RESUMEN
The fetal-to-adult hemoglobin switching at about the time of birth involves a shift in expression from γ-globin to ß-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and ß-thalassemia. Despite the physiological and clinical relevance of this switch, its posttranscriptional regulation is poorly understood. Here, we identify Pumilo 1 (PUM1), an RNA-binding protein with no previously reported functions in erythropoiesis, as a direct posttranscriptional regulator of ß-globin switching. PUM1, whose expression is regulated by the erythroid master transcription factor erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid differentiation, binds γ-globin messenger RNA (mRNA), and reduces γ-globin (HBG1) mRNA stability and translational efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase in fetal hemoglobin (â¼22% HbF) without affecting ß-globin levels in human erythroid cells. Importantly, targeting PUM1 does not limit the progression of erythropoiesis, which provides a potentially safe and effective treatment strategy for sickle cell anemia and ß-thalassemia. In support of this idea, we report elevated levels of HbF in the absence of anemia in an individual with a novel heterozygous PUM1 mutation in the RNA-binding domain (p.(His1090Profs∗16); c.3267_3270delTCAC), which suggests that PUM1-mediated posttranscriptional regulation is a critical player during human hemoglobin switching.
Asunto(s)
Anemia de Células Falciformes , Talasemia beta , Adulto , Humanos , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , gamma-Globinas/genética , gamma-Globinas/metabolismo , Talasemia beta/genética , Globinas beta/genética , Proteínas Portadoras , Anemia de Células Falciformes/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Erythroblastic islands are a specialized niche that contain a central macrophage surrounded by erythroid cells at various stages of maturation. However, identifying the precise genetic and transcriptional control mechanisms in the island macrophage remains difficult due to macrophage heterogeneity. Using unbiased global sequencing and directed genetic approaches focused on early mammalian development, we find that fetal liver macrophages exhibit a unique expression signature that differentiates them from erythroid and adult macrophage cells. The importance of erythroid Krüppel-like factor (EKLF)/KLF1 in this identity is shown by expression analyses in EKLF-/- and in EKLF-marked macrophage cells. Single-cell sequence analysis simplifies heterogeneity and identifies clusters of genes important for EKLF-dependent macrophage function and novel cell surface biomarkers. Remarkably, this singular set of macrophage island cells appears transiently during embryogenesis. Together, these studies provide a detailed perspective on the importance of EKLF in the establishment of the dynamic gene expression network within erythroblastic islands in the developing embryo and provide the means for their efficient isolation.
Asunto(s)
Eritropoyesis/genética , Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismoRESUMEN
Erythroid Krüppel-like Factor (EKLF/KLF1) is an erythroid-enriched transcription factor that plays a global role in all aspects of erythropoiesis, including cell cycle control and differentiation. We queried whether its mutation might play a role in red cell malignancies by genomic sequencing of the KLF1 transcription unit in cell lines, erythroid neoplasms, dysplastic disorders, and leukemia. In addition, we queried published databases from a number of varied sources. In all cases we only found changes in commonly notated SNPs. Our results suggest that if there are mutations in KLF1 associated with erythroid malignancies, they are exceedingly rare.
Asunto(s)
Genética de Población , Factores de Transcripción de Tipo Kruppel/metabolismo , Mutación , Transcripción Genética , Alelos , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Mapeo Cromosómico , Células Eritroides/citología , Células Eritroides/metabolismo , Eritropoyesis/genética , Variación Genética , Genómica/métodos , Humanos , Factores de Transcripción de Tipo Kruppel/clasificación , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
PURPOSE OF REVIEW: Transcriptional regulators provide the molecular and biochemical basis for the cell specific properties and characteristics that follow from their central role in establishing tissue-restricted expression. Precise and sequential control of terminal cell divisions, nuclear condensation, and enucleation are defining characteristics within erythropoietic differentiation. This review is focused on KLF1, a central global regulator of this process. RECENT FINDINGS: Studies in the past year have brought a number of proteins that are targets of KLF1 regulation into focus with respect to their roles in terminal erythroid differentiation. Many of these are involved in fine control of the cell cycle at both early (E2F2, Cyclin A2) and later (p18, p27, p19) stages of differentiation, or are directly involved in enucleation (p18, p27). Dramatic biophysical changes controlled at the nuclear lamin by caspase 3 enable histone release and nuclear condensation, whereas dematin association with structural proteins alters the timing of enucleation. Conditional ablation of mDia2 has established its role in late stage cell cycle and enucleation. SUMMARY: Transcription factors such as KLF1, along with epigenetic modifiers, play crucial roles in establishing the proper onset and progression of terminal differentiation events. Studies from the past year show a remarkable multifaceted convergence on cell cycle control, and establish that the orthochromatic erythroblast stage is a critical nodal point for many of the effects on enucleation. These studies are relevant to understanding the underlying causes of anemia and hematologic disease where defective enucleation predicts a poor clinical outcome.
