scholarly journals Genetic Heterogeneity of KLF1, a Master Regulator of Erythropoiesis, Revealed an Autosomal Recessive Ψβ-Thalassemia and a Very Strong Promoter In Vivo

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Laura Grech ◽  
Joseph Borg ◽  
Ruth Galdies ◽  
Carmen Attard ◽  
Christian A. Scerri ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Genetic Heterogeneity ◽  
Autosomal Recessive ◽  
Globin Gene ◽  
Constant Region ◽  
Zinc Finger Domain ◽  
Wild Type ◽  
Strong Promoter ◽  
The Family

The Erythroid Kruppel-like Factor 1 or KLF1 is a transcription factor that functioned in the early stage genetic programming of erythroid progenitors to promote physiological γ to β globin gene switching. Indeed, we showed that a truncation mutation (p.K288X) in KLF1 that deleted the DNA binding zinc finger domain resulted in marked KLF1 deficiency and a hematological condition that resembled a hereditary persistence of fetal hemoglobin (HbF) or a β thalassemia. Here, we describe five additional families with the same p.K288X mutation but varied hematological and HbF levels together with genetic and phenotypic data on a 600 data-set from the same Maltese population. The data accounted for a strong promoter embedded within a region of genetic heterogeneity of KLF1 that led to a ψβ thalassemia. Whole genome sequencing on 15 subjects of six families (FamF1 - FamF6) segregating (two) p.K288X frameworks of KLF1 had variable degrees of microcytosis (MCV; 76.1fL -77.4 fL) and HbF levels (HbF 2480 mg/dL - 802mg/dL) due to complex heterozygosity between promoter, coding and truncating mutations in KLF1. Case II-6 of FamF1 with the highest HbF (2480 mg/dL) had 2 promoter and 2 coding mutations in cis and in trans to the p.K288X truncation. Nine (9) KLF1 frameworks (A - I) were derived by transmission disequilibrium analyses of the family data, each assembled from 15 mutations and resulting in 7 genotypes among the families. The p.K288X truncation was found on a second rarer framework. Additional, rarer KLF1 frameworks were found with haploview in the population dataset. The population dataset was made up of 198 β thalassemia heterozygotes and 400 others from the clinic and the biobank and that had no β globin gene mutations, variable blood counts or hemoglobin profiles or both. They were older than 2 years of age, not pregnant and had normal iron levels. The number of KLF1 mutations differed from 0 in the wild-type framework A to 6 in one of the rare frameworks X. Six mutations were in the promoter region and 6 were in the coding region that defined a "KLF1 Variable Region" 5' genomic coordinate 12887183 - 12888273, whereas very few were found in the 3' (genomic coordinate 12884589 - 12884752) that defined the KLF1 "Constant Region" The Constant region has also been evolutionary conserved. It included the zinc finger domain and the proteasome binding site. The genotype - phenotype correlations and the family data were consistent with an autosomal recessive condition that resembled a β thalassemia, thus a ψβ thalassemia. It differed from a silent thalassemia because the β globin gene sequence was wild type. It provided a diagnosis for families with iron resistant microcytosis and borderline hemoglobin phenotypes suitable for counselling in the clinical setting. It was consistent with the observation among the families regarding the high strength of the KLF1 promoter. Multiple "hits" were necessary to suppress the biosynthesis of KLF1 for hemoglobin switching to escape perinatal suppression. The effect of the 6 promoter mutations were confirmed in vitro with native and induced K562 and Hek293T cell lines. Presumably, during normal development, the strong promoter served to rapidly drown KLF1 binding sites with KLF1 molecules to direct progenitors to erythropoiesis with the appropriate adult hemoglobin profile in the perinatal period. The diagnosis of the patients with selected genotypes due to compound and double heterozygosities in promoter and coding sequences shall further permit quantification of differential promoter function in vivo. Figure Disclosures No relevant conflicts of interest to declare.

A Novel EKLF Mutation in a Patient with Dyserythropoietic Anemia: The First Association of EKLF with Disease in Man.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 162-162 ◽  
Author(s):  
Belinda K Singleton ◽  
Victoria SS Fairweather ◽  
Winnie Lau ◽  
Stephen F Parsons ◽  
Nicholas M Burton ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Mrna Expression ◽  
Zinc Finger ◽  
Globin Gene ◽  
Promoter Sequence ◽  
Single Point Mutation ◽  
Zinc Finger Domain ◽  
Wild Type ◽  
Empty Vector ◽  
Significant Difference

