RESUMEN
BCL11A, a repressor of human fetal (γ-)globin expression, is required for immune and hematopoietic stem cell functions and brain development. Regulatory sequences within the gene, which are subject to genetic variation affecting fetal globin expression, display hallmarks of an erythroid enhancer in cell lines and transgenic mice. As such, this enhancer is a novel, attractive target for therapeutic gene editing. To explore the roles of such sequences in vivo, we generated mice in which the orthologous 10-kb intronic sequences were removed. Bcl11a enhancer-deleted mice, Bcl11a(Δenh), phenocopy the BCL11A-null state with respect to alterations of globin expression, yet are viable and exhibit no observable blood, brain, or other abnormalities. These preclinical findings provide strong in vivo support for genetic modification of the enhancer for therapy of hemoglobin disorders.
Asunto(s)
Proteínas Portadoras/metabolismo , Elementos de Facilitación Genéticos/genética , Células Eritroides/metabolismo , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Compartimento Celular , Proteínas de Unión al ADN , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Silenciador del Gen , Humanos , Ratones , Ratones Transgénicos , Proteínas RepresorasRESUMEN
Tyrosine kinase inhibitors (TKI) have revolutionized chronic myelogenous leukemia (CML) management. Disease eradication, however, is hampered by innate resistance of leukemia-initiating cells (LIC) to TKI-induced killing, which also provides the basis for subsequent emergence of TKI-resistant mutants. We report that EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is overexpressed in CML LICs and required for colony formation and survival and cell-cycle progression of CML cell lines. A critical role for EZH2 is supported by genetic studies in a mouse CML model. Inactivation of Ezh2 in conventional conditional mice and through CRISPR/Cas9-mediated gene editing prevents initiation and maintenance of disease and survival of LICs, irrespective of BCR-ABL1 mutational status, and extends survival. Expression of the EZH2 homolog EZH1 is reduced in EZH2-deficient CML LICs, creating a scenario resembling complete loss of PRC2. EZH2 dependence of CML LICs raises prospects for improved therapy of TKI-resistant CML and/or eradication of disease by addition of EZH2 inhibitors. SIGNIFICANCE: This work defines EZH2 as a selective vulnerability for CML cells and their LICs, regardless of BCR-ABL1 mutational status. Our findings provide an experimental rationale for improving disease eradication through judicious use of EZH2 inhibitors within the context of standard-of-care TKI therapy. Cancer Discov; 6(11); 1237-47. ©2016 AACR.See related article by Scott et al., p. 1248This article is highlighted in the In This Issue feature, p. 1197.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/biosíntesis , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 2/biosíntesis , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Complejo Represivo Polycomb 2/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Transducción de SeñalRESUMEN
The ß-hemoglobinopathies are inherited disorders resulting from altered coding potential or expression of the adult ß-globin gene. Impaired expression of ß-globin reduces adult hemoglobin (α2ß2) production, the hallmark of ß-thalassemia. A single-base mutation at codon 6 leads to formation of HbS (α2ßS2) and sickle cell disease. While the basis of these diseases is known, therapy remains largely supportive. Bone marrow transplantation is the only curative therapy. Patients with elevated levels of fetal hemoglobin (HbF, α2γ2) as adults exhibit reduced symptoms and enhanced survival. The ß-globin gene locus is a paradigm of cell- and developmental stage-specific regulation. Although the principal erythroid cell transcription factors are known, mechanisms responsible for silencing of the γ-globin gene were obscure until application of genome-wide association studies (GWAS). Here, we review findings in the field. GWAS identified BCL11A as a candidate negative regulator of γ-globin expression. Subsequent studies have established BCL11A as a quantitative repressor. GWAS-related single-nucleotide polymorphisms lie within an essential erythroid enhancer of the BCL11A gene. Disruption of a discrete region within the enhancer reduces BCL11A expression and induces HbF expression, providing the basis for gene therapy using gene editing tools. A recently identified, second silencing factor, leukemia/lymphoma-related factor/Pokemon, shares features with BCL11A, including interaction with the nucleosome remodeling deacetylase repressive complex. These findings suggest involvement of a common pathway for HbF silencing. In addition, we discuss other factors that may be involved in γ-globin gene silencing and their potential manipulation for therapeutic benefit in treating the ß-hemoglobinopathies.