Asunto(s)
Eritropoyesis , Factores de Transcripción de Tipo Kruppel/metabolismo , Animales , Ciclo Celular/genética , Diferenciación Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Células Eritroides/citología , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
The mechanisms regulating the sequential steps of terminal erythroid differentiation remain largely undefined, yet are relevant to human anemias that are characterized by ineffective red cell production. Erythroid Krüppel-like Factor (EKLF/KLF1) is a master transcriptional regulator of erythropoiesis that is mutated in a subset of these anemias. Although EKLF's function during early erythropoiesis is well studied, its role during terminal differentiation has been difficult to functionally investigate due to the impaired expression of relevant cell surface markers in Eklf(-/-) erythroid cells. We have circumvented this problem by an innovative use of imaging flow cytometry to investigate the role of EKLF in vivo and have performed functional studies using an ex vivo culture system that enriches for terminally differentiating cells. We precisely define a previously undescribed block during late terminal differentiation at the orthochromatic erythroblast stage for Eklf(-/-) cells that proceed beyond the initial stall at the progenitor stage. These cells efficiently decrease cell size, condense their nucleus, and undergo nuclear polarization; however, they display a near absence of enucleation. These late-stage Eklf(-/-) cells continue to cycle due to low-level expression of p18 and p27, a new direct target of EKLF. Surprisingly, both cell cycle and enucleation deficits are rescued by epistatic reintroduction of either of these 2 EKLF target cell cycle inhibitors. We conclude that the cell cycle as regulated by EKLF during late stages of differentiation is inherently critical for enucleation of erythroid precursors, thereby demonstrating a direct functional relationship between cell cycle exit and nuclear expulsion.
Asunto(s)
Núcleo Celular/metabolismo , Embrión de Mamíferos/metabolismo , Eritroblastos/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Animales , Sitios de Unión , Puntos de Control del Ciclo Celular , Diferenciación Celular , Células Cultivadas , Embrión de Mamíferos/citología , Eritroblastos/citología , Eritropoyesis/fisiología , Regulación del Desarrollo de la Expresión Génica , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production in vitro. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated Foxo3-/- erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued Foxo3-/- erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.
Asunto(s)
Eritrocitos/metabolismo , Eritropoyesis/genética , Factores de Transcripción Forkhead/genética , Biología de Sistemas , Animales , Autofagia/genética , Células de la Médula Ósea/metabolismo , Polaridad Celular/genética , Eritroblastos/metabolismo , Eritrocitos/citología , Citometría de Flujo , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Homeostasis , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/metabolismoRESUMEN
Alternative splicing has emerged as a vital way to expand the functional repertoire of a set number of mammalian genes. For example, such changes can dramatically alter the function and cellular localization of transcription factors. With this in mind, we addressed whether EKLF/KLF1 mRNA, coding for a transcription factor that plays a critical role in erythropoietic gene regulation, is alternatively spliced. We find that EKLF mRNA undergoes exon skipping only in primary tissues and that this splice variant (SV) remains at a very low level in both embryonic and adult erythroid cells, as well as during terminal differentiation. The resultant protein is truncated and partially encodes a non-erythroid Krüppel-like factor amino acid sequence. Its overexpression can alter full-length erythroid Krüppel-like factor function at selected promoters. We discuss these results in the context of stress and with respect to recent global studies on the role of alternative splicing during terminal erythroid differentiation.
