ABSTRACT
BNIP3 and NIX are proteins related to the BH3-only family, which induce both cell death and autophagy. Consistent with their ability to induce cell death, BNIP3 and NIX are implicated in the pathogenesis of cancer and heart disease. In tumor cells, BNIP3 and NIX are regulated by hypoxia, and the deregulation of BNIP3 or NIX expression is associated with tumor growth. In heart muscle, BNIP3 and NIX are regulated by hypoxia and Galphaq-dependent signaling, respectively, and their expression is associated with decreased myocardial function. Apart from their role in cell death, BNIP3 and NIX are also implicated in the induction of autophagy. In erythroid cells, NIX is required for a specialized type of autophagy that targets mitochondria for elimination (mitophagy). Similarly, BNIP3 regulates mitophagy in response to hypoxia. In this review, we will discuss possible mechanisms by which BNIP3 and NIX induce cell death and mitophagy. We will also consider the potential relationship between cell death pathways and autophagy in development and homeostasis.
Subject(s)
Autophagy/physiology , Membrane Proteins/metabolism , Mitochondria/physiology , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Death/physiology , Cell Hypoxia/physiology , Heart Diseases/metabolism , Humans , Membrane Proteins/genetics , Mice , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Neoplasms/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Rats , Tumor Suppressor Proteins/geneticsABSTRACT
The erythropoietin receptor (EpoR) is required for the proliferation and survival of committed erythroid lineage cells. Previous studies have utilized receptor mutations to show the requirement for the distal half of the cytoplasmic domain of the EpoR and receptor tyrosines for activation of signaling pathways potentially critical to Epo function. To extend these studies to in vivo erythropoiesis, we have created two mutant strains of mice. One strain (H) contains a truncation of the distal half of the cytoplasmic domain, while the second strain (HM) contains the same truncation as well as the mutation of the residual tyrosine (Y(343)) to a phenylalanine. Strikingly, both strains of mice are viable, with only slight alterations in constitutive erythropoiesis or in in vitro assays of red cell lineage function. Challenging H mutant mice with continuous injections of Epo results in an erythrocytosis that is not seen in HM mice. The results demonstrate that neither the distal region nor receptor tyrosines are essential for in vivo EpoR function, but contribute to receptor function in a subtle manner.
Subject(s)
Erythropoiesis/physiology , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Receptors, Erythropoietin/physiology , Animals , Binding Sites , Janus Kinase 2 , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolismABSTRACT
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 gene p45(NFE2). Frequent disruption of p45(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 in p45(NFE2) mutant mice. Since p45(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-type p45(NFE2) allele in 9 of 10 of these cell lines. The loss of a functional p45(NFE2) in these cell lines was associated with a marked reduction in globin gene expression. Expression of wild-type p45(NFE2) in the nonproducer erythroleukemic cells resulted in reduced cell growth and restored the expression of globin genes. Similarly, the expression of p45(NFE2) in these cells also slows tumor growth in vivo. These results indicate that p45(NFE2) functions as an inhibitor of erythroid cell growth and that perturbation of its expression contributes to the progression of Friend erythroleukemia.
Subject(s)
DNA-Binding Proteins/metabolism , Friend murine leukemia virus/physiology , Leukemia, Erythroblastic, Acute/pathology , Leukemia, Erythroblastic, Acute/virology , Transcription Factors/metabolism , Animals , Animals, Newborn , Cell Division , Clone Cells/metabolism , Clone Cells/pathology , Clone Cells/virology , DNA-Binding Proteins/genetics , Disease Progression , Erythroid-Specific DNA-Binding Factors , Gene Deletion , Gene Expression Regulation, Neoplastic , Genotype , Globins/genetics , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Erythroblastic, Acute/metabolism , Mice , Mice, Inbred Strains , Mice, Knockout , NF-E2 Transcription Factor, p45 Subunit , Transcription Factors/genetics , Transfection , Tumor Cells, CulturedSubject(s)
Friend murine leukemia virus/physiology , Leukemia, Erythroblastic, Acute/virology , Animals , Friend murine leukemia virus/chemistry , Friend murine leukemia virus/enzymology , Leukemia, Erythroblastic, Acute/enzymology , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Erythroblastic, Acute/physiopathology , Mice , Multigene Family , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/physiology , Receptors, Erythropoietin/metabolism , Receptors, Erythropoietin/physiology , Retroviridae Infections/enzymology , Retroviridae Infections/metabolism , Retroviridae Infections/physiopathology , Signal Transduction , Tumor Virus Infections/enzymology , Tumor Virus Infections/metabolism , Tumor Virus Infections/physiopathology , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/physiologyABSTRACT
The Friend virus susceptibility 2 (Fv2) locus encodes a dominant host factor that confers susceptibility to Friend virus-induced erythroleukaemia in mice. We mapped Fv2 to a 1.0-Mb interval that also contained the gene (Ron) encoding the stem cell kinase receptor (Stk). A truncated form of Stk (Sf-stk), which was the most abundant form of Stk in Fv2-sensitive (Fv2ss) erythroid cells, was not expressed in Fv2 resistant (Fv2rr) cells. Enforced expression of Sf-stk conferred susceptibility to Friend disease, whereas targeted disruption of Ron caused resistance. We conclude that the Fv2 locus encodes Ron, and that a naturally expressed, truncated form of Stk confers susceptibility to Friend virus-induced erythroleukaemia.