Asunto(s)
Hemoglobina Fetal/genética , Edición Génica , Estudio de Asociación del Genoma Completo , gamma-Globinas/genética , Proteínas Portadoras/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas RepresorasRESUMEN
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , Proteínas Portadoras/genética , Elementos de Facilitación Genéticos/genética , Ingeniería Genética , Mutagénesis/genética , Proteínas Nucleares/genética , Animales , Secuencia de Bases , Sistemas CRISPR-Cas/genética , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas de Unión al ADN , Eritroblastos/metabolismo , Hemoglobina Fetal/genética , Genoma/genética , Humanos , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Guía de Kinetoplastida/genética , Proteínas Represoras , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the ß-hemoglobinopathies.
Asunto(s)
Proteínas Portadoras/genética , Elementos de Facilitación Genéticos , Células Eritroides/metabolismo , Hemoglobina Fetal/biosíntesis , Regulación de la Expresión Génica , Hemoglobinopatías/genética , Proteínas Nucleares/genética , Animales , Línea Celular Tumoral , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Hemoglobina Fetal/genética , Marcación de Gen , Ingeniería Genética , Variación Genética , Estudio de Asociación del Genoma Completo , Hemoglobinopatías/terapia , Humanos , Ratones , Células Precursoras de Linfocitos B/metabolismo , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization.We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation.
Asunto(s)
Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/química , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Activación Enzimática , Humanos , Células Progenitoras de Megacariocitos/citología , Células Progenitoras de Megacariocitos/efectos de los fármacos , Células Progenitoras de Megacariocitos/metabolismo , Megacariocitos/efectos de los fármacos , Ratones , Multimerización de Proteína , Factor de Respuesta Sérica/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Trombopoyetina/farmacología , Transactivadores/metabolismoRESUMEN
Serum response factor and its transcriptional cofactor MKL1 are critical for megakaryocyte maturation and platelet formation. We show that MKL2, a homologue of MKL1, is expressed in megakaryocytes and plays a role in megakaryocyte maturation. Using a megakaryocyte-specific Mkl2 knockout (KO) mouse on the conventional Mkl1 KO background to produce double KO (DKO) megakaryocytes and platelets, a critical role for MKL2 is revealed. The decrease in megakaryocyte ploidy and platelet counts of DKO mice is more severe than in Mkl1 KO mice. Platelet dysfunction in DKO mice is revealed by prolonged bleeding times and ineffective platelet activation in vitro in response to adenosine 5'-diphosphate. Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. Surprisingly, the DKO mice have a more extreme thrombocytopenia than mice lacking serum response factor (SRF) expression in the megakaryocyte compartment. Comparison of gene expression reveals approximately 4400 genes whose expression is differentially affected in DKO compared with megakaryocytes deficient in SRF, strongly suggesting that MKL1 and MKL2 have both SRF-dependent and SRF-independent activity in megakaryocytopoiesis.
Asunto(s)
Plaquetas/citología , Plaquetas/metabolismo , Hematopoyesis , Megacariocitos/citología , Megacariocitos/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Adenosina Difosfato/metabolismo , Animales , Tiempo de Sangría , Plaquetas/ultraestructura , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Cruzamientos Genéticos , Citoplasma/metabolismo , Citoplasma/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Perfilación de la Expresión Génica , Megacariocitos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Activación Plaquetaria , Trombocitopenia/etiología , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
Lymphatic vessels (LVs) are important structures for antigen presentation, for lipid metabolism, and as conduits for tumor metastases, but they have been difficult to visualize in vivo. Prox1 is a transcription factor that is necessary for lymphangiogenesis in ontogeny and the maintenance of LVs. To visualize LVs in the lymph node of a living mouse in real time, we made the ProxTom transgenic mouse in a C57BL/6 background using red fluorescent LVs that are suitable for in vivo imaging. The ProxTom transgene contained all Prox1 regulatory sequences and was faithfully expressed in LVs coincident with endogenous Prox1 expression. The progenies of a ProxTom × Hec6stGFP cross were imaged using two-photon laser scanning microscopy, allowing the simultaneous visualization of LVs and high endothelial venules in a lymph node of a living mouse for the first time. We confirmed the expression of Prox1 in the adult liver, lens, and dentate gyrus. These intensely fluorescent mice revealed the expression of Prox1 in three novel sites: the neuroendocrine cells of the adrenal medulla, megakaryocytes, and platelets. The novel sites identified herein suggest previously unknown roles for Prox1. The faithful expression of the fluorescent reporter in ProxTom LVs indicates that these mice have potential utility in the study of diseases as diverse as lymphedema, filariasis, transplant rejection, obesity, and tumor metastasis.