Asunto(s)
Empalme Alternativo , Células Eritroides/metabolismo , Eritropoyesis/genética , Factores de Transcripción de Tipo Kruppel/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Linaje de la Célula , Femenino , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Células K562 , Factores de Transcripción de Tipo Kruppel/fisiología , Leucemia Eritroblástica Aguda/patología , Ratones , Datos de Secuencia Molecular , Flebotomía , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Bazo/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
The erythroblastic island provides an important nutritional and survival support niche for efficient erythropoietic differentiation. Island integrity is reliant on adhesive interactions between erythroid and macrophage cells. We show that erythroblastic islands can be formed from single progenitor cells present in differentiating embryoid bodies, and that these correspond to erythro-myeloid progenitors (EMPs) that first appear in the yolk sac of the early developing embryo. Erythroid Krüppel-like factor (EKLF; KLF1), a crucial zinc finger transcription factor, is expressed in the EMPs, and plays an extrinsic role in erythroid maturation by being expressed in the supportive macrophage of the erythroblastic island and regulating relevant genes important for island integrity within these cells. Together with its well-established intrinsic contributions to erythropoiesis, EKLF thus plays a coordinating role between two different cell types whose interaction provides the optimal environment to generate a mature red blood cell.
Asunto(s)
Eritrocitos/citología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Nicho de Células Madre , Animales , Adhesión Celular , Diferenciación Celular , Membrana Celular/metabolismo , Células Madre Embrionarias/citología , Eritroblastos/citología , Células Precursoras Eritroides/citología , Eritropoyesis/fisiología , Macrófagos/citología , Ratones , Regiones Promotoras Genéticas , Reticulocitos/citología , Células Madre/citología , Saco Vitelino/fisiología , Dedos de ZincRESUMEN
KLF1 is an erythroid specific transcription factor that is involved in erythroid lineage commitment, globin switching and terminal red blood cell maturation. Various mutations of KLF1 have been identified in humans, which have led to both benign and pathological phenotypes. The E325K mutation, within the second zinc finger of the KLF1 gene, has been shown to cause a new form of congenital dyserythropoietic anemia (CDA) now labeled as CDA type IV. We report the fourth documented case of this mutation, and propose a clinical diagnostic model to better identify this disease in other patients. Our patient is a Taiwanese child who presented to us at 8years of age with severe hemolytic anemia, splenomegaly, elevated fetal hemoglobin (HbF), iron overload, and dyserythropoiesis in the bone marrow. KLF1 sequence analysis revealed a G-to-A transition in one allele of exon 3, which resulted in the substitution of a glutamate 325 by a lysine. Flow cytometry analysis revealed decreased protein expression of CD44 on the red blood cells, and decreased red blood cell deformability as measured using an ektacytometer. Blood typing revealed his red blood cells to be Co(a-b-), In(b-), LW(ab-) and Lu(b+), even though DNA testing predicted that he would be Co(a+b-) and LW(a+b-). This newly discovered CDA combines features of a hemoglobinopathy, RBC membrane defect and hereditary persistence of HbF (HPFH) which are not seen in the previous types of CDA. Increased awareness of this phenotype may improve the more prompt and accurate diagnosis of these patients.
Asunto(s)
Anemia Diseritropoyética Congénita/diagnóstico , Anemia Diseritropoyética Congénita/genética , Pueblo Asiatico/genética , Factores de Transcripción de Tipo Kruppel/genética , Mutación , Anemia Diseritropoyética Congénita/terapia , Médula Ósea/patología , Niño , Análisis Mutacional de ADN , Membrana Eritrocítica/metabolismo , Humanos , Masculino , Fragilidad Osmótica/genética , TaiwánRESUMEN
An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate ß-type globin gene disorders such as sickle cell anemia and ß-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD, play a role in γ-globin gene silencing, and Mi2ß (CHD4) is a critical component of NuRD complexes. We observed that knockdown of Mi2ß relieves γ-globin gene silencing in ß-YAC transgenic murine chemical inducer of dimerization hematopoietic cells and in CD34(+) progenitor-derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2ß binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during ß-type globin gene switching. Remarkably, <50% knockdown of Mi2ß is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34(+) progenitors. These results indicate that Mi2ß is a potential target for therapeutic induction of fetal hemoglobin.
