Publications by authors named "Urban Gullberg"

52 Publications

Depletion of the transcriptional coactivators CREB-binding protein or EP300 downregulates CD20 in diffuse large B-cell lymphoma cells and impairs the cytotoxic effects of anti-CD20 antibodies.

Exp Hematol 2019 11 25;79:35-46.e1. Epub 2019 Oct 25.

Department of Hematology and Transfusion Medicine, Lund University, Lund, Sweden; Clinic of Oncology, Skåne University Hospital, Lund, Sweden. Electronic address:

Monoclonal antibodies targeting CD20 are central in the treatment of B-cell lymphomas. In diffuse large B-cell lymphoma (DLBCL), inactivating mutations of the histone acetyltransferases CREB-binding protein (CBP) and EP300 are common. Moreover, knockdown of CBP in DLBCL has been shown to result in aberrant transcriptional silencing. Expression of CD20 is sensitive to epigenetic manipulation, and histone deacetylase inhibitors have been found to potentiate treatment with anti-CD20 antibodies. Therefore, we studied the role of CBP and EP300 depletion on CD20 expression and effects of the anti-CD20 antibodies rituximab and obinutuzumab in DLBCL cells. Levels of CBP and EP300 were reduced by shRNA in the germinal centre-derived diffuse large B-cell lymphoma cell line SU-DHL4. The levels of CD20 mRNA and protein were determined by quantitative polymerase chain reaction, Western blot, and flow cytometry. Binding of the transcription factors PU.1 and FOXO1 to the CD20 promoter was determined by chromatin immunoprecipitation coupled with quantitative polymerase chain reaction. Response to the monoclonal anti-CD20 antibodies rituximab and obinutuzumab in CBP- or EP300-depleted cells was assessed by complement-dependent cell death, direct cell death, and antibody-dependent cellular cytotoxicity (ADCC). Our results suggest that depletion of CBP and EP300 levels leads to a strong reduction of CD20 expression, accompanied by reduced binding of PU.1 to the CD20 promoter. In CBP-depleted, but not EP300-depleted cells, increased binding of FOXO1 to the CD20 promoter was observed. Interestingly, CBP or EP300 depletion leads to decreased complement-dependent cell death and direct cell death in response to rituximab and obinutuzumab, which was most pronounced in response to rituximab in CBP-depleted cells. Our data suggest that inactivating mutations of CBP, and to a lesser extent EP300, may impair the response to anti-CD20 antibodies. However, these observations should be analyzed in future clinical trials.
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http://dx.doi.org/10.1016/j.exphem.2019.10.004DOI Listing
November 2019

Author Correction: Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma.

Nat Commun 2019 01 10;10(1):213. Epub 2019 Jan 10.

Institute for Medical Informatics, Biometry and Epidemiology, University Hospital Essen, University of Duisburg-Essen, Essen, D-45147, Germany.

The original version of this Article contained an error in the spelling of a member of the PRACTICAL Consortium, Manuela Gago-Dominguez, which was incorrectly given as Manuela Gago Dominguez. This has now been corrected in both the PDF and HTML versions of the Article. Furthermore, in the original HTML version of this Article, the order of authors within the author list was incorrect. The PRACTICAL consortium was incorrectly listed after Richard S. Houlston and should have been listed after Nora Pashayan. This error has been corrected in the HTML version of the Article; the PDF version was correct at the time of publication.
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http://dx.doi.org/10.1038/s41467-018-08107-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328616PMC
January 2019

Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma.

Nat Commun 2018 09 13;9(1):3707. Epub 2018 Sep 13.

Institute for Medical Informatics, Biometry and Epidemiology, University Hospital Essen, University of Duisburg-Essen, Essen, D-45147, Germany.

Genome-wide association studies (GWAS) have transformed our understanding of susceptibility to multiple myeloma (MM), but much of the heritability remains unexplained. We report a new GWAS, a meta-analysis with previous GWAS and a replication series, totalling 9974 MM cases and 247,556 controls of European ancestry. Collectively, these data provide evidence for six new MM risk loci, bringing the total number to 23. Integration of information from gene expression, epigenetic profiling and in situ Hi-C data for the 23 risk loci implicate disruption of developmental transcriptional regulators as a basis of MM susceptibility, compatible with altered B-cell differentiation as a key mechanism. Dysregulation of autophagy/apoptosis and cell cycle signalling feature as recurrently perturbed pathways. Our findings provide further insight into the biological basis of MM.
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http://dx.doi.org/10.1038/s41467-018-04989-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137048PMC
September 2018

The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression.

