RESUMO
The human ankyrin-1 gene (ANK1) contains 3 tissue-specific alternative promoters. We have shown previously that the erythroid-specific ankyrin 1 (ANK1E) core promoter contains a 5' DNase I hypersensitive site (HS) with barrier insulator function that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and hereditary spherocytosis. In this report, we demonstrate a second ANK1E regulatory element located in an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter at the 3' boundary of an erythroid-specific DNase I-sensitive chromatin domain. The 3' regulatory element exhibits enhancer activity in vitro and in transgenic mice, and it has the histone modifications associated with an enhancer element. One of the ANK1E 3'HS contains an NF-E2 binding site that is required for enhancer function. We show that a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including the ANK1E core promoter. These observations demonstrate a model for the tissue-specific activation of alternative promoters that may be applicable to the â¼ 30% of mammalian genes with alternative promoters that exhibit distinct expression patterns.
Assuntos
Anquirinas/genética , Cromatina/genética , Elementos Facilitadores Genéticos , Elementos Isolantes , Subunidade p45 do Fator de Transcrição NF-E2/genética , Regiões Promotoras Genéticas , Esferocitose Hereditária/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Animais , Anquirinas/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Camundongos , Camundongos Transgênicos , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Especificidade de Órgãos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esferocitose Hereditária/metabolismoRESUMO
The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or beta-spectrin in mouse fetal liver cells. When linked to a human gamma-globin gene, two different AE1 promoters directed erythroid-specific expression of gamma-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of gamma-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of gamma-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the insulator element from 5'HS4 of the chicken beta-globin cluster to the AE1/gamma-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/gamma-globin transgene mapped to the start site of the endogenous AE1 mRNA, and gamma-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken beta-globin 5'HS4 element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , beta-Globulinas/genética , Elementos Isolantes , Sítio de Iniciação de Transcrição , Transcrição Gênica , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Núcleo Celular/genética , Galinhas/genética , Membrana Eritrocítica/genética , Regulação da Expressão Gênica , Globinas/genética , Humanos , Fígado/embriologia , Fígado/fisiologia , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
Defects of the ankyrin-1 gene are the most common cause in humans of hereditary spherocytosis, an inherited anemia that affects patients of all ethnic groups. In some kindreds, linked -108/-153 nucleotide substitutions have been found in the upstream region of the ankyrin gene promoter that is active in erythroid cells. In vivo, the ankyrin erythroid promoter and its upstream region direct position-independent, uniform expression, a property of barrier insulators. Using human erythroid cell lines and primary cells and transgenic mice, here we have demonstrated that a region upstream of the erythroid promoter is a barrier insulator in vivo in erythroid cells. The region exhibited both functional and structural characteristics of a barrier, including prevention of gene silencing in an in vivo functional assay, appropriate chromatin configuration, and occupancy by barrier-associated proteins. Fragments with the -108/-153 spherocytosis-associated mutations failed to function as barrier insulators in vivo and demonstrated perturbations in barrier-associated chromatin configuration. In transgenic mice, flanking a mutant -108/-153 ankyrin gene promoter with the well-characterized chicken HS4 barrier insulator restored position-independent, uniform expression at levels comparable to wild-type. These data indicate that an upstream region of the ankyrin-1 erythroid promoter acts as a barrier insulator and identify disruption of the barrier element as a potential pathogenetic mechanism of human disease.
Assuntos
Anquirinas/genética , Elementos Isolantes , Proteínas Mutantes/genética , Mutação , Esferocitose Hereditária/genética , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Cromatina/genética , Primers do DNA/genética , Desoxirribonuclease I , Células Eritroides/metabolismo , Expressão Gênica , Inativação Gênica , Humanos , Células K562 , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Regiões Promotoras Genéticas , Esferocitose Hereditária/sangueRESUMO
The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a beta-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa beta-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Anquirinas/sangue , Eritrócitos/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Sítios de Ligação , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Citoplasma/metabolismo , Deformação Eritrocítica , Membrana Eritrocítica/ultraestrutura , Eritrócitos/citologia , Éxons , Glicina , Camundongos , Camundongos Transgênicos , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
Hmgb3 is an X-linked member of a family of chromatin-binding proteins that is expressed in primitive hematopoietic cells capable of long-term hematopoietic repopulation. To examine the role of Hmgb3 in adult hematopoiesis, we generated Hmgb3-deficient (Hmgb3(-/Y)) mice, which are viable but erythrocythemic. Hmgb3(-/Y) mice contain normal numbers of hematopoietic stem cells (HSCs), which generate fewer than normal numbers of common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs) and greater than normal numbers of more mature progenitors. Although fewer Hmgb3(-/Y) primitive progenitor cells are in the G2/M cell cycle phase, bromodeoxyuridine (BrdU) incorporation demonstrated enhanced proliferation compared with their wild-type counterparts. Hmgb3(-/Y) HSCs have increased levels of Gata-2 and c-myb mRNA. We propose that Hmgb3 deficiency leads to a failure of HSCs to expand into normal numbers of CLPs and CMPs. This defect is compensated for by the ability of Hmgb3(-/Y) progenitors to expand rapidly and differentiate into normal numbers of hematopoietic cells.
Assuntos
Linfócitos B/citologia , Proteína HMGB3/genética , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/fisiologia , Linfócitos T/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA2 , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/farmacologia , Proteína HMGB3/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Proteínas Proto-Oncogênicas c-myb/genética , Fatores de Transcrição/genéticaRESUMO
alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Precursoras Eritroides/metabolismo , Regiões Promotoras Genéticas , Espectrina/genética , Fatores de Transcrição/metabolismo , Região 5'-Flanqueadora , Animais , Sequência de Bases , Sítios de Ligação , DNA Complementar/química , DNA Complementar/isolamento & purificação , Fatores de Ligação de DNA Eritroide Específicos , Eritropoese , Fator de Transcrição GATA1 , Células HeLa , Humanos , Células K562 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2RESUMO
Hmgb3 is a member of a family of chromatin-binding proteins that can alter DNA structure to facilitate transcription factor binding. We identified the Hmgb3 cDNA in a subtractive hybridization screen for transcripts that are preferentially expressed in hematopoietic stem cells. We inserted an internal ribosomal entry site-green fluorescence protein cassette into the 3' untranslated region of the X-linked Hmgb3 locus to identify Hmgb3-expressing cells. In adult mice, Hmgb3 mRNA is detected in bone marrow cells, primitive Lin-, c-kit+, Sca-1+, IL-7Ralpha- cells, and Ter119+ erythroid cells. We observed that long-term repopulating ability is entirely contained in the subpopulation of Lin-, c-kitHI cells that express Hmgb3. Most common lymphoid and myeloid progenitors express Hmgb3. Introduction of a retrovirus containing the Hmgb3 cDNA into mouse bone marrow stem cells demonstrated that enforced expression of Hmgb3 inhibited B-cell and myeloid differentiation. We conclude that down-regulation of Hmgb3 protein levels is an important step for myeloid and B-cell differentiation.