Subject(s)
Friend murine leukemia virus , Leukemia, Erythroblastic, Acute/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/genetics , 3T3 Cells , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Contig Mapping , Gene Expression , Genetic Predisposition to Disease , Mice , Mice, Inbred AKR , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred CBA , Mice, Inbred NZB , Mice, Inbred Strains , Molecular Sequence Data , Muridae , Protein Isoforms/genetics , Retroviridae Infections/genetics , Species Specificity , Spleen/cytology , Spleen/metabolism , Spleen/pathology , Tumor Virus Infections/geneticsABSTRACT
Tandem binding sites for the hematopoietic transcription factor NF-E2 in the beta-globin locus control region activate high-level beta-globin gene expression in transgenic mice. NF-E2 is a heterodimer consisting of a hematopoietic subunit p45 and a ubiquitous subunit p18. Gavva et al. [Gavva, N. R., Gavva, R., Ermekova, K., Sudol, M., and Shen, J. C. (1997) J. Biol. Chem. 272, 24105-24108] reported that human p45 contains a PPXY motif that binds WW domains. We show that murine NF-E2, which contains two PPXY motifs (PPXY-1 and -2) within its transactivation domain, differentially interacted with nine GST-WW domain fusion proteins. Quantitative analysis revealed high-affinity binding (KD = 5.7 nM) of p45 to a WW domain from a novel human ubiquitin ligase homologue (WWP1) expressed in hematopoietic tissues. The amino-terminal WW domain of WWP1 formed a multimeric complex with DNA-bound NF-E2. A WWP1 ligand peptide, isolated by phage display, and a peptide spanning PPXY-1 inhibited p45 binding, whereas an SH3 domain-interacting peptide and a peptide spanning PPXY-2 did not. Mutation of PPXY-1, but not PPXY-2, inhibited the transactivation function of NF-E2, providing support for the hypothesis that WW domain interactions are important for NF-E2-mediated transactivation.
Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Peptide Fragments/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/physiology , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/physiology , Dimerization , Erythroid-Specific DNA-Binding Factors , Erythropoiesis/genetics , Humans , Intracellular Signaling Peptides and Proteins , Ligases/metabolism , Macromolecular Substances , MafK Transcription Factor , Mice , Mice, Transgenic , Molecular Sequence Data , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Organ Specificity/genetics , Peptide Fragments/chemistry , Peptide Fragments/physiology , Protein Binding , Protein Structure, Tertiary , RNA, Messenger/biosynthesis , Trans-Activators , Transcription Factors/chemistry , Transcription Factors/physiology , Transcriptional Activation/physiology , Ubiquitin-Protein LigasesABSTRACT
NF-E2 binding sites, located in distant regulatory sequences, may be important for high level alpha- and beta-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2-related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2-deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of alpha- and beta-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2-deficient mice is not the result of compensation by Nrf-2.