Nat Commun 2018 04 25;9(1):1649. Epub 2018 Apr 25.

Department of Laboratory Medicine, Hematology and Transfusion Medicine, SE 221 84, Lund, Sweden.

Recently, we identified ELL2 as a susceptibility gene for multiple myeloma (MM). To understand its mechanism of action, we performed expression quantitative trait locus analysis in CD138 plasma cells from 1630 MM patients from four populations. We show that the MM risk allele lowers ELL2 expression in these cells (P = 2.5 × 10; β = -0.24 SD), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause-effect relationship. Our results provide mechanistic insight into MM predisposition.
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http://dx.doi.org/10.1038/s41467-018-04082-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917026PMC
April 2018

Direct evidence for a polygenic etiology in familial multiple myeloma.

Blood Adv 2017 Apr 7;1(10):619-623. Epub 2017 Apr 7.

Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden.

Although common risk alleles for multiple myeloma (MM) were recently identified, their contribution to familial MM is unknown. Analyzing 38 familial cases identified primarily by linking Swedish nationwide registries, we demonstrate an enrichment of common MM risk alleles in familial compared with 1530 sporadic cases ( = 4.8 × 10 and 6.0 × 10, respectively, for 2 different polygenic risk scores) and 10 171 population-based controls ( = 1.5 × 10 and 1.3 × 10, respectively). Using mixture modeling, we estimate that about one-third of familial cases result from such enrichments. Our results provide the first direct evidence for a polygenic etiology in a familial hematologic malignancy.
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http://dx.doi.org/10.1182/bloodadvances.2016003111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728350PMC
April 2017

DNA and RNA binding by the Wilms' tumour gene 1 (WT1) protein +KTS and -KTS isoforms-From initial observations to recent global genomic analyses.

Eur J Haematol 2018 Mar 10;100(3):229-240. Epub 2018 Jan 10.

Department of Haematology and Transfusion Medicine, Lund University, Lund, Sweden.

The Wilms' tumour gene 1 protein (WT1) is a zinc finger transcription factor found indispensable for foetal development. WT1 has also been implicated in the development of tumours in several organ systems, including acute myeloid leukaemia (AML). Four main WT1 isoforms come from 2 alternative splice events. One alternative splice results in the inclusion or exclusion of 3 amino acids, KTS, between zinc fingers 3 and 4 in the WT1 protein. The KTS insert has been extensively investigated due to the functional implications for DNA and RNA binding. In this review, we provide an overview of the research into the isoforms containing or lacking the KTS insert in leukaemic cells, as well as the research into the binding patterns of the WT1 -KTS and +KTS isoforms to DNA and RNA. Finally, we connect the results of the DNA binding research to the ChIP-CHIP and ChIP-Seq investigations into the global genomic binding of the WT1 protein that have recently been performed.
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http://dx.doi.org/10.1111/ejh.13010DOI Listing
March 2018

The transcriptional coregulator is a target gene for the Wilms' tumor gene 1 protein (WT1) in leukemic cells.

Oncotarget 2017 Oct 3;8(50):87136-87150. Epub 2017 Aug 3.

Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden.

The Wilms' tumor gene 1 () is recurrently mutated in acute myeloid leukemia. Mutations and high expression of associate with a poor prognosis. In mice, WT1 cooperates with the () fusion gene in the induction of acute leukemia, further emphasizing a role for WT1 in leukemia development. Molecular mechanisms for WT1 are, however, incompletely understood. Here, we identify the transcriptional coregulator as a target gene of WT1. Analysis of gene expression profiles of leukemic samples revealed a positive correlation between the expression of and , as well as a non-zero partial correlation. Overexpression of in hematopoietic cells resulted in increased levels, while suppression of decreased expression. WT1 bound and transactivated the proximal promoter, as shown by ChIP and reporter experiments, respectively. ChIP experiments also revealed that WT1 can recruit NAB2 to the promoter, thus modulating the transcriptional activity of WT1, as shown by reporter experiments. Our results implicate as a previously unreported target gene of WT1 and that NAB2 acts as a transcriptional cofactor of WT1.
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http://dx.doi.org/10.18632/oncotarget.19896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675621PMC
October 2017

Identification of sequence variants influencing immunoglobulin levels.

Nat Genet 2017 Aug 19;49(8):1182-1191. Epub 2017 Jun 19.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, the Netherlands.

Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10, β = -0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10, β = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.
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http://dx.doi.org/10.1038/ng.3897DOI Listing
August 2017

The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo.

Oncotarget 2017 Jun;8(23):37409-37422

Department of Hematology and Transfusion Medicine, Lund University, Lund, Sweden.

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies.
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http://dx.doi.org/10.18632/oncotarget.16964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514918PMC
June 2017

Anti-apoptotic quinolinate phosphoribosyltransferase (QPRT) is a target gene of Wilms' tumor gene 1 (WT1) protein in leukemic cells.

Biochem Biophys Res Commun 2017 Jan 23;482(4):802-807. Epub 2016 Nov 23.

Department of Hematology and Transfusion Medicine, Lund University, Lund, Sweden. Electronic address:

Wilms' tumor gene 1 (WT1) is a zinc finger transcription factor that has been implicated as an oncogene in leukemia and several other malignancies. When investigating possible gene expression network partners of WT1 in a large acute myeloid leukemia (AML) patient cohort, one of the genes with the highest correlation to WT1 was quinolinate phosphoribosyltransferase (QPRT), a key enzyme in the de novo nicotinamide adenine dinucleotide (NAD+) synthesis pathway. To investigate the possible relationship between WT1 and QPRT, we overexpressed WT1 in hematopoietic progenitor cells and cell lines, resulting in an increase of QPRT expression. WT1 knock-down gave a corresponding decrease in QPRT gene and protein expression. Chromatin-immunoprecipitation revealed WT1 binding to a conserved site in the first intron of the QPRT gene. Upon overexpression in leukemic K562 cells, QPRT conferred partial resistance to the anti-leukemic drug imatinib, indicating possible anti-apoptotic functions, consistent with previous reports on glioma cells. Interestingly, the rescue effect of QPRT overexpression was not correlated to increased NAD + levels, suggesting NAD + independent mechanisms. We conclude that QPRT, encoding a protein with anti-apoptotic properties, is a novel and direct target gene of WT1 in leukemic cells.
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http://dx.doi.org/10.1016/j.bbrc.2016.11.114DOI Listing
January 2017

Distinct global binding patterns of the Wilms tumor gene 1 (WT1) -KTS and +KTS isoforms in leukemic cells.

Haematologica 2017 02 9;102(2):336-345. Epub 2016 Sep 9.

Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Huddinge, Sweden

The zinc finger transcription factor Wilms tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia. A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (±KTS), is believed to change the DNA binding affinity of WT1, although there are conflicting data regarding the binding affinity and motifs of the different isoforms. Increased expression of the WT1 -KTS isoform at the expense of the WT1 +KTS isoform is associated with poor prognosis in acute myeloid leukemia. We determined the genome-wide binding pattern of WT1 -KTS and WT1 +KTS in leukemic K562 cells by chromatin immunoprecipitation and deep sequencing. We discovered that the WT1 -KTS isoform predominantly binds close to transcription start sites and to enhancers, in a similar fashion to other transcription factors, whereas WT1 +KTS binding is enriched within gene bodies. We observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. Additional analyses showed that both WT1 -KTS and WT1 +KTS target genes are more likely to be transcribed than non-targets, and are involved in cell proliferation, cell death, and development. Our study provides evidence that WT1 -KTS and WT1 +KTS share target genes yet still bind distinct locations, indicating isoform-specific regulation in transcription of genes related to cell proliferation and differentiation, consistent with the involvement of WT1 in acute myeloid leukemia.
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http://dx.doi.org/10.3324/haematol.2016.149815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286941PMC
February 2017

Genome-wide association study identifies multiple susceptibility loci for multiple myeloma.

Nat Commun 2016 07 1;7:12050. Epub 2016 Jul 1.

Division of Genetics and Epidemiology, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK.

