RESUMEN
GATA factors establish transcriptional networks that control fundamental developmental processes. Whereas the regulator of hematopoiesis GATA-1 is subject to multiple posttranslational modifications, how these modifications influence GATA-1 function at endogenous loci is unknown. We demonstrate that sumoylation of GATA-1 K137 promotes transcriptional activation only at target genes requiring the coregulator Friend of GATA-1 (FOG-1). A mutation of GATA-1 V205G that disrupts FOG-1 binding and K137 mutations yielded similar phenotypes, although sumoylation was FOG-1 independent, and FOG-1 binding did not require sumoylation. Both mutations dysregulated GATA-1 chromatin occupancy at select sites, FOG-1-dependent gene expression, and were rescued by tethering SUMO-1. While FOG-1- and SUMO-1-dependent genes migrated away from the nuclear periphery upon erythroid maturation, FOG-1- and SUMO-1-independent genes persisted at the periphery. These results illustrate a mechanism that controls trans-acting factor function in a locus-specific manner, and differentially regulated members of the target gene ensemble reside in distinct subnuclear compartments.
Asunto(s)
Factor de Transcripción GATA1/metabolismo , Hematopoyesis/fisiología , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Factor de Transcripción GATA1/genética , Regulación de la Expresión Génica , Ratones , Mutación , Proteínas Nucleares/genética , Unión Proteica , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The unremitting demand to replenish differentiated cells in tissues requires efficient mechanisms to generate and regulate stem and progenitor cells. Although master regulatory transcription factors, including GATA binding protein-2 (GATA-2), have crucial roles in these mechanisms, how such factors are controlled in developmentally dynamic systems is poorly understood. Previously, we described five dispersed Gata2 locus sequences, termed the -77, -3.9, -2.8, -1.8, and +9.5 GATA switch sites, which contain evolutionarily conserved GATA motifs occupied by GATA-2 and GATA-1 in hematopoietic precursors and erythroid cells, respectively. Despite common attributes of transcriptional enhancers, targeted deletions of the -2.8, -1.8, and +9.5 sites revealed distinct and unpredictable contributions to Gata2 expression and hematopoiesis. Herein, we describe the targeted deletion of the -3.9 site and mechanistically compare the -3.9 site with other GATA switch sites. The -3.9(-/-) mice were viable and exhibited normal Gata2 expression and steady-state hematopoiesis in the embryo and adult. We established a Gata2 repression/reactivation assay, which revealed unique +9.5 site activity to mediate GATA factor-dependent chromatin structural transitions. Loss-of-function analyses provided evidence for a mechanism in which a mediator of long-range transcriptional control [LIM domain binding 1 (LDB1)] and a chromatin remodeler [Brahma related gene 1 (BRG1)] synergize through the +9.5 site, conferring expression of GATA-2, which is known to promote the genesis and survival of hematopoietic stem cells.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Células Madre/citología , Animales , Secuencia de Bases , Diferenciación Celular/genética , Células Cultivadas , Elementos de Facilitación Genéticos , Hematopoyesis , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Células Madre/metabolismoRESUMEN
Disease mutations provide unique opportunities to decipher protein and cell function. Mutations in the master regulator of hematopoiesis GATA-2 underlie an immunodeficiency associated with myelodysplastic syndrome and leukemia. We discovered that a GATA-2 disease mutant (T354M) defective in chromatin binding was hyperphosphorylated by p38 mitogen-activated protein kinase. p38 also induced multisite phosphorylation of wild-type GATA-2, which required a single phosphorylated residue (S192). Phosphorylation of GATA-2, but not T354M, stimulated target gene expression. While crosstalk between oncogenic Ras and GATA-2 has been implicated as an important axis in cancer biology, its mechanistic underpinnings are unclear. Oncogenic Ras enhanced S192-dependent GATA-2 phosphorylation, nuclear foci localization, and transcriptional activation. These studies define a mechanism that controls a key regulator of hematopoiesis and a dual mode of impairing GATA-2-dependent genetic networks: mutational disruption of chromatin occupancy yielding insufficient GATA-2, and oncogenic Ras-mediated amplification of GATA-2 activity.