Subject(s)
DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Erythropoiesis , Globins/genetics , Transcription Factors/metabolism , Transcription Factors/physiology , Amino Acid Sequence , Animals , Dimerization , Erythroid Precursor Cells/cytology , Erythroid-Specific DNA-Binding Factors , GA-Binding Protein Transcription Factor , Gene Expression Regulation, Developmental , MafK Transcription Factor , Mice , Mice, Knockout/embryology , Molecular Sequence Data , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Peptides/immunology , Stem CellsABSTRACT
Regulated expression of genes in the beta-globin cluster depends upon sequences located between 5 and 20 kb upstream of the epsilon gene, known as the locus control region (LCR). beta-Globin expression in murine erythroleukemia (MEL) cells depends on NF-E2, a transcription factor which binds to enhancer sequences in the LCR. To gain insight into the mechanism of globin gene activation by NF-E2, an NF-E2 null MEL cell line was used to map regions of NF-E2 required for beta-globin expression. Within the transactivation domain, two discrete proline-rich regions were required for rescue of beta-globin expression. The first was located at the N-terminus of NF-E2, while the second was located N-terminal of the cap 'n collar (CNC) domain. Other proline-rich sequences were dispensable, indicating that proline content per se does not determine NF-E2 activity. Mutations within the conserved CNC domain markedly diminished rescue of beta-globin expression. This domain was required, in addition to the basic leucine zipper domain, for DNA binding activity. The requirement for discrete proline-rich sequences within the transactivation domain suggests that globin gene expression in MEL cells depends on specific interactions between NF-E2 and downstream effector molecules.
Subject(s)
DNA-Binding Proteins/metabolism , Globins/biosynthesis , Transcription Factors/metabolism , Amino Acid Sequence , Animals , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/chemistry , Erythroid-Specific DNA-Binding Factors , Gene Expression Regulation, Neoplastic , Leukemia, Erythroblastic, Acute , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Nuclear Proteins/metabolism , Point Mutation , Polymerase Chain Reaction , Proline , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Deletion , Transcription Factors/biosynthesis , Transcription Factors/chemistry , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
Mice homozygous for the mk mutation have a severe hypochromic, microcytic anemia that is characterized by a decreased mean-corpuscular hemoglobin concentration and balanced alpha- and beta-globin-chain synthesis. Transplantation studies have shown that the defect in homozygous mk/mk mice is intrinsic to both the hematopoietic system and the gut. The gene for the hematopoietic-specific transcription factor, p45 NF-E2, has been found to cosegregate with the mk phenotype and contain a point mutation in mk/mk mice that results in an amino acid substitution (173V-->A). In order to test the hypothesis that this amino acid substitution is responsible for the mk phenotype, we have used recombinant retroviruses to introduce wild-type p45 NF-E2 into the bone marrow of mk/mk mice. Despite gene transfer and expression of p45 NF-E2 in erythroid cells, we found no evidence for correction of the phenotype in mk/mk mice. These results indicate that the mk mutation cannot be corrected by enforced expression of wild-type p45 NF-E2 and suggest that the 173V-->A mutation of the p45 NF-E2 gene is not the cause of anemia in mk/mk mice.
Subject(s)
Anemia/therapy , DNA-Binding Proteins/genetics , Gene Transfer Techniques , Genetic Therapy , Retroviridae/genetics , Transcription Factors/genetics , Actins/genetics , Animals , Base Sequence , Bone Marrow/metabolism , DNA, Recombinant/therapeutic use , Erythroid-Specific DNA-Binding Factors , Gene Expression , Genetic Vectors , Globins/genetics , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Molecular Sequence Data , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Point MutationABSTRACT
A cis-acting DNA regulatory element 3' to the A gamma-globin gene contains eight distinct regions of DNA-protein interaction distributed over 750 bp of DNA. The sequences of two foot-printed regions (sites I and IV) are A-T rich and generate a highly retarded complex on gel shift analysis with nuclear extract from human erythroleukemia (K562) cells. We have purified a 98-kD protein that reproduces this gel shift. Tryptic cleavage and peptide sequence analysis demonstrated that the 98-kD protein is identical to a recently cloned protein, special A-T-rich binding protein 1 (SATB1), that binds selectively to nuclear matrix/scaffold-associated regions of DNA (MARs/SARs). We have shown by functional analysis that the 3' A gamma regulatory element associates with the nuclear matrix. SATB1 mRNA was identified in K562 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated its transcript in several other hematopoietic lines. Antisera to SATB1 caused ablation of the gel shift complex generated by both the crude nuclear extract and the purified 98-kD protein with the site I oligonucleotide. Furthermore, oligonucleotides that bind SATB1 inhibited formation of the site I gel shift complex when added as excess unlabeled competitor. An immunoblot analysis of the site I gel shift complex documented the presence of SATB1. Binding of SATB1 to two sites within the 3' A gamma regulatory element and its MAR/SAR activity suggests that this element may influence gene expression through interaction with the nuclear matrix.