Asunto(s)
Autoantígenos/metabolismo , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Regulación de la Expresión Génica , Silenciador del Gen , Células Madre Hematopoyéticas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , gamma-Globinas/genética , Adulto , Animales , Autoantígenos/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Células Eritroides/citología , Hemoglobina Fetal/antagonistas & inhibidores , Hemoglobina Fetal/metabolismo , Células Madre Hematopoyéticas/citología , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Ratones Transgénicos , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/genética , Proteínas Represoras , gamma-Globinas/antagonistas & inhibidores , gamma-Globinas/metabolismoRESUMEN
Methyl cytosine binding domain protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where repatterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable short hairpin RNA (shRNA)-mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak antiproliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in Bagg Albino (BALB)/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by derepression of tumor suppressor genes, including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing show MBD2 binding directly to the hyper methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG island-associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and resilencing of the MBD2-dependent tumor suppressor genes. Our data suggest that uncoupling CpG methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results show a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Unión al ADN/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Islas de CpG/genética , Metilación de ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Estadificación de Neoplasias , ARN Interferente Pequeño , Células Tumorales CultivadasRESUMEN
Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal ß-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epigénesis Genética/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Modelos Moleculares , Proteínas Represoras/metabolismo , Western Blotting , Metilación de ADN/genética , Cartilla de ADN/genética , Silenciador del Gen , Humanos , Inmunoprecipitación , Interferencia de ARNRESUMEN
During erythroid development, the embryonic ε-globin gene becomes silenced as erythropoiesis shifts from the yolk sac to the fetal liver where γ-globin gene expression predominates. Previous studies have shown that the ε-globin gene is autonomously silenced through promoter proximal cis-acting sequences in adult erythroid cells. We have shown a role for the methylcytosine binding domain protein 2 (MBD2) in the developmental silencing of the avian embryonic ρ-globin and human fetal γ-globin genes. To determine the roles of MBD2 and DNA methylation in human ε-globin gene silencing, transgenic mice containing all sequences extending from the 5' hypersensitive site 5 (HS5) of the ß-globin locus LCR to the human γ-globin gene promoter were generated. These mice show correct developmental expression and autonomous silencing of the transgene. Either the absence of MBD2 or treatment with the DNA methyltransferase inhibitor 5-azacytidine increases ε-globin transgene expression by 15-20 fold in adult mice. Adult mice containing the entire human ß-globin locus also show an increase in expression of both the ε-globin gene transgene and endogenous ε(Y) and ß(H1) genes in the absence of MBD2. These results indicate that the human ε-globin gene is subject to multilayered silencing mediated in part by MBD2.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Globinas épsilon/genética , Animales , Azacitidina/farmacología , Metilación de ADN , Eritroblastos/metabolismo , Eritrocitos/metabolismo , Femenino , Orden Génico , Hemoglobinas Anormales/metabolismo , Humanos , Región de Control de Posición/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Globinas beta/metabolismo , gamma-Globinas/metabolismoRESUMEN
The sequence complexity of the known vertebrate genomes alone is insufficient to account for the diversity between individuals of a species. Although our knowledge of vertebrate biology has evolved substantially with the growing compilation of sequenced genomes, understanding the temporal and spatial regulation of genes remains fundamental to fully exploiting this information. The importance of epigenetic factors in gene regulation was first hypothesized decades ago when biologists posited that methylation of DNA could heritably alter gene expression [Holliday and Pugh, 1975. Science 187(4173), 226-232; Riggs, 1975. Cytogenet. and Cell Genet.14(1), 9-25; Scarano et al., 1967. Proc. Natl. Acad. Sci. USA 57(5), 1394-1400)]. It was subsequently shown that vertebrate DNA methylation, almost exclusively at the 5' position of cytosine in the dinucleotide CpG, played a role in a number of processes including embryonic development, genetic imprinting, cell differentiation, and tumorigenesis. At the time of this writing, a large and growing list of genes is known to exhibit DNA methylation-dependent regulation, and we understand in some detail the mechanisms employed by cells in using methylation as a regulatory modality. In this context, we revisit one of the original systems in which the role of DNA methylation in vertebrate gene regulation during development was described and studied: erythroid cells. We briefly review the recent advances in our understanding of DNA methylation and, in particular, its regulatory role in red blood cells during differentiation and development. We also address DNA methylation as a component of erythroid chromatin architecture, and the interdependence of CpG methylation and histone modification.