Asunto(s)
Factor de Transcripción GATA2/biosíntesis , Genes ras/genética , Leucemia/genética , Proteínas Quinasas p38 Activadas por Mitógenos/biosíntesis , Línea Celular Tumoral , Cromatina/genética , Factor de Transcripción GATA2/genética , Regulación Leucémica de la Expresión Génica/genética , Hematopoyesis/genética , Humanos , Leucemia/patología , Mutación , Síndromes Mielodisplásicos , Fosforilación , Regiones Promotoras Genéticas , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The establishment and maintenance of cell type-specific transcriptional programs require an ensemble of broadly expressed chromatin remodeling and modifying enzymes. Many questions remain unanswered regarding the contributions of these enzymes to specialized genetic networks that control critical processes, such as lineage commitment and cellular differentiation. We have been addressing this problem in the context of erythrocyte development driven by the transcription factor GATA-1 and its coregulator Friend of GATA-1 (FOG-1). As certain GATA-1 target genes have little to no FOG-1 requirement for expression, presumably additional coregulators can mediate GATA-1 function. Using a genetic complementation assay and RNA interference in GATA-1-null cells, we demonstrate a vital link between GATA-1 and the histone H4 lysine 20 methyltransferase PR-Set7/SetD8 (SetD8). GATA-1 selectively induced H4 monomethylated lysine 20 at repressed, but not activated, loci, and endogenous SetD8 mediated GATA-1-dependent repression of a cohort of its target genes. GATA-1 used different combinations of SetD8, FOG-1, and the FOG-1-interacting nucleosome remodeling and deacetylase complex component Mi2ß to repress distinct target genes. Implicating SetD8 as a context-dependent GATA-1 corepressor expands the repertoire of coregulators mediating establishment/maintenance of the erythroid cell genetic network, and provides a biological framework for dissecting the cell type-specific functions of this important coregulator. We propose a coregulator matrix model in which distinct combinations of chromatin regulators are required at different GATA-1 target genes, and the unique attributes of the target loci mandate these combinations.
Asunto(s)
Cromatina/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Células Madre Hematopoyéticas/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Cricetinae , Cricetulus , Células Eritroides/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Perfilación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A poorly understood problem in genetics is how the three-dimensional organization of the nucleus contributes to establishment and maintenance of transcriptional networks. Genetic loci can reside in chromosome "territories" and undergo dynamic changes in subnuclear positioning. Such changes appear to be important for regulating transcription, although many questions remain regarding how loci reversibly transit in and out of their territories and the functional significance of subnuclear transitions. We addressed this issue using GATA-1, a master regulator of hematopoiesis implicated in human leukemogenesis, which often functions with the coregulator Friend of GATA-1 (FOG-1). In a genetic complementation assay in GATA-1-null cells, GATA-1 expels FOG-1-dependent target genes from the nuclear periphery during erythroid maturation, but the underlying mechanisms are unknown. We demonstrate that GATA-1 induces extrusion of the ß-globin locus away from its chromosome territory at the nuclear periphery, and extrusion precedes the maturation-associated transcriptional surge and morphological transition. FOG-1 and its interactor Mi-2ß, a chromatin remodeling factor commonly linked to repression, were required for locus extrusion. Erythroid Krüppel-like factor, a pivotal regulator of erythropoiesis that often co-occupies chromatin with GATA-1, also promoted locus extrusion. Disruption of transcriptional maintenance did not restore the locus subnuclear position that preceded activation. These results lead to a model for how a master developmental regulator relocalizes a locus into a new subnuclear neighborhood that is permissive for high level transcription as an early step in establishing a cell type-specific genetic network. Alterations in the regulatory milieu can abrogate maintenance without reversion of locus residency back to its original neighborhood.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Factor de Transcripción GATA1/metabolismo , Sitios Genéticos/fisiología , Modelos Biológicos , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Globinas beta/biosíntesis , Animales , Células CHO , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Cricetinae , Cricetulus , Factor de Transcripción GATA1/genética , Hematopoyesis/fisiología , Humanos , Proteínas Nucleares/genética , Factores de Transcripción/genética , Transcripción Genética/fisiología , Globinas beta/genéticaRESUMEN
Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However, the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a "neuropeptide," neurokinin-B (NK-B), reversibly inhibits endothelial cell vascular network assembly and opposes angiogenesis in the chicken chorioallantoic membrane. Disruption of endogenous NK-B signaling promoted angiogenesis. Mechanistic analyses defined a multicomponent pathway in which NK-B signaling converges upon cellular processes essential for angiogenesis. NK-B-mediated ablation of Ca2+ oscillations and elevation of 3'-5' [corrected] cyclic adenosine monophosphate (cAMP) reduced cellular proliferation, migration, and vascular endothelial growth factor receptor expression and induced the antiangiogenic protein calreticulin. Whereas NK-B initiated certain responses, other activities required additional stimuli that increase cAMP. Although NK-B is a neurotransmitter/ neuromodulator and NK-B overexpression characterizes the pregnancy-associated disorder preeclampsia, NK-B had not been linked to vascular remodeling. These results establish a conserved mechanism in which NK-B instigates multiple activities that collectively oppose vascular remodeling.