Subject(s)
DNA-Binding Proteins/metabolism , Globins/genetics , Matrix Attachment Region Binding Proteins , Nuclear Matrix/metabolism , Regulatory Sequences, Nucleic Acid , Amino Acid Sequence , Base Sequence , Binding Sites , Cell Line , DNA-Binding Proteins/isolation & purification , Electrophoresis, Polyacrylamide Gel , Globins/metabolism , Humans , Leukemia, Erythroblastic, Acute , Molecular Sequence Data , Mutagenesis , Oligodeoxyribonucleotides , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Tumor Cells, CulturedABSTRACT
Erythroid transcription factor NF-E2 is a tissue-restricted heterodimeric protein which recognizes an extended AP-1 motif [(T/C)TGCTGA(C/G)TCA(T/C)] found in the upstream locus control regions of the alpha- and beta-globin gene clusters. A cDNA clone encoding a cell-type-specific subunit of NF-E2, designated p45 NF-E2, has previously been characterized and shown to encode a basic-leucine zipper DNA-binding protein. Here we describe protein purification and cloning of cDNA that encodes the second basic-leucine zipper subunit of the native NF-E2 heterodimer. This polypeptide, designated p18, is widely expressed. It displays extensive homology to the v-maf oncogene product and a human retinal-specific protein, NRL. Unusual features in the basic region shared by v-Maf, NRL, and p18 place them in a distinct subfamily of AP-1-like proteins.
Subject(s)
DNA-Binding Proteins/biosynthesis , Eye Proteins , Oncogene Proteins, Viral/biosynthesis , Oncogenes , Transcription Factors/biosynthesis , Viral Proteins , Amino Acid Sequence , Animals , Base Sequence , Basic-Leucine Zipper Transcription Factors , Cell Line , DNA-Binding Proteins/chemistry , Erythroid-Specific DNA-Binding Factors , Globins/genetics , Humans , Leucine Zippers/genetics , Leukemia, Erythroblastic, Acute , Macromolecular Substances , MafK Transcription Factor , Mice , Molecular Sequence Data , Multigene Family , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Oligodeoxyribonucleotides , Oncogene Protein v-maf , Oncogene Proteins, Viral/chemistry , Proto-Oncogene Proteins c-jun/biosynthesis , Proto-Oncogene Proteins c-jun/genetics , Sequence Homology, Amino Acid , Transcription Factors/chemistry , Transfection , Tumor Cells, Cultured , Zinc Fingers/geneticsABSTRACT
The use of recombinant adeno-associated virus (rAAV) vectors provides a new strategy to investigate the role of specific regulatory elements and trans-acting factors in globin gene expression. We linked hypersensitivity site 2 (HS2) from the locus control region (LCR) to a A gamma-globin gene (A gamma*) mutationally marked to allow its transcript to be distinguished from endogenous gamma-globin mRNA. The vector also contains the phosphotransferase gene that confers resistance to neomycin (NeoR). HS2 region mutations within the NF-E2 motifs prevented NF-E2 binding while preserving AP-1 binding. Another set in the GATA-1 motif prevented binding of the factor. Several NeoR K562 clones containing a single unrearranged RAAV genome with the A gamma* gene linked to the native HS2 core fragment (WT), mutant NF-E2 HS2 (mut-NFE2), mutant GATA-1 HS2 (mut-GATA1), or no HS [(-)HS] were identified. In uninduced K562 cells, mut-NFE2 clones expressed A gamma* mRNA at the same level as the WT clones, compared with a lack of A gamma* signal in the (-)HS2 clones. However, hemin induction of mut-NFE2 clones did not result in an increase in the A gamma* signal above the level seen in uninduced cells. Mut-GATA1 clones expressed the A gamma* mRNA at the same level as WT clones in both uninduced and induced cells. Thus, GATA-1 binding to this site does not appear to be required for the enhancing function of HS2 in this context. This single-copy rAAV transduction model is useful for evaluating the effects of specific mutations in regulatory elements on the transcription of linked genes.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Globins/biosynthesis , Transcription Factors/metabolism , Zinc Fingers/physiology , Binding Sites , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Dependovirus/genetics , Electrophoresis, Polyacrylamide Gel , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Expression , Gene Rearrangement , Genetic Vectors , Globins/genetics , Globins/isolation & purification , Humans , Leukemia, Erythroblastic, Acute , Mutagenesis, Site-Directed , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Recombination, Genetic , Transcription Factors/genetics , Transcription Factors/isolation & purification , Tumor Cells, Cultured , Zinc Fingers/geneticsABSTRACT
The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.
