RESUMEN
Our current understanding of the kinetics and dynamics of erythroid differentiation is based almost entirely on the ex vivo expansion of cultured hematopoietic progenitor cells. In this study, we used an erythroid-specific, inducible transgenic mouse line to investigate for the first time, the in vivo erythroid differentiation kinetics under steady-state conditions. We demonstrated that bipotent premegakaroycyte/erythroid (PreMegE) progenitor cells differentiate into erythroid-committed proerythroblast/basophilic erythroblasts (ProBasoE) after 6.6 days under steady-state conditions. During this process, each differentiation phase (from PreMegE to precolony forming unit-erythroid [PreCFU-E], PreCFU-E to CFU-E, and CFU-E to ProBasoE) took â¼2 days in vivo. Upon challenge with 5-flurouracil (5-FU), which leads to the induction of stress erythropoiesis, erythroid maturation time was reduced from 6.6 to 4.7 days. Furthermore, anemia induced in 5-FU-treated mice was shown to be due not only to depleted bone marrow erythroid progenitor stores but also to a block in reticulocyte exit from the bone marrow into the circulation, which differed from the mechanism induced by acute blood loss.
Asunto(s)
Anemia , Ratones , Animales , Células Madre Hematopoyéticas , Médula Ósea , Diferenciación Celular , FluorouraciloRESUMEN
Ribosomal protein dysfunction causes diverse human diseases, including Diamond-Blackfan anemia (DBA). Despite the universal need for ribosomes in all cell types, the mechanisms underlying ribosomopathies, which are characterized by tissue-specific defects, are still poorly understood. In the present study, we analyzed the transcriptomes of single purified erythroid progenitors isolated from the bone marrow of DBA patients. These patients were categorized into untreated, glucocorticoid (GC)-responsive and GC-non-responsive groups. We found that erythroid progenitors from untreated DBA patients entered S-phase of the cell cycle under considerable duress, resulting in replication stress and the activation of P53 signaling. In contrast, cell cycle progression was inhibited through induction of the type 1 interferon pathway in treated, GC-responsive patients, but not in GC-non-responsive patients. Notably, a low dose of interferon alpha treatment stimulated the production of erythrocytes derived from DBA patients. By linking the innately shorter cell cycle of erythroid progenitors to DBA pathogenesis, we demonstrated that interferon-mediated cell cycle control underlies the clinical efficacy of glucocorticoids. Our study suggests that interferon administration may constitute a new alternative therapeutic strategy for the treatment of DBA. The trial was registered at www.chictr.org.cn as ChiCTR2000038510.
RESUMEN
Human γ-globin is predominantly expressed in fetal liver erythroid cells during gestation from 2 nearly identical genes, HBG1 and HBG2, that are both perinatally silenced. Reactivation of these fetal genes in adult red blood cells can ameliorate many symptoms associated with the inherited ß-globinopathies, sickle cell disease, and Cooley anemia. Although promising genetic strategies to reactivate the γ-globin genes to treat these diseases have been explored, there are significant barriers to their effective implementation worldwide; alternatively, pharmacological induction of γ-globin synthesis could readily reach the majority of affected individuals. In this study, we generated a CRISPR knockout library that targeted all erythroid genes for which prospective or actual therapeutic compounds already exist. By probing this library for genes that repress fetal hemoglobin (HbF), we identified several novel, potentially druggable, γ-globin repressors, including VHL and PTEN. We demonstrate that deletion of VHL induces HbF through activation of the HIF1α pathway and that deletion of PTEN induces HbF through AKT pathway stimulation. Finally, we show that small-molecule inhibitors of PTEN and EZH induce HbF in both healthy and ß-thalassemic human primary erythroid cells.
Asunto(s)
Talasemia beta , gamma-Globinas , Adulto , Células Eritroides/metabolismo , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Humanos , Estudios Prospectivos , Talasemia beta/genética , Talasemia beta/terapia , gamma-Globinas/genética , gamma-Globinas/metabolismoRESUMEN
Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.
Asunto(s)
Tipificación del Cuerpo , Cara/embriología , Factor de Transcripción GATA3/metabolismo , Animales , Región Branquial/citología , Región Branquial/embriología , Región Branquial/metabolismo , Muerte Celular , Proliferación Celular , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/metabolismo , Embrión de Mamíferos , Factor de Transcripción GATA3/genética , Regulación del Desarrollo de la Expresión Génica , Mandíbula/citología , Mandíbula/embriología , Maxilar/citología , Maxilar/embriología , Ratones , Morfogénesis , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismoRESUMEN
Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of ß-globinopathies (sickle cell disease and ß-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor-like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.