Asunto(s)
Inhibidores de la Angiogénesis/fisiología , Neuroquinina B/fisiología , Transducción de Señal/fisiología , Tromboxano A2/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Inhibidores de la Angiogénesis/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Ratones , Modelos Biológicos , Músculo Liso Vascular/fisiología , Neuroquinina B/farmacología , Neurotransmisores/farmacología , Neurotransmisores/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Transducción de Señal/efectos de los fármacos , Tromboxano A2/farmacología , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Elucidating mechanisms controlling nuclear processes requires an understanding of the nucleoprotein structure of genes at endogenous chromosomal loci. Traditional approaches to measuring protein-DNA interactions in vitro have often failed to provide insights into physiological mechanisms. Given that most transcription factors interact with simple DNA sequence motifs, which are abundantly distributed throughout a genome, it is essential to pinpoint the small subset of sites bound by factors in vivo. Signaling mechanisms induce the assembly and modulation of complex patterns of histone acetylation, methylation, phosphorylation, and ubiquitination, which are crucial determinants of chromatin accessibility. These seemingly complex issues can be directly addressed by a powerful methodology termed the chromatin immunoprecipitation (ChIP) assay. ChIP analysis involves covalently trapping endogenous proteins at chromatin sites, thereby yielding snapshots of protein-DNA interactions and histone modifications within living cells. The chromatin is sonicated to generate small fragments, and an immunoprecipitation is conducted with an antibody against the desired factor or histone modification. Crosslinks are reversed, and polymerase chain reaction (PCR) is used to assess whether DNA sequences are recovered immune-specifically. Chromatin-domain scanning coupled with quantitative analysis is a powerful means of dissecting mechanisms by which signaling pathways target genes within a complex genome.
Asunto(s)
Cromatina/química , Proteínas de Unión al ADN/análisis , Pruebas de Precipitina/métodos , Transducción de Señal , Factores de Transcripción/análisis , Acetilación , Animales , Núcleo Celular/genética , Cromatina/metabolismo , ADN/análisis , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Histonas/química , Histonas/inmunología , Histonas/metabolismo , Metilación , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismoRESUMEN
The generation of hematopoietic stem cells (HSCs) from hemogenic endothelium within the aorta, gonad, mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. We described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia/leukemia. Here, we demonstrate that the +9.5 element enhances Gata2 expression and is required to generate long-term repopulating HSCs in the AGM. Deletion of the +9.5 element abrogated the capacity of hemogenic endothelium to generate HSC-containing clusters in the aorta. Genomic analyses indicated that the +9.5 element regulated a rich ensemble of genes that control hemogenic endothelium and HSCs, as well as genes not implicated in hematopoiesis. These results reveal a mechanism that controls stem cell emergence from hemogenic endothelium to establish the adult hematopoietic system.