Subject(s)
Chromosomes, Human, Pair 12 , DNA-Binding Proteins/genetics , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Erythroid-Specific DNA-Binding Factors , Gene Expression Regulation , Globins/genetics , Hematopoiesis , Humans , Leucine Zippers , MafK Transcription Factor , Molecular Sequence Data , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Nuclear Proteins/genetics , Oligodeoxyribonucleotides/chemistry , RNA, Messenger/genetics , Sequence AlignmentABSTRACT
The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S.M. Jane, P.A. Ney, E.F. Vanin, D.L. Gumucio, and A.W. Nienhuis. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ubiquitous activator Sp1 and a protein previously known as -50 gamma and now named the stage selector protein (SSP). Binding of the second protein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the -202(C-->G) mutation that causes hereditary persistence of fetal hemoglobin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 cells. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for the transcriptional regulator Sp1. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HPFH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Purified Sp1 binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPFH probe. The methylation-induced preferential binding of Sp1 to the SSE at the expense of SSP may be part of the mechanism by which the gamma genes are repressed in normal adult erythroid cells. In cells containing the -202 HPFH mutation, the inability of Sp1 to displace SSP in the methylated state may explain the persistence of gamma-promoter activity and gamma-gene expression observed in adults with this mutation.
Subject(s)
DNA/metabolism , Fetal Hemoglobin/genetics , Gene Expression Regulation , Globins/genetics , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , Adult , Aging/genetics , Animals , Base Sequence , Binding Sites , DNA/genetics , Hemoglobinopathies , Humans , Leukemia, Erythroblastic, Acute , Methylation , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Restriction Mapping , Transfection , Tumor Cells, CulturedABSTRACT
The erythroid-specific enhancer within hypersensitivity site 2 (HS2) of the human beta-globin locus control region is required for high level globin gene expression. We investigated interaction between HS2 and the gamma- and beta-promoters using reporter constructs in transient assays in human erythroleukemia (K562) cells. The beta-promoter, usually silent in K562 cells, was activated by HS2. This activity was abolished when a gamma-promoter was linked in cis. Analysis of truncation mutants suggested that sequences conveying the competitive advantage of the gamma-promoter for HS2 included those between positions -53 and -35 relative to the transcriptional start site. This sequence, when used to replace the corresponding region of the beta-promoter, increased beta-promoter activity 10-fold when linked to HS2. The modified beta-promoter was also capable of competing with a gamma-promoter modified internally in the -53 to -35 region, when the two promoters were linked to HS2 in a single plasmid. The corresponding sequences from the Galago gamma-promoter, a species which lacks fetal gamma-gene expression, were inactive in analogous assays. We have identified and partially purified a nuclear protein found in human (fetal stage) erythroleukemia cells, but present in much lower concentration in murine (adult stage) erythroleukemia cells, that binds the -53 to -35 sequence of the gamma-promoter. We speculate that this region of the gamma-promoter functions as a stage selector element in the regulation of hemoglobin switching in humans.