Multiple myeloma (MM) is a plasma cell malignancy with a significant heritable basis. Genome-wide association studies have transformed our understanding of MM predisposition, but individual studies have had limited power to discover risk loci. Here we perform a meta-analysis of these GWAS, add a new GWAS and perform replication analyses resulting in 9,866 cases and 239,188 controls. We confirm all nine known risk loci and discover eight new loci at 6p22.3 (rs34229995, P=1.31 × 10(-8)), 6q21 (rs9372120, P=9.09 × 10(-15)), 7q36.1 (rs7781265, P=9.71 × 10(-9)), 8q24.21 (rs1948915, P=4.20 × 10(-11)), 9p21.3 (rs2811710, P=1.72 × 10(-13)), 10p12.1 (rs2790457, P=1.77 × 10(-8)), 16q23.1 (rs7193541, P=5.00 × 10(-12)) and 20q13.13 (rs6066835, P=1.36 × 10(-13)), which localize in or near to JARID2, ATG5, SMARCD3, CCAT1, CDKN2A, WAC, RFWD3 and PREX1. These findings provide additional support for a polygenic model of MM and insight into the biological basis of tumour development.
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http://dx.doi.org/10.1038/ncomms12050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932178PMC
July 2016

The hematopoietic tumor suppressor interferon regulatory factor 8 (IRF8) is upregulated by the antimetabolite cytarabine in leukemic cells involving the zinc finger protein ZNF224, acting as a cofactor of the Wilms' tumor gene 1 (WT1) protein.

Leuk Res 2016 Jan 2;40:60-7. Epub 2015 Nov 2.

Department of Hematology and Transfusion Medicine, Medical Faculty, University of Lund, Lund, Sweden. Electronic address:

The transcription factor interferon regulatory factor-8 (IRF8) is highly expressed in myeloid progenitors, while most myeloid leukemias show low or absent expression. Loss of IRF8 in mice leads to a myeloproliferative disorder, indicating a tumor-suppressive role of IRF8. The Wilms tumor gene 1 (WT1) protein represses the IRF8-promoter. The zinc finger protein ZNF224 can act as a transcriptional co-factor of WT1 and potentiate the cytotoxic response to the cytostatic drug cytarabine. We hypothesized that cytarabine upregulates IRF8 and that transcriptional control of IRF8 involves WT1 and ZNF224. Treatment of leukemic K562 cells with cytarabine upregulated IRF8 protein and mRNA, which was correlated to increased expression of ZNF224. Knock down of ZNF224 with shRNA suppressed both basal and cytarabine-induced IRF8 expression. While ZNF224 alone did not affect IRF8 promoter activity, ZNF224 partially reversed the suppressive effect of WT1 on the IRF8 promoter, as judged by luciferase reporter experiments. Coprecipitation revealed nuclear binding of WT1 and ZNF224, and by chromatin immunoprecipitation (ChIP) experiments it was demonstrated that WT1 recruits ZNF224 to the IRF8 promoter. We conclude that cytarabine-induced upregulation of the IRF8 in leukemic cells involves increased levels of ZNF224, which can counteract the repressive activity of WT1 on the IRF8-promoter.
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http://dx.doi.org/10.1016/j.leukres.2015.10.014DOI Listing
January 2016

WT1-mediated repression of the proapoptotic transcription factor ZNF224 is triggered by the BCR-ABL oncogene.

Oncotarget 2015 Sep;6(29):28223-37

Department of Molecular Medicine and Medical Biotechnology University of Naples Federico II, Naples, Italy.

The Kruppel-like protein ZNF224 is a co-factor of the Wilms' tumor 1 protein, WT1. We have previously shown that ZNF224 exerts a specific proapoptotic role in chronic myelogenous leukemia (CML) K562 cells and contributes to cytosine arabinoside-induced apoptosis, by modulating WT1-dependent transcription of apoptotic genes. Here we demonstrate that ZNF224 gene expression is down-regulated both in BCR-ABL positive cell lines and in primary CML samples and is restored after imatinib and second generation tyrosine kinase inhibitors treatment. We also show that WT1, whose expression is positively regulated by BCR-ABL, represses transcription of the ZNF224 gene. Finally, we report that ZNF224 is significantly down-regulated in patients with BCR-ABL positive chronic phase-CML showing poor response or resistance to imatinib treatment as compared to high-responder patients. Taken as a whole, our data disclose a novel pathway activated by BCR-ABL that leads to inhibition of apoptosis through the ZNF224 repression. ZNF224 could thus represent a novel promising therapeutic target in CML.
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http://dx.doi.org/10.18632/oncotarget.4950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695056PMC
September 2015

Variants in ELL2 influencing immunoglobulin levels associate with multiple myeloma.

Nat Commun 2015 May 26;6:7213. Epub 2015 May 26.

Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, BMC B13, SE-221 84 Lund, Sweden.