Asunto(s)
Células Eritroides/citología , Eritropoyesis , Histona Demetilasas/metabolismo , Células Mieloides/citología , Animales , Línea Celular , Células Cultivadas , Células Eritroides/metabolismo , Eliminación de Gen , Histona Demetilasas/genética , Humanos , Ratones , Células Mieloides/metabolismoRESUMEN
Mutations in the transcription factor GATA2 cause lymphedema. GATA2 is necessary for the development of lymphatic valves and lymphovenous valves, and for the patterning of lymphatic vessels. Here, we report that GATA2 is not necessary for valvular endothelial cell (VEC) differentiation. Instead, GATA2 is required for VEC maintenance and morphogenesis. GATA2 is also necessary for the expression of the cell junction molecules VE-cadherin and claudin 5 in lymphatic vessels. We identified miR-126 as a target of GATA2, and miR-126-/- embryos recapitulate the phenotypes of mice lacking GATA2. Primary human lymphatic endothelial cells (HLECs) lacking GATA2 (HLECΔGATA2) have altered expression of claudin 5 and VE-cadherin, and blocking miR-126 activity in HLECs phenocopies these changes in expression. Importantly, overexpression of miR-126 in HLECΔGATA2 significantly rescues the cell junction defects. Thus, our work defines a new mechanism of GATA2 activity and uncovers miR-126 as a novel regulator of mammalian lymphatic vascular development.
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Células Endoteliales/metabolismo , Factor de Transcripción GATA2/metabolismo , MicroARNs/metabolismo , Mutación , Angiopoyetina 2/metabolismo , Animales , Sistemas CRISPR-Cas , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Línea Celular , Claudina-5/metabolismo , Familia de Proteínas EGF/metabolismo , Endotelio Vascular/metabolismo , Femenino , Eliminación de Gen , Humanos , Vasos Linfáticos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , RNA-SeqRESUMEN
Human globin gene production transcriptionally "switches" from fetal to adult synthesis shortly after birth and is controlled by macromolecular complexes that enhance or suppress transcription by cis elements scattered throughout the locus. The DRED (direct repeat erythroid-definitive) repressor is recruited to the ε-globin and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA-binding and epigenetic enzyme components (e.g., DNA methyltransferase 1 [DNMT1] and lysine-specific demethylase 1 [LSD1]) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: The deubiquitinase BRCA1-associated protein-1 (BAP1) is a component of the repressor complex whose activity maintains NCoR1 at sites in the ß-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression.
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Regulación de la Expresión Génica/genética , Co-Represor 1 de Receptor Nuclear/genética , Co-Represor 1 de Receptor Nuclear/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , gamma-Globinas/genética , Sitios de Unión , Línea Celular , Activación Enzimática/genética , Epigénesis Genética/genética , Células Eritroides/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Células K562 , Miembro 1 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Dominios Proteicos , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismoRESUMEN
Transcription factor GATA3 plays vital roles in inner ear development, while regulatory mechanisms controlling its inner ear-specific expression are undefined. We demonstrate that a cis-regulatory element lying 571 kb 3' to the Gata3 gene directs inner ear-specific Gata3 expression, which we refer to as the Gata3 otic vesicle enhancer (OVE). In transgenic murine embryos, a 1.5-kb OVE-directed lacZ reporter (TgOVE-LacZ) exhibited robust lacZ expression specifically in the otic vesicle (OV), an inner ear primordial tissue, and its derivative semicircular canal. To further define the regulatory activity of this OVE, we generated Cre transgenic mice in which Cre expression was directed by a 246-bp core sequence within the OVE element (TgcoreOVE-Cre). TgcoreOVE-Cre successfully marked the OV-derived inner ear tissues, including cochlea, semicircular canal and spiral ganglion, when crossed with ROSA26 lacZ reporter mice. Furthermore, Gata3 conditionally mutant mice, when crossed with the TgcoreOVE-Cre, showed hypoplasia throughout the inner ear tissues. These results demonstrate that OVE has a sufficient regulatory activity to direct Gata3 expression specifically in the otic vesicle and semicircular canal and that Gata3 expression driven by the OVE is crucial for normal inner ear development.
Asunto(s)
Oído Interno/crecimiento & desarrollo , Factor de Transcripción GATA3/genética , Regulación del Desarrollo de la Expresión Génica/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.