Subject(s)
Enhancer Elements, Genetic , Globins/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Binding Sites , Binding, Competitive , Cell Line , Cell Nucleus/physiology , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Kinetics , Leukemia, Experimental , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Luciferases/genetics , Luciferases/metabolism , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/metabolism , Restriction Mapping , TransfectionABSTRACT
An erythroid specific, inducible enhancer associated with hypersensitive site II (HS II) plays a central role in the function of the human beta globin dominant control region. The HS II enhancer consists of tandem AP-1 binding sites and has been shown to bind members of the ubiquitous jun and fos families of proteins. The same sites are now shown to bind the erythroid specific protein, NF-E2. Inducibility of the HS II enhancer depends on NF-E2 binding, even in the presence of another hypersensitive site. Further, increased activity of the enhancer in induced K562 cells correlates with the presence of NF-E2, which appears to be present in a modified form. NF-E2 is distinct from some enhancer binding proteins in K562 nuclear extracts, in that it does not contain Fos or Fra-1 protein. Thus, binding by NF-E2 may be the mechanism, whereby tandem AP-1 binding sites confer erythroid specificity on the HS II enhancer.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Globins/genetics , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Base Sequence , Cell Line , Erythroid-Specific DNA-Binding Factors , Globins/biosynthesis , Humans , Leukemia, Erythroblastic, Acute , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Oligonucleotide Probes , Plasmids , Promoter Regions, Genetic , TransfectionABSTRACT
A powerful enhancer has been mapped to an 18-bp DNA segment located 11 kb 5' to the human epsilon-globin gene within the dominant control or locus-activating region. This enhancer is inducible in K562 human erythroleukemia cells, increasing linked gamma-globin promoter/luciferase gene expression to 170-fold over an enhancerless construct. The enhancer consists of tandem AP-1-binding sites, phased 10 bp apart, which are both required for full activity. DNA-protein binding assays with nuclear extracts from induced cells demonstrate a high molecular weight complex on the enhancer. The formation of this complex also requires both AP-1 sites and correlates with maximal enhancer activity. Induction of the enhancer may have a role in the increase in globin gene transcription that characterizes erythroid maturation. Enhancer activity appears to be mediated by the binding of a complex of proteins from the jun and fos families to tandem AP-1 consensus sequences.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Erythrocytes/metabolism , Globins/genetics , Transcription Factors/metabolism , Base Sequence , Binding Sites , DNA/genetics , Gene Expression Regulation , HeLa Cells , Humans , Leukemia, Erythroblastic, Acute , Molecular Sequence Data , Proto-Oncogene Proteins c-jun , Restriction Mapping , Tumor Cells, CulturedABSTRACT
Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. We have developed an amphotropic producer clone that generates greater than 10(10) recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone-marrow cells were cocultured with either high (2 x 10(10) colony-forming units/ml) or low (5 x 10(6) colony-forming units/ml) titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer.
Subject(s)
Hematopoietic Stem Cells/cytology , Retroviridae/genetics , Transfection , Animals , Base Sequence , Cell Line , Cells, Cultured , Culture Techniques/methods , DNA, Viral/genetics , DNA, Viral/isolation & purification , HeLa Cells/cytology , Macaca mulatta , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction , RNA, Viral/genetics , RNA, Viral/isolation & purification , Retroviridae/growth & developmentABSTRACT
The normal red blood cell (RBC) membrane is remarkable for its durability (eg, preservation of permeability barrier function) despite its need to remain deformable for the benefit of microvascular blood flow. Yet, it may be hypothesized that the membrane's tolerance of deformation might be compromised under certain pathologic conditions. We studied this by subjecting normal RBC in viscous suspending medium (20% dextran) to elliptical deformation induced by application of shear stress under physiologic conditions (290 mOsm/L, 37 degrees C, pH 7.40) in the presence of ouabain and furosemide. Measurement of resulting net passive K efflux ("K leak") demonstrated that shear-induced RBC deformation causes K leak in a dose-dependent fashion at shear stresses far below the hemolytic threshold, an effect shown to be due to deformation per se. To model the specific hypothesis that oxidatively perturbed RBC membranes would be abnormally susceptible to this potentially adverse effect of deformation, we treated normal RBC with the lipid peroxidant t-butylhydroperoxide. Under conditions inducing only minimal K leak due to either oxidation alone or deformation alone, deformation of peroxidant-pretreated RBC showed a markedly enhanced K leak (P less than .001). This highly synergistic oxidation-plus-deformation leak pathway is less active at low pH, is neither chloride-dependent nor calcium-dependent, and allows K efflux to be balanced by Na influx so there is no change in total monovalent cation content or cell density. Moreover, it is fully reversible since deformation-induced K leak terminates on cessation of shear stress (even for oxidant-treated RBC). Control experiments showed that our results are not explained simply by hemolysis, RBC vesiculation, or development of prelytic pores. We conclude that oxidation and deformation individually promote passive leak of monovalent cation through RBC membranes and that a markedly synergistic effect is exerted when the two stresses are combined. We hypothesize that these findings may help explain the abnormal monovalent cation leak stimulated by deoxygenation of sickle RBC.