Multiple myeloma (MM) is characterized by an uninhibited, clonal growth of plasma cells. While first-degree relatives of patients with MM show an increased risk of MM, the genetic basis of inherited MM susceptibility is incompletely understood. Here we report a genome-wide association study in the Nordic region identifying a novel MM risk locus at ELL2 (rs56219066T; odds ratio (OR)=1.25; P=9.6 × 10(-10)). This gene encodes a stoichiometrically limiting component of the super-elongation complex that drives secretory-specific immunoglobulin mRNA production and transcriptional regulation in plasma cells. We find that the MM risk allele harbours a Thr298Ala missense variant in an ELL2 domain required for transcription elongation. Consistent with a hypomorphic effect, we find that the MM risk allele also associates with reduced levels of immunoglobulin A (IgA) and G (IgG) in healthy subjects (P=8.6 × 10(-9) and P=6.4 × 10(-3), respectively) and, potentially, with an increased risk of bacterial meningitis (OR=1.30; P=0.0024).
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http://dx.doi.org/10.1038/ncomms8213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4455110PMC
May 2015

Myeloid translocation gene-16 co-repressor promotes degradation of hypoxia-inducible factor 1.

PLoS One 2015 14;10(5):e0123725. Epub 2015 May 14.

Department of Hematology, Lund University, Lund, Sweden.

The myeloid translocation gene 16 (MTG16) co-repressor down regulates expression of multiple glycolytic genes, which are targets of the hypoxia-inducible factor 1 (HIF1) heterodimer transcription factor that is composed of oxygen-regulated labile HIF1α and stable HIF1β subunits. For this reason, we investigated whether MTG16 might regulate HIF1 negatively contributing to inhibition of glycolysis and stimulation of mitochondrial respiration. A doxycycline Tet-On system was used to control levels of MTG16 in B-lymphoblastic Raji cells. Results from co-association studies revealed MTG16 to interact with HIF1α. The co-association required intact N-terminal MTG16 residues including Nervy Homology Region 1 (NHR1). Furthermore, electrophoretic mobility shift assays demonstrated an association of MTG16 with hypoxia response elements (HREs) in PFKFB3, PFKFB4 and PDK1 promoters in-vitro. Results from chromatin immunoprecipitation assays revealed co-occupancy of these and other glycolytic gene promoters by HIF1α, HIF1β and MTG16 in agreement with possible involvement of these proteins in regulation of glycolytic target genes. In addition, MTG16 interacted with prolyl hydroxylase D2 and promoted ubiquitination and proteasomal degradation of HIF1α. Our findings broaden the area of MTG co-repressor functions and reveal MTG16 to be part of a protein complex that controls the levels of HIF1α.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123725PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4431712PMC
April 2016

Pharmacologically relevant doses of valproate upregulate CD20 expression in three diffuse large B-cell lymphoma patients in vivo.

Exp Hematol Oncol 2015 26;4. Epub 2015 Jan 26.

Department of Hematology and Transfusion Medicine, Lund University, Lund, Sweden.

Background: Epigenetic code modifications by histone deacetylase inhibitors (HDACi) have been proposed as potential new therapies for lymphoid malignancies. Diffuse large B-cell lymphoma (DLBCL) is the most common type of aggressive lymphoma for which standard first line treatment is the chemotherapy regimen CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) combined with the monoclonal anti-CD20 antibody rituximab (R-CHOP). The HDACi valproate, which has for long been utilized in anti-convulsive therapy, has been shown to sensitize to chemotherapy in vitro. Valproate upregulates expression of CD20 in lymphoma cell lines; therefore, 48 hour pre-treatment with valproate before first line R-CHOP in DLBCL stages II-IV is evaluated in the phase I clinical trial VALFRID; Valproate as First line therapy in combination with Rituximab and CHOP in Diffuse large B-cell lymphoma.

Findings: Pretreatment with valproate at oral doses comparable to anti-convulsive therapy, resulted in upregulation of CD20 mRNA and CD20 protein on the cell surface as measured by qPCR and FACS analysis in lymphoma biopsies from three evaluated patients from the VALFRID study. Valproate-treatment corresponded to increased acetylation of Histone3Lysine9 (H3K9ac) in peripheral blood mononuclear cells (PBMCs), which were employed as surrogate tissue for valproate-related epigenetic modifications.

Conclusions: Valproate treatment at pharmacologically relevant doses resulted in upregulation of CD20 in vivo, and also in expected epigenetic modifications. This suggests that pre-treatment with valproate or other HDACis before anti-CD20 therapy could be advantageous in CD20-low B-cell lymphomas. Further studies are warranted to evaluate this conclusion.
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http://dx.doi.org/10.1186/2162-3619-4-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429466PMC
May 2015

The DEK oncoprotein is upregulated by multiple leukemia-associated fusion genes.