Asunto(s)
Cromosomas Artificiales de Levadura/genética , Factor de Transcripción GATA3/genética , Mesangio Glomerular/patología , Enfermedades Renales/genética , Animales , Modelos Animales de Enfermedad , Factor de Transcripción GATA3/metabolismo , Mesangio Glomerular/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Ratones , Ratones Transgénicos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , TransgenesRESUMEN
Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.
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Vasos Linfáticos/embriología , Linfedema/embriología , Linfedema/patología , Válvulas Venosas/embriología , Animales , Animales Recién Nacidos , Diferenciación Celular , Modelos Animales de Enfermedad , Células Endoteliales/patología , Células Endoteliales/ultraestructura , Vasos Linfáticos/ultraestructura , Ratones Endogámicos C57BL , Morfogénesis , Penetrancia , Fenotipo , Válvulas Venosas/ultraestructuraRESUMEN
Protein abundance must be precisely regulated throughout life, and nowhere is the stringency of this requirement more evident than during T-cell development: A twofold increase in the abundance of transcription factor GATA3 results in thymic lymphoma, while reduced GATA3 leads to diminished T-cell production. GATA3 haploinsufficiency also causes human HDR (hypoparathyroidism, deafness, and renal dysplasia) syndrome, often accompanied by immunodeficiency. Here we show that loss of one Gata3 allele leads to diminished expansion (and compromised development) of immature T cells as well as aberrant induction of myeloid transcription factor PU.1. This effect is at least in part mediated transcriptionally: We discovered that Gata3 is monoallelically expressed in a parent of origin-independent manner in hematopoietic stem cells and early T-cell progenitors. Curiously, half of the developing cells switch to biallelic Gata3 transcription abruptly at midthymopoiesis. We show that the monoallelic-to-biallelic transcriptional switch is stably maintained and therefore is not a stochastic phenomenon. This unique mechanism, if adopted by other regulatory genes, may provide new biological insights into the rather prevalent phenomenon of monoallelic expression of autosomal genes as well as into the variably penetrant pathophysiological spectrum of phenotypes observed in many human syndromes that are due to haploinsufficiency of the affected gene.
Asunto(s)
Alelos , Factor de Transcripción GATA3/genética , Regulación de la Expresión Génica/genética , Linfocitos T/metabolismo , Animales , Médula Ósea/metabolismo , Proliferación Celular/genética , Células Cultivadas , Factor de Transcripción GATA3/metabolismo , Ratones , Proteínas Proto-Oncogénicas/genética , Timocitos/citología , Timocitos/metabolismo , Transactivadores/genéticaRESUMEN
Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD.
Asunto(s)
Anemia de Células Falciformes/prevención & control , Hemoglobina Fetal/biosíntesis , Histona Demetilasas/antagonistas & inhibidores , Rodaminas/farmacología , Compuestos de Espiro/farmacología , Esplenomegalia/prevención & control , Tiofenos/farmacología , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/patología , Animales , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Hemoglobina Fetal/efectos de los fármacos , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Ratones , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esplenomegalia/sangre , Esplenomegalia/patología , Globinas beta/genética , Globinas beta/metabolismoRESUMEN
The orphan nuclear receptors TR2 and TR4 have been shown to play key roles in repressing the embryonic and fetal globin genes in erythroid cells. However, combined germline inactivation of Tr2 and Tr4 leads to periimplantation lethal demise in inbred mice. Hence, we have previously been unable to examine the consequences of their dual loss of function in adult definitive erythroid cells. To circumvent this issue, we generated conditional null mutants in both genes and performed gene inactivation in vitro in adult bone marrow cells. Compound Tr2/Tr4 loss of function led to induced expression of the embryonic εy and ßh1 globins (murine counterparts of the human ε- and γ-globin genes). Additionally, TR2/TR4 function is required for terminal erythroid cell maturation. Loss of TR2/TR4 abolished their occupancy on the εy and ßh1 gene promoters, and concurrently impaired co-occupancy by interacting corepressors. These data strongly support the hypothesis that the TR2/TR4 core complex is an adult stage-specific, gene-selective repressor of the embryonic globin genes. Detailed mechanistic understanding of the roles of TR2/TR4 and their cofactors in embryonic and fetal globin gene repression may ultimately enhance the discovery of novel therapeutic agents that can effectively inhibit their transcriptional activity and be safely applied to the treatment of ß-globinopathies.