Abstract Abstract 162 We describe a single-point mutation in the transcription factor EKLF associated with dyserythropoietic anemia. The female Danish patient was extensively studied in the early 1990's (Wickramasinghe et al. Br J Haem 1991,79:322; Tang et al. Blood 1993,81:1636; Parsons et al. Blood 1994,83:860; Agre et al. J Clin Invest 1994,94:1050). The patient was severely anemic at birth and required repeated transfusions during childhood. Notable features included persistent expression of epsilon and zeta embryonic globins, an HbF level of 40%, novel intra-erythroblastic and intra-erythrocytic inclusions and deficiency of erythroid proteins CD44 and Aquaporin 1. Evidence that EKLF plays a major role in globin gene regulation in particular and erythroid gene expression in general led us to examine EKLF in this patient, her unaffected sister, and her parents. The coding sequence of EKLF was normal wild-type in both healthy parents and sister, but the patient had a mutation on one allele of EKLF in the second zinc finger domain (Glu325Lys). This mutation is at a site that is central to the expected site of interaction of EKLF with DNA. However, modelling revealed that reversal of the charge at this position in a critical DNA-binding domain was likely to create a novel direct contact between Lys325 and a phosphate on the DNA backbone, hence maintaining rather than disrupting binding of EKLF to promoter regions. To test this, we created recombinant forms of the EKLF zinc finger domain, corresponding to the wild-type, Glu325Lys and 3 presumed loss-of-function mutations previously associated with the rare blood group In(Lu) phenotype (Singleton et al. Blood 2008,112:2081). Using fluorescence-based binding assays, mutant EKLF proteins Arg328Leu, Arg328His, and Arg331Gly showed virtually no binding to the beta globin (HBB) promoter sequence, as expected. In contrast, there was no significant difference in the binding of EKLF Glu325Lys and wild-type EKLF to the promoter sequence. We then transfected full-length wild-type and mutated EKLF into K562 cells and measured the effect on expression of several genes by quantitative real-time PCR. Transfectants with wild-type EKLF showed an average 13.7 fold increase in EKLF mRNA expression (SD 4.8, n=5) compared with a clone transfected with the empty vector. This was associated with an elevation in HBB and CD44H mRNA expression (average 12.8 (SD 10.9, n=5) and 27.0 (SD 21.7, n=5) fold respectively, compared with the empty vector clone). In contrast, transfectants with EKLF Glu325Lys, although expressing slightly lower levels of EKLF mRNA than the wild-type clones (average 8.0 fold compared with the empty vector clone, SD 5.2, n=11), showed much reduced HBB and CD44H expression (average 1.9 (SD 1.6, n=11) and 1.4 (SD 1.4, n=11) fold respectively, compared with the empty vector clone). Our findings indicate that EKLF Glu325Lys has a reduced ability to activate HBB and CD44H expression, thus establishing a link between the mutation and the patient's phenotype. This reduction, however, does not appear to be explained by differences in the ability of the mutant EKLF to bind to the HBB promoter, implying that other mechanisms of gene regulation must be affected in the patient. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Unanticipated Repression Function Linked to Erythroid Krüppel-Like Factor

2001 ◽  
Vol 21 (9) ◽  
pp. 3118-3125 ◽  
Author(s):  
Xiaoyong Chen ◽  
James J. Bieker
Keyword(s):  
Gene Expression ◽  
Zinc Finger ◽  
Trichostatin A ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Zinc Finger Domain ◽  
Transcription Activator ◽  
Histone Deacetylase 1 ◽  
Globin Promoter

ABSTRACT The erythroid cell-specific transcription factor erythroid Krüppel-like factor (EKLF) is an important activator of β-globin gene expression. It achieves this by binding to the CACCC element at the β-globin promoter via its zinc finger domain. The coactivators CBP and P300 interact with, acetylate, and enhance its activity, helping to explain its role as a transcription activator. Here we show that EKLF can also interact with the corepressors mSin3A and HDAC1 (histone deacetylase 1) through its zinc finger domain. When linked to a GAL4 DNA binding domain, full-length EKLF or its zinc finger domain alone can repress transcription in vivo. This repressive activity can be relieved by the HDAC inhibitor trichostatin A. Although recruitment of EKLF to a promoter is required to show repression, its zinc finger domain cannot bind directly to DNA and repress transcription simultaneously. In addition, the target promoter configuration is important for enabling EKLF to exhibit any repressive activity. These results suggest that EKLF may function in vivo as a transcription repressor and play a previously unsuspected additional role in regulating erythroid gene expression and differentiation.

scholarly journals Zinc Finger Domain of Murine Leukemia Virus Nucleocapsid Protein Enhances the Rate of Viral DNA Synthesis in Vivo

2002 ◽  
Vol 76 (15) ◽  
pp. 7473-7484 ◽  
Author(s):  
Wen-hui Zhang ◽  
Carey K. Hwang ◽  
Wei-Shau Hu ◽  
Robert J. Gorelick ◽  
Vinay K. Pathak
Keyword(s):  
Secondary Structure ◽  
Dna Synthesis ◽  
Zinc Finger ◽  
Rna Secondary Structure ◽  
Leukemia Virus ◽  
Template Switching ◽  
Zinc Finger Domain ◽  
Wild Type ◽  
Murine Leukemia