Asunto(s)
Elementos de Facilitación Genéticos , Factor de Transcripción GATA2/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Animales , Aorta/embriología , Separación Celular , Femenino , Citometría de Flujo , Redes Reguladoras de Genes , Genómica , Gónadas/embriología , Masculino , Mesonefro/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Células Madre/citologíaRESUMEN
Haploinsufficiency for GATA2 causes human immunodeficiency syndromes characterized by mycobacterial infection, myelodysplasia, lymphedema, or aplastic anemia that progress to myeloid leukemia. GATA2 encodes a master regulator of hematopoiesis that is also linked to endothelial biology. Though the disease-causing mutations commonly occur in the GATA-2 DNA binding domain, we identified a patient with mycobacterial infection and myelodysplasia who had an uncharacterized heterozygous deletion in a GATA2 cis-element consisting of an E-box and a GATA motif. Targeted deletion of the equivalent murine element to yield homozygous mutant mice revealed embryonic lethality later than occurred with global Gata2 knockout, hematopoietic stem/progenitor cell depletion, and impaired vascular integrity. Heterozygous mutant mice were viable, but embryos exhibited deficits in definitive, but not primitive, hematopoietic stem/progenitor activity and reduced expression of Gata2 and its target genes. Mechanistic analysis revealed disruption of the endothelial cell transcriptome and loss of vascular integrity. Thus, the composite element disrupted in a human immunodeficiency is essential for establishment of the murine hematopoietic stem/progenitor cell compartment in the fetal liver and for essential vascular processes.
Asunto(s)
Vasos Sanguíneos/embriología , Elementos E-Box , Factor de Transcripción GATA2/deficiencia , Factor de Transcripción GATA2/fisiología , Hematopoyesis/genética , Síndromes de Inmunodeficiencia/genética , Síndromes Mielodisplásicos/genética , Elementos Reguladores de la Transcripción , Animales , Secuencia de Bases , Vasos Sanguíneos/patología , Desarrollo Embrionario/genética , Endotelio Vascular/metabolismo , Factor de Transcripción GATA2/química , Factor de Transcripción GATA2/genética , Genes Letales , Predisposición Genética a la Enfermedad , Genotipo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/patología , Hemorragia/embriología , Hemorragia/genética , Humanos , Hígado/citología , Hígado/embriología , Ratones , Datos de Secuencia Molecular , Infecciones por Mycobacterium/etiología , Síndromes Mielodisplásicos/complicaciones , Eliminación de SecuenciaRESUMEN
Whereas the transcription factors GATA-1 and GATA-2 function both uniquely and redundantly to control blood cell development, the process termed hematopoiesis, mechanisms underlying their unique versus common functions are poorly understood. We used two independent assays to demonstrate that GATA-1 is considerably more stable than GATA-2 in multiple cellular contexts, even though both factors are subject to degradation via the ubiquitin-proteasome system. Studies with GATA factor mutants and novel chimeric GATA factors provided evidence that both GATA-1 and GATA-2 have highly unstable zinc finger core modules. The GATA-1 and GATA-2 N-termini both confer stabilization to their respective zinc finger core modules. In contrast, the GATA-1 and GATA-2 C-termini confer stabilization and destabilization, respectively. As GATA-2 stabilization via proteasome inhibition impairs the capacity of GATA-1 to displace GATA-2 from endogenous chromatin sites, we propose that differential GATA factor stability is an important determinant of chromatin target site occupancy and therefore the establishment of genetic networks that control hematopoiesis.
Asunto(s)
Cromatina/metabolismo , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA2/genética , Expresión Génica/efectos de los fármacos , Humanos , Células K562 , Leupeptinas/farmacología , Ratones , Mutación/fisiología , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Tamoxifeno/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Combinatorial interactions among trans-acting factors establish transcriptional circuits that orchestrate cellular differentiation, survival, and development. Unlike circuits instigated by individual factors, efforts to identify gene ensembles controlled by multiple factors simultaneously are in their infancy. A paradigm has emerged in which the important regulators of hematopoiesis GATA-1 and GATA-2 function combinatorially with Scl/TAL1, another key regulator of hematopoiesis. The underlying mechanism appears to involve preferential assembly of a multimeric complex on a composite DNA element containing WGATAR and E-box motifs. Based on this paradigm, one would predict that GATA-2 and Scl/TAL1 would commonly co-occupy such composite elements in cells. However, chromosome-wide analyses indicated that the vast majority of conserved composite elements were occupied by neither GATA-2 nor Scl/TAL1. Intriguingly, the highly restricted set of GATA-2-occupied composite elements had characteristic molecular hallmarks, specifically Scl/TAL1 occupancy, a specific epigenetic signature, specific neighboring cis elements, and preferential enhancer activity in GATA-2-expressing cells. Genes near the GATA-2-Scl/TAL1-occupied composite elements were regulated by GATA-2 or GATA-1, and therefore these fundamental studies on combinatorial transcriptional mechanisms were also leveraged to discover novel GATA factor-mediated cell regulatory pathways.