Asunto(s)
Embrión de Mamíferos/metabolismo , Células Eritroides/citología , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Miembro 1 del Grupo C de la Subfamilia 2 de Receptores Nucleares/fisiología , Receptores de Esteroides/fisiología , Receptores de Hormona Tiroidea/fisiología , Globinas beta/metabolismo , Animales , Western Blotting , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Células Eritroides/metabolismo , Citometría de Flujo , Silenciador del Gen , Humanos , Integrasas/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Globinas beta/genéticaRESUMEN
Notch1-Delta-like 4 (Dll4) signalling controls vascular development by regulating endothelial cell (EC) targets that modulate vessel wall remodelling and arterial-venous specification. The molecular effectors that modulate Notch signalling during vascular development remain largely undefined. Here we demonstrate that the transcriptional repressor, Snail1, acts as a VEGF-induced regulator of Notch1 signalling and Dll4 expression. EC-specific Snail1 loss-of-function conditional knockout mice die in utero with defects in vessel wall remodelling in association with losses in mural cell investment and disruptions in arterial-venous specification. Snail1 loss-of-function conditional knockout embryos further display upregulated Notch1 signalling and Dll4 expression that is partially reversed by inhibiting γ-secretase activity in vivo with Dll4 identified as a direct target of Snail1-mediated transcriptional repression. These results document a Snail1-Dll4/Notch1 axis that controls embryonic vascular development.
Asunto(s)
Vasos Sanguíneos/embriología , Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Transición Epitelial-Mesenquimal , Retroalimentación Fisiológica , Técnicas In Vitro , Ratones , Ratones Noqueados , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Remodelación Vascular/genéticaRESUMEN
We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34(+) hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.
Asunto(s)
Células Eritroides/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Sitios de Unión , Células Cultivadas , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Genoma Humano , Humanos , Proteínas Nucleares/química , Proteínas Represoras/químicaRESUMEN
To globally survey the changes in transcriptional landscape during terminal erythroid differentiation, we performed RNA sequencing (RNA-seq) on primary human CD34(+) cells after ex vivo differentiation from the earliest into the most mature erythroid cell stages. This analysis identified thousands of novel intergenic and intronic transcripts as well as novel alternative transcript isoforms. After rigorous data filtering, 51 (presumptive) novel protein-coding transcripts, 5326 long and 679 small non-coding RNA candidates remained. The analysis also revealed two clear transcriptional trends during terminal erythroid differentiation: first, the complexity of transcript diversity was predominantly achieved by alternative splicing, and second, splicing junctional diversity diminished during erythroid differentiation. Finally, 404 genes that were not known previously to be differentially expressed in erythroid cells were annotated. Analysis of the most extremely differentially expressed transcripts revealed that these gene products were all closely associated with hematopoietic lineage differentiation. Taken together, this study will serve as a comprehensive platform for future in-depth investigation of human erythroid development that, in turn, may reveal new insights into multiple layers of the transcriptional regulatory hierarchy that controls erythropoiesis.
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Eritropoyesis/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Adulto , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Sistemas de Lectura Abierta/genética , Isoformas de Proteínas/metabolismo , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , Análisis de Secuencia de ARN , Globinas beta/metabolismoRESUMEN
Peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1α (PGC-1α) and PGC-1ß have been shown to be intimately involved in the transcriptional regulation of cellular energy metabolism as well as other biological processes, but both coactivator proteins are expressed in many other tissues and organs in which their function is, in essence, unexplored. Here, we found that both PGC-1 proteins are abundantly expressed in maturing erythroid cells. PGC-1α and PGC-1ß compound null mutant (Pgc-1(c)) animals express less ß-like globin mRNAs throughout development; consequently, neonatal Pgc-1(c) mice exhibit growth retardation and profound anemia. Flow cytometry shows that the number of mature erythrocytes is markedly reduced in neonatal Pgc-1(c) pups, indicating that erythropoiesis is severely compromised. Furthermore, hematoxylin and eosin staining revealed necrotic cell death and cell loss in Pgc-1(c) livers and spleen. Chromatin immunoprecipitation studies revealed that both PGC-1α and -1ß, as well as two nuclear receptors, TR2 and TR4, coordinately bind to the various globin gene promoters. In addition, PGC-1α and -1ß can interact with TR4 to potentiate transcriptional activation. These data provide new insights into our understanding of globin gene regulation and raise the interesting possibility that the PGC-1 coactivators can interact with TR4 to elicit differential stage-specific effects on globin gene transcription.