Blood Cells Mol Dis 2015 Mar 25;54(3):284-5. Epub 2014 Nov 25.

Department of Hematology, Lund University, Lund, Sweden.

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http://dx.doi.org/10.1016/j.bcmd.2014.11.014DOI Listing
March 2015

The DEK oncoprotein binds to highly and ubiquitously expressed genes with a dual role in their transcriptional regulation.

Mol Cancer 2014 Sep 12;13:215. Epub 2014 Sep 12.

Department of Hematology, Lund University, BMC B13, Klinikgatan 26, 221 84 Lund, Sweden.

Background: The DEK gene is highly expressed in a wide range of cancer cells, and a recurrent translocation partner in acute myeloid leukemia. While DEK has been identified as one of the most abundant proteins in human chromatin, its function and binding properties are not fully understood.

Methods: We performed ChIP-seq analysis in the myeloid cell line U937 and coupled it with epigenetic and gene expression analysis to explore the genome-wide binding pattern of DEK and its role in gene regulation.

Results: We show that DEK preferentially binds to open chromatin, with a low degree of DNA methylation and scarce in the heterochromatin marker H3K9me(3) but rich in the euchromatin marks H3K4me(2/3), H3K27ac and H3K9ac. More specifically, DEK binding is predominantly located at the transcription start sites of highly transcribed genes and a comparative analysis with previously established transcription factor binding patterns shows a similarity with that of RNA polymerase II. Further bioinformatic analysis demonstrates that DEK mainly binds to genes that are ubiquitously expressed across tissues. The functional significance of DEK binding was demonstrated by knockdown of DEK by shRNA, resulting in both significant upregulation and downregulation of DEK-bound genes.

Conclusions: We find that DEK binds to transcription start sites with a dual role in activation and repression of highly and ubiquitously expressed genes.
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http://dx.doi.org/10.1186/1476-4598-13-215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175287PMC
September 2014

Robust isolation of malignant plasma cells in multiple myeloma.

Blood 2014 Feb 2;123(9):1336-40. Epub 2014 Jan 2.

Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Sweden;

Molecular characterization of malignant plasma cells is increasingly important for diagnostic and therapeutic stratification in multiple myeloma. However, the malignant plasma cells represent a relatively small subset of bone marrow cells, and need to be enriched prior to analysis. Currently, the cell surface marker CD138 (SDC1) is used for this enrichment, but has an important limitation in that its expression decreases rapidly after sampling. Seeking alternatives to CD138, we performed a computational screen for myeloma plasma cell markers and systematically evaluated 7 candidates. Our results conclusively show that the markers CD319 (SLAMF7/CS1) and CD269 (TNFRSF17/BCMA) are considerably more robust than CD138 and enable isolation of myeloma plasma cells under more diverse conditions, including the samples that have been delayed or frozen. Our results form the basis of improved procedures for characterizing cases of multiple myeloma in clinical practice.
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http://dx.doi.org/10.1182/blood-2013-09-529800DOI Listing
February 2014

Forced expression of the DEK-NUP214 fusion protein promotes proliferation dependent on upregulation of mTOR.

BMC Cancer 2013 Sep 27;13:440. Epub 2013 Sep 27.

Department of Hematology, Lund University, BMC B13, Klinikgatan 26, 221 84 Lund, Sweden.

Background: The t(6;9)(p23;q34) chromosomal translocation is found in 1% of acute myeloid leukemia and encodes the fusion protein DEK-NUP214 (formerly DEK-CAN) with largely uncharacterized functions.

Methods: We expressed DEK-NUP214 in the myeloid cell lines U937 and PL-21 and studied the effects on cellular functions.

Results: In this study, we demonstrate that expression of DEK-NUP214 increases cellular proliferation. Western blot analysis revealed elevated levels of one of the key proteins regulating proliferation, the mechanistic target of rapamycin, mTOR. This conferred increased mTORC1 but not mTORC2 activity, as determined by the phosphorylation of their substrates, p70 S6 kinase and Akt. The functional importance of the mTOR upregulation was determined by assaying the downstream cellular processes; protein synthesis and glucose metabolism. A global translation assay revealed a substantial increase in the translation rate and a metabolic assay detected a shift from glycolysis to oxidative phosphorylation, as determined by a reduction in lactate production without a concomitant decrease in glucose consumption. Both these effects are in concordance with increased mTORC1 activity. Treatment with the mTORC1 inhibitor everolimus (RAD001) selectively reversed the DEK-NUP214-induced proliferation, demonstrating that the effect is mTOR-dependent.