Asunto(s)
Cromatina/metabolismo , Hematopoyesis/genética , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células CHO , Línea Celular , Cromosomas de los Mamíferos/metabolismo , Secuencia Conservada , Cricetinae , Cricetulus , Epigénesis Genética , Factor de Transcripción GATA2/metabolismo , Ratones , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción GenéticaRESUMEN
GATA factors are fundamental components of developmentally important transcriptional networks. By contrast to common mechanisms in which transacting factors function directly at promoters, the hematopoietic GATA factors GATA-1 and GATA-2 often assemble dispersed complexes over broad chromosomal regions. For example, GATA-1 and GATA-2 occupy five conserved regions over approximately 100 kb of the Gata2 locus in the transcriptionally repressed and active states, respectively, in erythroid cells. Since it is unknown whether the individual complexes exert qualitatively distinct or identical functions to regulate Gata2 transcription in vivo, we compared the activity of the -3.9 and +9.5 kb sites of the Gata2 locus in transgenic mice. The +9.5 site functioned as an autonomous enhancer in the endothelium and fetal liver of embryonic day 11 embryos, whereas the -3.9 site lacked such activity. Mechanistic studies demonstrated critical requirements for a GATA motif and a neighboring E-box within the +9.5 site for enhancer activity in endothelial and hematopoietic cells. Surprisingly, whereas this GATA-E-box composite motif was sufficient for enhancer activity in an erythroid precursor cell line, its enhancer function in primary human endothelial cells required additional regulatory modules. These results identify the first molecular determinant of Gata2 transcription in vascular endothelium, composed of a core enhancer module active in both endothelial and hematopoietic cells and regulatory modules preferentially required in endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica , Sistema Hematopoyético/metabolismo , Transcripción Genética , Animales , Aorta/citología , Aorta/metabolismo , Secuencia de Bases , Células Cultivadas , Endotelio Vascular/citología , Elementos de Facilitación Genéticos , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA2/metabolismo , Sistema Hematopoyético/citología , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Homología de Secuencia de Ácido Nucleico , Activación Transcripcional , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
The GATA-1-interacting protein Friend Of GATA-1 (FOG-1) is essential for the proper transcriptional activation and repression of numerous GATA-1 target genes. Although FOG-1-independent activation by GATA-1 has been described, all known examples of GATA-1-mediated repression are FOG-1 dependent. In the GATA-1-null G1E cell line, estrogen receptor ligand binding domain (ER) chimeras of either wild-type GATA-1 or a FOG-1-binding defective mutant of GATA-1 repressed several genes similarly upon activation with beta-estradiol. Repression also occurred in a FOG-1-null cell line expressing ER-GATA-1 and during ex vivo erythropoiesis. At the Lyl1 and Rgs18 loci, we found highly restricted occupancy by GATA-1 and GATA-2, indicating that these genes are direct targets of GATA factor regulation. The identification of genes repressed by GATA-1 independent of FOG-1 defines a novel mode of GATA-1-mediated transcriptional regulation.