Asunto(s)
Eritropoyesis/genética , Miembro 1 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo C de la Subfamilia 2 de Receptores Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Globinas beta/genética , Anemia/genética , Animales , Apoptosis/genética , Recuento de Eritrocitos , Células Eritroides/metabolismo , Retardo del Crecimiento Fetal/genética , Regulación de la Expresión Génica , Hígado/citología , Ratones , Ratones Noqueados , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Regiones Promotoras Genéticas , Bazo/citología , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional/genética , Globinas alfaRESUMEN
BACKGROUND & AIMS: Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3. METHODS: We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively. RESULTS: Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development. CONCLUSIONS: Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Proteínas de Homeodominio/metabolismo , Desarrollo de Músculos/fisiología , Músculo Liso/embriología , Miocitos del Músculo Liso/metabolismo , Píloro/embriología , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Proteína Homeótica Nkx-2.5 , Ratones , Músculo Liso/metabolismo , Píloro/metabolismoRESUMEN
The transcription factor GATA-2 plays vital roles in quite diverse developmental programs, including hematopoietic stem cell (HSC) survival and proliferation. We previously identified a vascular endothelial (VE) enhancer that regulates GATA-2 activity in pan-endothelial cells. To more thoroughly define the in vivo regulatory properties of this enhancer, we generated a tamoxifen-inducible Cre transgenic mouse line using the Gata2 VE enhancer (Gata2 VECre) and utilized it to temporally direct tissue-specific conditional loss of Gata2. Here, we report that Gata2 VECre-mediated loss of GATA-2 led to anemia, hemorrhage, and eventual death in edematous embryos. We further determined that the etiology of anemia in conditional Gata2 mutant embryos involved HSC loss in the fetal liver, as demonstrated by in vitro colony-forming and immunophenotypic as well as in vivo long-term competitive repopulation experiments. We further documented that the edema and hemorrhage in conditional Gata2 mutant embryos were due to defective lymphatic development. Thus, we unexpectedly discovered that in addition to its contribution to endothelial cell development, the VE enhancer also regulates GATA-2 expression in definitive fetal liver and adult BM HSCs, and that GATA-2 function is required for proper lymphatic vascular development during embryogenesis.
Asunto(s)
Anemia/genética , Elementos de Facilitación Genéticos , Muerte Fetal/genética , Factor de Transcripción GATA2/fisiología , Hematopoyesis/genética , Hemorragia/genética , Sistema Linfático/embriología , Anemia/embriología , Animales , División Celular , Supervivencia Celular , Ensayo de Unidades Formadoras de Colonias , Femenino , Factor de Transcripción GATA2/deficiencia , Factor de Transcripción GATA2/genética , Genes Reporteros , Células Madre Hematopoyéticas/patología , Hemorragia/embriología , Inmunofenotipificación , Hígado/citología , Hígado/embriología , Sistema Linfático/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Especificidad de Órganos , Embarazo , Tamoxifeno/farmacologíaRESUMEN
During renal development, the proper emergence of the ureteric bud (UB) from the Wolffian duct is essential for formation of the urinary system. Previously, we showed that expression of transcription factor GATA-2 in the urogenital primordium was demarcated anteroposteriorly into two domains that were regulated by separate enhancers. While GATA-2 expression in the caudal urogenital mesenchyme is controlled by the UG4 enhancer, its more-rostral expression is regulated by UG2. We found that anteriorly displaced budding led to obstructed megaureters in Gata2 hypomorphic mutant mice, possibly due to reduced expression of the downstream effector bone morphogenetic protein 4 (BMP4). Here, we report that UG4-driven, but not UG2-driven, GATA-2 expression in the urogenital mesenchyme significantly reverts the uropathy observed in the Gata2 hypomorphic mutant mice. Furthermore, the data show that transgenic rescue by GATA-2 reverses the rostral outgrowth of the UB. We also provide evidence for a GATA-2-BMP4 epistatic relationship by demonstrating that reporter gene expression from a Bmp4 bacterial artificial chromosome (BAC) transgene is altered in Gata2 hypomorphs; furthermore, UG4-directed BMP4 expression in the mutants leads to reduced incidence of megaureters. These results demonstrate that GATA-2 expression in the caudal urogenital mesenchyme as directed by the UG4 enhancer is crucial for proper development of the urinary tract and that its regulation of BMP4 expression is a critical aspect of this function.