ABSTRACT In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A trans-complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in trans. We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals MRG15 Regulates Embryonic Development and Cell Proliferation

2005 ◽  
Vol 25 (8) ◽  
pp. 2924-2937 ◽  
Author(s):  
Kaoru Tominaga ◽  
Bhakti Kirtane ◽  
James G. Jackson ◽  
Yuji Ikeno ◽  
Takayoshi Ikeda ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Embryonic Development ◽  
Chromatin Remodeling ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Immunohistochemical Analysis ◽  
Wild Type ◽  
Proliferating Cells ◽  
Globin Promoter

ABSTRACT MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15 − / − embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15 − / − mouse embryonic fibroblasts. The hearts of the Mrg15 − / − embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15 − / − embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation.

scholarly journals The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
V. Helfinger ◽  
K. Palfi ◽  
A. Weigert ◽  
K. Schröder
Keyword(s):  
Ex Vivo ◽  
Macrophage Polarization ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Superoxide Anions ◽  
Wild Type ◽  
Macrophage Population ◽  
The Family ◽  
High Level

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

The Role of p38 MAPK/CREB1 Signaling In γ-Globin Gene Regulation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1012-1012
Author(s):  
Valya Ramakrishnan ◽  
Li Liu ◽  
Subhradip Karmakar ◽  
Milind Mahajan ◽  
Sherman M. Weissman ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Luciferase Activity ◽  
Globin Gene ◽  
Mapk Signaling ◽  
Wild Type ◽  
Camp Response Element ◽  
Globin Promoter ◽  
Sirna Knockdown

Abstract Abstract 1012 The clinical symptoms of sickle cell disease can be ameliorated by increased fetal hemoglobin (HbF) levels. Previous work from our laboratory demonstrated that Trichostatin A (TSA) and sodium butyrate (NaB) activate γ-globin expression via p38 MAPK signaling. In addition, cAMP response element binding protein 1 (CREB1) was shown to trans-activate the -1222 Gγ-globin cAMP response element (G-CRE) in a transient assay system. To study the role of p38 MAPK signaling in γ-globin regulation, loss of function siRNA studies were performed in K562 cells. siRNA-mediated knockdown of p38 MAPK resulted in 72% loss of γ-globin transcription. Furthermore, enforced stable expression of MKK3/6 increased the phosphorylated form of p38 MAPK by 70%, which in turn produced a 2- to 3-fold increase in γ-globin mRNA and HbF levels. Likewise, siCREB1 treatment reduced CREB1 levels by 62% and down regulated γ-globin expression 59%. In the same vein, stable expression of recombinant CREB1 activated HbF by 2-fold. These findings were subsequently confirmed in human primary erythroid cells grown in a two-phase liquid culture system. On day 11, we observed 50–70% γ-globin silencing after CREB1 and p38 MAPK siRNA knockdown with 60% target gene silencing. CREB1 enforced expression trans-activated γ-globin 4.5-fold which was accompanied by a 90% increase in HbF-FITC positive cells and HbF levels. Collectively, these data demonstrate that p38 MAPK and CREB1 are required for steady-state γ-globin gene transcription. To determine the role of the G-CRE in γ-globin regulation, the Gγ-globin promoter (-1500 to +36) was cloned into pGL4.17 Luc2/neo to produce pGγLuc2 (wild type) and mutant plasmids: -1225 G/A (m1), -1227 AC/TG (m2) and a scrambled G-CRE (m3s). Five K562 stable lines including KLuc2 (promoterless), KGγLuc2, KGγLuc2(m1), KGγLuc2(m2) and KGγLuc2(m3s) were established. Luciferase activity in KGγLuc2 was 1000-fold higher than in the control Kluc2 line; all mutations produced >90% loss of luciferase activity and a loss of γ-globin trans-activation by TSA and NaB. Next, siRNA studies were completed to determine if the G-CRE is required for γ-globin activation. A dose-dependent loss of promoter activity was observed after p38 MAPK and CREB1 siRNA knockdown of the KGγLuc2 cell line; however, promoter silencing was not observed in the mutant lines supporting a role for the G-CRE in p38 MAPK/CREB1 mediated γ-globin regulation. To study in vivo binding, chromatin immunoprecipitation (ChIP) assays were performed with CREB1 antibody in the KGγLuc2 stable line. We observed comparable 2- to 3-fold chromatin enrichment with CREB1 compared to the control IgG in the G-CRE regions of the pGγLuc2 plasmid and endogenous Gγ-globin promoter. To determine if an enhanceosome complex is bound to the G-CRE, we performed affinity column pull-down/mass spectrometry analysis. K562 nuclear extract was purified on a Heparin Sepharose column, following which fractions eluting at 0.6M NaCl showing peak gel shift binding activities with the G-CRE oligo were loaded into a size selecting Suprose 6 gel exclusion column. G-CRE eluting fractions were then identified by protein microsequencing (MS/MS). We identified CREB1, ATF2, c-Jun, BRG-1, hnRNPC1/C2, and the TCP-1 complex as major components. To determine protein co-localization, promoter pull-down assays were performed using biotinylated wild type and mutant (AC/TG) G-CRE probes and K562 nuclear extracts. We observed simultaneous CREB1, ATF-2 and cJun binding to the G-CRE which was abolished in the mutant probe. However, Brg1 was bound after NaB (2mM) induction. Subsequent co-IP studies showed interactions between ATF-2 and Brg1, CREB1, cJun, and hnRNPC1C2, which was further confirmed by co-elution profile of these molecules observed by sucrose gradient centrifugation, thus implying association as one complex. These data support complex protein-protein interactions in the G-CRE, which modulate γ-globin gene expression. Additional studies will be performed in primary erythroid cells using siRNA-based gene silencing and ChIP assays to determine novel mechanisms of γ-globin regulation and to define in vivo binding of proteins identified in the G-CRE enhanceosome complex. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reduced Fertility in Male Mice Deficient in the Zinc Metallopeptidase NL1

