proteins for binding to their overlapping binding sites may thus play a role in the maintenance of the asTF / flTF ratio; however, a physical association of SC35 and SRp40 with their putative sites in exon 5 was not demonstrated. Objective. To determine whether SC35 and/or SRp40 physically associate with their putative binding sites. Results. To evaluate interaction of the SR proteins SC35 and SRp40 with their putative binding sites in TF exon 5, we employed RNA mobility shift assay methodology using freshly prepared nuclear extracts of THP-1 cells, a monocytic cell line, and in vitro transcribed, uniformly labeled RNA probes comprising the two regions of interest – one spanning SC35 site 33 and SRp40 site 44, and the other spanning the SC35 site 81. As expected, these RNA probes yielded reproducible band shift products, confirming RNA-protein complex formation. We then developed three counterpart mutant RNA probes in which the SC35 and SRp40 binding motifs were selectively weakened by targeted site-directed mutagenesis, and performed RNA gel shift assays alongside the corresponding wild-type probes. Each mutant probe exhibited a significantly weaker interaction with the SR proteins compared to its wild-type counterpart: the SC35 site 33 mutant probe produced a 31.1% reduction in complex formation relative to wild-type (p = 0.011), the SRp40 site 44 mutant probe also produced a 31.1% reduction in complex formation relative to wild-type (p = 0.0001), and the SC35 site 81 mutant probe produced a 33.0% reduction in complex formation relative to wild-type (p = 0.0151). Conclusions. We show for the first time that SR proteins SC35 and SRp40 physically associate with functional binding sites within TF exon 5. The SC35 and SRp40 binding sites overlap with the binding sites for ASF/SF2 and SRp55, the SR proteins that promote exon 5 inclusion. The opposing effects of distinct SR proteins on TF exon 5 processing reveal a heretofore unknown mechanism governing regulated TF biosynthesis. Further studies of SR protein-mediated effects on the TF profile of human monocytes are likely to aid in the development of novel therapeutic strategies aimed at selective targeting of biologically distinct TF forms. Disclosures: No relevant conflicts of interest to declare.

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A Therapeutic TCR Mimic Monoclonal Antibody for Intracellular PRAME Protein in Leukemias

Blood ◽

10.1182/blood.v126.23.2527.2527 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2527-2527 ◽

Cited By ~ 2

Author(s):

Aaron Chang ◽

Tao Dao ◽

Andrew Scott ◽

Leonid Dubrovsky ◽

Cheng Liu ◽

...

Keyword(s):

Monoclonal Antibody ◽

Retinoic Acid ◽

Conflicts Of Interest ◽

Specific Binding ◽

Retinoic Acid Receptor ◽

Pharmacokinetic Studies ◽

Breast Cancers ◽

Biodistribution Studies

Abstract Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics. Disclosures No relevant conflicts of interest to declare.

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Retinoic Acid Receptor Alpha Expression Drives Cell-Cycle Progression and Is Associated with Increased Sensitivity to Retinoids in Peripheral T-Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.1749.1749 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1749-1749

Author(s):

Rebecca L Boddicker ◽

Xueju Wang ◽

Surendra Dasari ◽

Grzegorz S. Nowakowski ◽

Konstantinos N Lazaridis ◽

...

Keyword(s):

Cell Cycle ◽

Retinoic Acid ◽

T Cell ◽

Cell Growth ◽

Cell Lines ◽

Research Funding ◽

Retinoic Acid Receptor ◽

Wild Type ◽

T Cell Lymphomas

Abstract Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined. Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells. Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p<0.001), with a greater increase observed from mutant versus wild-type RARA in Karpas 299 (136% of control versus 122%; p=0.04). Accordingly, knockdown of wild-type RARA in the RARAhigh cell line, Mac-1, resulted in a 22% inhibition of cell growth (p=0.0002). This inhibition specifically was associated with G1 cell cycle arrest (120% of control; p=0.004) and decreased protein expression of the G1-S-associated cyclin-dependent kinases, CDK2, CDK4, and CDK6. These kinases were up-regulated by overexpression of RARA in RARAlow HuT78 cells. The relatively RARA-specific retinoid, AM80 (tamibarotene), and the less specific retinoid, all-trans retinoic acid (ATRA), resulted in RARA protein degradation, cell growth inhibition that was both dose-dependent and proportional to baseline RARA expression, G1 arrest, and CDK protein up-regulation. Gene-set enrichment analysis (GSEA) of transcriptome data confirmed that genes down-regulated by AM80 were highly enriched for regulators of cell cycle and particularly G1-S transition. Finally, overexpressing RARA in RARAlow Karpas 299 and HuT78 cell lines significantly increased the ability of AM80 to inhibit CDK2/4/6 expression and cell growth (16% to 23% greater growth inhibition than control; p<0.05). Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. Disclosures Nowakowski: Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

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The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties

Blood ◽

10.1182/blood.v95.8.2683 ◽

2000 ◽

Vol 95 (8) ◽

pp. 2683-2690 ◽

Cited By ~ 43

Author(s):

Robert L. Redner ◽

J. Don Chen ◽

Elizabeth A. Rush ◽

Hui Li ◽

Sheri L. Pollock

Keyword(s):

Retinoic Acid ◽

Acute Promyelocytic Leukemia ◽

Transcriptional Activation ◽

Rna Splicing ◽

Fusion Proteins ◽

Retinoic Acid Receptor ◽

Promyelocytic Leukemia ◽

Dominant Negative ◽

Wild Type ◽

Retinoic Acid Response Element

Abstract The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.

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