Asunto(s)
Factor de Transcripción GATA1/fisiología , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Sitios de Unión , Factor de Transcripción GATA1/genética , Ratones , Mutación , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión , Transcripción GenéticaRESUMEN
Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, chromatin immunoprecipitation analysis has revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the -2.8-kilobase (kb) region of the GATA-2 locus in the active state despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high resolution chromatin immunoprecipitation to show that GATA-1 and GATA-2 occupy two additional regions, -3.9 and -1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the -3.9- and -1.8-kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and GATA-2 displacement at the sites. In the transcriptionally active state, DNase I hypersensitive sites (HSs) were detected at the -3.9- and -1.8-kb regions, with a weak HS at the -2.8-kb region. Whereas ER-GATA-1-instigated repression abolished the -1.8-kb HS, the -3.9-kb HS persisted in the repressed state. Transient transfection analysis provided evidence that the -3.9-kb region functions distinctly from the -2.8- and -1.8-kb regions. We propose that GATA-2 transcription is regulated via the collective actions of complexes assembled at the -2.8- and -1.8-kb regions, which share similar properties, and through a qualitatively distinct activity of the -3.9-kb complex.
Asunto(s)
Proteínas de Unión al ADN/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Factor de Transcripción GATA2 , Regulación de la Expresión Génica , Ratones , Células 3T3 NIH , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine beta-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the betamajor promoter. ER-GATA-1 increased erythroid Krüppel-like factor and SWI/SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of beta-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial/temporal regulation and underlies the multistep activation mechanism.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , ADN/metabolismo , Factor de Transcripción GATA1/fisiología , Animales , Línea Celular , Cromatina/química , Secuencia Conservada , ADN Helicasas , Factor de Transcripción GATA2/metabolismo , Globinas/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Histone H3 methylated at lysine 4 (H3-meK4) co-localizes with hyperacetylated histones H3 and H4 in transcriptionally active chromatin, but mechanisms that establish H3-meK4 are poorly understood. Previously, we showed that the hematopoietic-specific activator NF-E2, which is required for beta-globin transcription in erythroleukemia cells, induces histone H3 hyperacetylation and H3-meK4 at the adult beta-globin genes (betamajor and betaminor). Chromatin immunoprecipitation analysis indicated that NF-E2 occupies hypersensitive site two (HS2) of the beta-globin locus control region. The mechanism of NF-E2-mediated chromatin modification was investigated by complementation analysis in NF-E2-null CB3 erythroleukemia cells. The activation domain of the hematopoietic-specific subunit of NF-E2 (p45/NF-E2) contains two WW domain-binding motifs (PXY-1 and PXY-2). PXY-1 is required for activation of beta-globin transcription. Here, we determined which step in NF-E2-dependent transactivation is PXY-1-dependent. A p45/NF-E2 mutant lacking 42 amino acids of the activation domain, including both PXY motifs, and a mutant lacking only PXY-1 were impaired in inducing histone H3 hyperacetylation, H3-meK4, and RNA polymerase II recruitment. The PXY motifs were not required for transactivation in the context of a GAL4 DNA-binding domain fusion to p45/NF-E2 in transient transfection assays. As the PXY-1 mutant occupied HS2 normally, the chromatin modification defect occurred post-DNA binding. PXY-1 was not required for recruitment of the histone acetyltransferases cAMP-responsive element-binding protein-binding protein (CBP) and p300 to HS2. These results indicate that PXY-1 confers chromatin-specific transcriptional activation via interaction with a co-regulator distinct from CBP/p300 or by regulating CBP/p300 function.