2004 ◽  
Vol 24 (10) ◽  
pp. 4428-4437 ◽  
Author(s):  
Mélanie Carpentier ◽  
Christine Guillemette ◽  
Janice L. Bailey ◽  
Guy Boileau ◽  
Lucie Jeannotte ◽  
...  
Keyword(s):  
Male Fertility ◽  
Protein A ◽  
Physiological Role ◽  
Sperm Function ◽  
Secreted Protein ◽  
Wild Type ◽  
The Family ◽  
Fertility Problems ◽  
Targeting Strategy

ABSTRACT Members of the M13 family of zinc metalloendopeptidases have been shown to play critical roles in the metabolism of various neuropeptides and peptide hormones, and they have been identified as important therapeutic targets. Recently, a mouse NL1 protein, a novel member of the family, was identified and shown to be expressed mainly in the testis as a secreted protein. To define its physiological role(s), we used a gene targeting strategy to disrupt the endogenous murine Nl1 gene by homologous recombination and generate Nl1 mutant mice. The Nl1−/− mice were viable and developed normally, suggesting that zygotic expression of Nl1 is not required for development. However, Nl1−/− males produced smaller litters than their wild-type siblings, indicating specific male fertility problems. Reduced fertility may be explained by two impaired processes, decreased egg fertilization and perturbed early development of fertilized eggs. These two phenotypes did not result from gross anatomical modifications of the testis or from impaired spermatogenesis. Basic sperm parameters were also normal. Thus, our findings suggest that one of the roles of NL1 in mice is related to sperm function and that NL1 modulates the processes of fertilization and early embryonic development in vivo.

scholarly journals p45NFE2 Is a Negative Regulator of Erythroid Proliferation Which Contributes to the Progression of Friend Virus-Induced Erythroleukemias

2001 ◽  
Vol 21 (1) ◽  
pp. 73-80 ◽  
Author(s):  
You-Jun Li ◽  
Rachel R. Higgins ◽  
Brian J. Pak ◽  
Ramesh A. Shivdasani ◽  
Paul A. Ney ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Globin Gene ◽  
Negative Regulator ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Globin Genes ◽  
Friend Virus ◽  
Wild Type

ABSTRACT In previous studies, we identified a common site of retroviral integration designated Fli-2 in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia cell lines. Insertion of F-MuLV at the Fli-2 locus, which was associated with the loss of the second allele, resulted in the inactivation of the erythroid cell- and megakaryocyte-specific genep45 NFE2 . Frequent disruption ofp45 NFE2 due to proviral insertion suggests a role for this transcription factor in the progression of Friend virus-induced erythroleukemias. To assess this possibility, erythroleukemia was induced by F-MuLV inp45 NFE2 mutant mice. Sincep45 NFE2 homozygous mice mostly die at birth, erythroleukemia was induced in +/− and +/+ mice. We demonstrate that +/− mice succumb to the disease moderately but significantly faster than +/+ mice. In addition, the spleens of +/− mice were significantly larger than those of +/+ mice. Of the 37 tumors generated from the +/− and +/+ mice, 10 gave rise to cell lines, all of which were derived from +/− mice. Establishment in culture was associated with the loss of the remaining wild-typep45 NFE2 allele in 9 of 10 of these cell lines. The loss of a functional p45NFE2 in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-typep45 NFE2 in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression ofp45 NFE2 in these cells also slows tumor growth in vivo. These results indicate thatp45 NFE2 functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.

Sign in / Sign up

Export Citation Format

Share Document