Conclusions: Our study shows that the DEK-NUP214 fusion gene increases proliferation by upregulation of mTOR, suggesting that patients with leukemias carrying DEK-NUP214 may benefit from treatment with mTOR inhibitors.
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http://dx.doi.org/10.1186/1471-2407-13-440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849736PMC
September 2013

Leukemia associated mutant Wilms' tumor gene 1 protein promotes expansion of human hematopoietic progenitor cells.

Leuk Res 2013 Oct 19;37(10):1341-9. Epub 2013 Jul 19.

Division of Hematology and Transfusion Medicine, Lund University, Lund, Sweden.

The transcription factor Wilms' tumor gene 1 (WT1) is highly expressed in the majority of leukemias, suggesting a role in leukemogenesis. Acquired WT1 mutations are reported as an independent predictor of poor clinical outcome, and mutations resulting in deletion of the entire DNA-binding zinc-finger domain (WT1delZ), is the most common type. The aim of this study was to study cellular effects of WT1(delZ) that may contribute to an oncogenic phenotype. We found that expression of WT1(delZ) supported proliferation of human hematopoietic CD34(+) progenitor cells. Moreover, WT1(delZ) transduced cells expressed erythroid markers, including raised levels of STAT5, independently of addition of erythropoietin. At the global gene expression level, WT1(delZ) caused upregulation of genes related to cell division and genes associated with erythroid maturation, in the absence of added erythropoietin. Our results indicate that WT1(delZ) promotes cell proliferation and expansion of progenitor cells, consistent with a possible role in leukemogenesis.
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http://dx.doi.org/10.1016/j.leukres.2013.06.018DOI Listing
October 2013

The transcriptional co-repressor myeloid translocation gene 16 inhibits glycolysis and stimulates mitochondrial respiration.

PLoS One 2013 1;8(7):e68502. Epub 2013 Jul 1.

Department of Hematology, Lund University, Lund, Sweden.

The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068502PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698176PMC
February 2014

Lentivirus-induced dendritic cells for immunization against high-risk WT1(+) acute myeloid leukemia.

Hum Gene Ther 2013 Feb;24(2):220-37

Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany.

Wilms' tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML), a high-risk neoplasm warranting development of novel immunotherapeutic approaches. Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. With the rationale of stimulating multiantigenic responses against WT1, we genetically programmed long-lasting dendritic cells capable of producing and processing endogenous WT1 epitopes. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) was used to transduce human monocytes ex vivo. Overnight transduction induced self-differentiation of monocytes into immunophenotypically stable "SmartDC/tWT1" (GM-CSF(+), IL-4(+), tWT1(+), IL-6(+), IL-8(+), TNF-α(+), MCP-1(+), HLA-DR(+), CD86(+), CCR2(+), CCR5(+)) that were viable for 3 weeks in vitro. SmartDC/tWT1 were produced with peripheral blood mononuclear cells (PBMC) obtained from an FLT3-ITD(+) AML patient and surplus material from a donor lymphocyte infusion (DLI) and used to expand CD8(+) T cells in vitro. Expanded cytotoxic T lymphocytes (CTLs) showed antigen-specific reactivity against WT1 and against WT1(+) leukemia cells. SmartDC/tWT1 injected s.c. into Nod.Rag1(-/-).IL2rγc(-/-) mice were viable in vivo for more than three weeks. Migration of human T cells (huCTLs) to the immunization site was demonstrated following adoptive transfer of huCTLs into mice immunized with SmartDC/tWT1. Furthermore, SmartDC/tWT1 immunization plus adoptive transfer of T cells reactive against WT1 into mice resulted in growth arrest of a WT1(+) tumor. Gene array analyses of SmartDC/tWT1 demonstrated upregulation of several genes related to innate immunity. Thus, SmartDC/tWT1 can be produced in a single day of ex vivo gene transfer, are highly viable in vivo, and have great potential for use as immunotherapy against malignant transformation overexpressing WT1.
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http://dx.doi.org/10.1089/hum.2012.128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696945PMC
February 2013

Ultranet: efficient solver for the sparse inverse covariance selection problem in gene network modeling.