Asunto(s)
Proteínas de Unión al ADN/química , Histonas/metabolismo , Factores de Transcripción/química , Acetilación , Animales , Sitios de Unión/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Factores de Unión al ADN Específico de las Células Eritroides , Eliminación de Gen , Globinas/genética , Hematopoyesis , Histonas/química , Humanos , Leucemia Eritroblástica Aguda , Megacariocitos , Ratones , Mutagénesis , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , ARN Polimerasa II/metabolismo , Proteínas Recombinantes de Fusión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Transcripción Genética , Activación Transcripcional , Transfección , Células Tumorales CultivadasRESUMEN
Interplay among GATA transcription factors is an important determinant of cell fate during hematopoiesis. Although GATA-2 regulates hematopoietic stem cell function, mechanisms controlling GATA-2 expression are undefined. Of particular interest is the repression of GATA-2, because sustained GATA-2 expression in hematopoietic stem and progenitor cells alters hematopoiesis. GATA-2 transcription is derepressed in erythroid precursors lacking GATA-1, but the underlying mechanisms are unknown. Using chromatin immunoprecipitation analysis, we show that GATA-1 binds a highly restricted upstream region of the approximately 70-kb GATA-2 domain, despite >80 GATA sites throughout the domain. GATA-2 also binds this region in the absence of GATA-1. Genetic complementation studies in GATA-1-null cells showed that GATA-1 rapidly displaces GATA-2, which is coupled to transcriptional repression. GATA-1 also displaces CREB-binding protein (CBP), despite the fact that GATA-1 binds CBP in other contexts. Repression correlates with reduced histone acetylation domain-wide, but not altered methylation of histone H3 at lysine 4. The GATA factor-binding region exhibited cell-type-specific enhancer activity in transient transfection assays. We propose that GATA-1 instigates GATA-2 repression by means of disruption of positive autoregulation, followed by establishment of a domain-wide repressive chromatin structure. Such a mechanism is predicted to be critical for the control of hematopoiesis.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Cromatina/química , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Factor de Transcripción GATA2 , Hematopoyesis , Histonas/química , Histonas/metabolismo , Homeostasis , Humanos , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Posttranslational modification of histones through acetylation, methylation, and phosphorylation is a common mode of regulating chromatin structure and, therefore, diverse nuclear processes. One such modification, methylated histone H3 at lysine-4 (H3-meK4), colocalizes with hyperacetylated histones H3 and H4 in mammalian chromatin. Whereas activators directly recruit acetyltransferases, the process whereby H3-meK4 is established is unknown. We tested whether the hematopoietic-specific activators NF-E2 and GATA-1, which mediate transactivation of the beta-globin genes, induce both histone acetylation and H3-meK4. Through the use of NF-E2- and GATA-1-null cell lines, we show that both activators induce H3 acetylation at the promoter upon transcriptional activation. However, analysis of H3-mek4 revealed that NF-E2 and GATA-1 differentially regulate chromatin modifications at the betamajor promoter. NF-E2, but not GATA-1, induces H3-meK4 at the promoter. Thus, under conditions in which NF-E2 and GATA-1 activate the transcription of an endogenous gene at least 570-fold, these activators differ in their capacity to induce H3-meK4. Despite strong H3-meK4 at hypersensitive site 2 of the upstream locus control region, neither factor was required to establish H3-meK4 at this site. These results support a model in which multiple tissue-specific activators collectively function to assemble a composite histone modification pattern, consisting of overlapping histone acetylation and methylation. As GATA-1 induced H3 acetylation, but not H3-meK4, at the promoter, H3 acetylation and H3-meK4 components of a composite histone modification pattern can be established independently.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Sitios de Unión , Proteínas de Unión al ADN/genética , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Hematopoyesis , Mamíferos , Metilación , Ratones , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Transactivadores/genética , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
The hematopoietic transcription factor GATA-1 regulates erythropoiesis and beta-globin expression. Although consensus GATA-1 binding sites exist throughout the murine beta-globin locus, we found that GATA-1 discriminates among these sites in vivo. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the beta-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner.
Asunto(s)
Médula Ósea/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Globinas/genética , Ratones , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
Coregulator recruitment by DNA-bound factors results in chromatin modification and protein-protein interactions, which regulate transcription. However, the mechanism by which the Friend of GATA (FOG) coregulator mediates GATA factor-dependent transcription is unknown. We showed previously that GATA-1 replaces GATA-2 at an upstream region of the GATA-2 locus, and that this GATA switch represses GATA-2. Genetic complementation analysis in FOG-1-null hematopoietic precursors revealed that FOG-1 is not required for establishment or maintenance of the active GATA-2 domain, but is critical for the GATA switch. Analysis of GATA factor binding to additional loci also revealed FOG-1-dependent GATA switches. Thus, FOG-1 facilitates chromatin occupancy by GATA-1 at sites bound by GATA-2. We propose that FOG-1 is a prototype of a new class of coregulators termed chromatin occupancy facilitators, which confer coregulation in certain contexts via enhancing trans-acting factor binding to chromatin in vivo.