Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Ther Nucleic Acids ; 31: 452-465, 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36852088

RESUMO

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the ß-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) ß-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the ß-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human ß-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

2.
Nat Commun ; 11(1): 5913, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33219211

RESUMO

Over the last 3 decades ATP-dependent chromatin remodelers have been thought to recognize chromatin at the level of single nucleosomes rather than higher-order organization of more than one nucleosome. We show the yeast ISW1a remodeler has such higher-order structural specificity, as manifested by large allosteric changes that activate the nucleosome remodeling and spacing activities of ISW1a when bound to dinucleosomes. Although the ATPase domain of Isw1 docks at the SHL2 position when ISW1a is bound to either mono- or di-nucleosomes, there are major differences in the interactions of the catalytic subunit Isw1 with the acidic pocket of nucleosomes and the accessory subunit Ioc3 with nucleosomal DNA. By mutational analysis and uncoupling of ISW1a's dinucleosome specificity, we find that dinucleosome recognition is required by ISW1a for proper chromatin organization at promoters; as well as transcription regulation in combination with the histone acetyltransferase NuA4 and histone H2A.Z exchanger SWR1.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Animais , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Salmão , Fatores de Transcrição/metabolismo , Xenopus
3.
Cell Rep ; 28(1): 282-294.e6, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269447

RESUMO

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/genética , Histonas/metabolismo , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico/genética , Simulação por Computador , Pegada de DNA , Histonas/química , Espectrometria de Massas , Modelos Moleculares , Nucleossomos/química , Ligação Proteica , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
4.
Blood Adv ; 3(10): 1586-1597, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31126914

RESUMO

Reactivation of fetal hemoglobin (HbF) production benefits patients with sickle cell disease and ß-thalassemia. To identify new HbF regulators that might be amenable to pharmacologic control, we screened a protein domain-focused CRISPR-Cas9 library targeting chromatin regulators, including BTB domain-containing proteins. Speckle-type POZ protein (SPOP), a substrate adaptor of the CUL3 ubiquitin ligase complex, emerged as a novel HbF repressor. Depletion of SPOP or overexpression of a dominant negative version significantly raised fetal globin messenger RNA and protein levels with minimal detrimental effects on normal erythroid maturation, as determined by transcriptome and proteome analyses. SPOP controls HbF expression independently of the major transcriptional HbF repressors BCL11A and LRF. Finally, pharmacologic HbF inducers cooperate with SPOP depletion during HbF upregulation. Our study implicates SPOP and the CUL3 ubiquitin ligase system in controlling HbF production in human erythroid cells and may offer new therapeutic strategies for the treatment of ß-hemoglobinopathies.


Assuntos
Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Feminino , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Adulto Jovem
5.
Cytotherapy ; 20(10): 1278-1287, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30249524

RESUMO

BACKGROUND AIMS: γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. METHODS: We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. RESULTS: Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups (P < 0.01). DISCUSSION: In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.


Assuntos
Técnicas de Cultura de Células/métodos , Terapia Genética/métodos , Células-Tronco Hematopoéticas/citologia , gama-Globinas/genética , Animais , Diferenciação Celular , Cromatografia Líquida de Alta Pressão/métodos , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/terapia , Hemoglobinas/análise , Humanos , Proteínas com Domínio LIM/genética , Macaca mulatta , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Transdução Genética , Dedos de Zinco/genética , gama-Globinas/análise
6.
Science ; 361(6399): 285-290, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30026227

RESUMO

Increasing fetal hemoglobin (HbF) levels in adult red blood cells provides clinical benefit to patients with sickle cell disease and some forms of ß-thalassemia. To identify potentially druggable HbF regulators in adult human erythroid cells, we employed a protein kinase domain-focused CRISPR-Cas9-based genetic screen with a newly optimized single-guide RNA scaffold. The screen uncovered the heme-regulated inhibitor HRI (also known as EIF2AK1), an erythroid-specific kinase that controls protein translation, as an HbF repressor. HRI depletion markedly increased HbF production in a specific manner and reduced sickling in cultured erythroid cells. Diminished expression of the HbF repressor BCL11A accounted in large part for the effects of HRI depletion. Taken together, these results suggest HRI as a potential therapeutic target for hemoglobinopathies.


Assuntos
Anemia Falciforme/genética , Proteínas de Transporte/genética , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , eIF-2 Quinase/genética , Anemia Falciforme/tratamento farmacológico , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , Linhagem Celular , Testes Genéticos , Humanos , Terapia de Alvo Molecular , Proteínas Nucleares/metabolismo , RNA Guia de Cinetoplastídeos , Proteínas Repressoras , eIF-2 Quinase/metabolismo
7.
Nat Commun ; 8: 15616, 2017 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-28604691

RESUMO

ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3-H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A-H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A-H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z-H2B dimers out of nucleosomes and replacement by H2A-H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.


Assuntos
Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina/genética , DNA Fúngico/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Saccharomyces cerevisiae/metabolismo
8.
Nat Struct Mol Biol ; 20(2): 222-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23334290

RESUMO

The ISWI family of ATP-dependent chromatin remodelers represses transcription by changing nucleosome positions. ISWI regulates nucleosome positioning by requiring a minimal length of extranucleosomal DNA for moving nucleosomes. ISW2 from Saccharomyces cerevisiae, a member of the ISWI family, has a conserved domain called SLIDE (SANT-like ISWI domain) that binds to extranucleosomal DNA ~19 base pairs from the edge of nucleosomes. Loss of SLIDE binding does not perturb binding of the ATPase domain or the initial movement of DNA inside of nucleosomes. Not only is extranucleosomal DNA required to help recruit ISW2, but also the interactions of the SLIDE domain with extranucleosomal DNA are functionally required to move nucleosomes.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Centrifugação com Gradiente de Concentração , Montagem e Desmontagem da Cromatina/genética , Transferência Ressonante de Energia de Fluorescência , Regulação Fúngica da Expressão Gênica/genética , Radical Hidroxila/metabolismo , Marcadores de Fotoafinidade , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...