Bioinformatics 2013 Feb 24;29(4):511-2. Epub 2012 Dec 24.

Department of Hematology and Transfusion Medicine, Lund University Hospital, SE-221 85 Lund, Sweden.

Summary: Graphical Gaussian models (GGMs) are a promising approach to identify gene regulatory networks. Such models can be robustly inferred by solving the sparse inverse covariance selection (SICS) problem. With the high dimensionality of genomics data, fast methods capable of solving large instances of SICS are needed. We developed a novel network modeling tool, Ultranet, that solves the SICS problem with significantly improved efficiency. Ultranet combines a range of mathematical and programmatical techniques, exploits the structure of the SICS problem and enables computation of genome-scale GGMs without compromising analytic accuracy.

Availability And Implementation: Ultranet is implemented in C++ and available at www.broadinstitute.org/ultranet.
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http://dx.doi.org/10.1093/bioinformatics/bts717DOI Listing
February 2013

Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma.

BMC Cancer 2012 Jul 28;12:318. Epub 2012 Jul 28.

Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, S- 751 85, Sweden.

Background: Multiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM.

Methods: To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS).

Results: To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered.

Conclusion: We conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro-apoptotic genes. However, this shift alone is not sufficient to alter apoptosis sensitivity in MM cells, suggesting that Stat1 independent pathways are operative in IFN mediated apoptosis sensitization.
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http://dx.doi.org/10.1186/1471-2407-12-318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488543PMC
July 2012

The p53 target gene TRIM22 directly or indirectly interacts with the translation initiation factor eIF4E and inhibits the binding of eIF4E to eIF4G.

Biol Cell 2012 Aug 31;104(8):462-75. Epub 2012 May 31.

Department of Haematology and Transfusion Medicine, BioMedical Centre, Lund University, 221 84 Lund, Sweden.

Background Information: The interferon (IFN)-inducible protein TRIM22 (Staf50) is a member of the tripartite motif protein family and has been suggested a role in the regulation of viral replication as well as of protein ubiquitylation. In addition, we have previously shown that TRIM22 is a direct target gene for the tumour suppressor p53. Consistently, over-expression of TRIM22 inhibits the clonogenic growth of monoblastic U937 cells, suggesting anti-proliferative or cell death-inducing effects.

Results: Here, we demonstrate that TRIM22 directly or indirectly interacts with the eukaryotic translation initiation factor (eIF)4E, and inhibits the binding of eIF4E to eIF4G, thus disturbing the assembly of the eIF4F complex, which is necessary for cap-dependent translation. Furthermore, TRIM22 exerts a repressive effect on luciferase reporter protein levels and to some extent on radiolabelled methionine incorporation. Even though all nuclear mRNAs are capped, some are more dependent on eIF4F than others for translation. The translation of one of these mRNAs, IRF-7C, was indeed found to be repressed in the presence of TRIM22.

Conclusions: Our data suggest TRIM22 to repress protein translation preferably of some specific mRNAs. Taken together, we show that TRIM22 represses translation by inhibiting the binding of eIF4E to eIF4G, suggesting a mechanism for regulation of protein translation, which may be of importance in response to p53 and/or IFN signalling.
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http://dx.doi.org/10.1111/boc.201100099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161774PMC
August 2012

The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells.

BMC Mol Biol 2012 Mar 23;13:11. Epub 2012 Mar 23.

Department of Hematology, C14, BMC, S-221 84, Lund, Sweden.

Background: MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis.

Results: 5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16/MTGR1 promoters by the leukemia associated AML1-ETO fusion gene may have a role in hematopoietic dysfunction of leukemia.

Conclusions: An evolutionary conserved GATA binding site is critical in transcriptional regulation of the MTG16 promoter. In contrast, the MTGR1 gene depends on a GC-box-rich sequence for transcriptional regulation and possible ubiquitous expression. Our results demonstrate that the ETO homologue promoters are regulated differently consistent with hematopoietic cell-type- specific expression and function.
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http://dx.doi.org/10.1186/1471-2199-13-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364894PMC
March 2012

The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells.

BMC Mol Biol 2010 May 20;11:38. Epub 2010 May 20.

1Department of Hematology, C14, BMC, S-221 84 Lund, Sweden.

Background: The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.

Results: A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells.

Conclusions: We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression.
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http://dx.doi.org/10.1186/1471-2199-11-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882371PMC
May 2010
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