RESUMEN
The limited proliferative capacity of erythroid precursors is a major obstacle to generate sufficient numbers of in vitro-derived red blood cells (RBC) for clinical purposes. We and others have determined that BMI1, a member of the polycomb repressive complex 1 (PRC1), is both necessary and sufficient to drive extensive proliferation of self-renewing erythroblasts (SREs). However, the mechanisms of BMI1 action remain poorly understood. BMI1 overexpression led to 10 billion-fold increase BMI1-induced (i)SRE self-renewal. Despite prolonged culture and BMI1 overexpression, human iSREs can terminally mature and agglutinate with typing reagent monoclonal antibodies against conventional RBC antigens. BMI1 and RING1B occupancy, along with repressive histone marks, were identified at known BMI1 target genes, including the INK-ARF locus, consistent with an altered cell cycle following BMI1 inhibition. We also identified upregulated BMI1 target genes with low repressive histone modifications, including key regulator of cholesterol homeostasis. Functional studies suggest that both cholesterol import and synthesis are essential for BMI1-associated self-renewal. These findings support the hypothesis that BMI1 regulates erythroid self-renewal not only through gene repression but also through gene activation and offer a strategy to expand the pool of immature erythroid precursors for eventual clinical uses.
RESUMEN
Human erythropoiesis is a complex process leading to the production of 2.5 million red blood cells per second. Following commitment of hematopoietic stem cells to the erythroid lineage, this process can be divided into three distinct stages: erythroid progenitor differentiation, terminal erythropoiesis, and reticulocyte maturation. We recently resolved the heterogeneity of erythroid progenitors into four different subpopulations termed EP1-EP4. Here, we characterized the growth factor(s) responsiveness of these four progenitor populations in terms of proliferation and differentiation. Using mass spectrometry-based proteomics on sorted erythroid progenitors, we quantified the absolute expression of ~5500 proteins from EP1 to EP4. Further functional analyses highlighted dynamic changes in cell cycle in these populations with an acceleration of the cell cycle during erythroid progenitor differentiation. The finding that E2F4 expression was increased from EP1 to EP4 is consistent with the noted changes in cell cycle. Finally, our proteomic data suggest that the protein machinery necessary for both oxidative phosphorylation and glycolysis is present in these progenitor cells. Together, our data provide comprehensive insights into growth factor-dependence of erythroid progenitor proliferation and the proteome of four distinct populations of human erythroid progenitors which will be a useful framework for the study of erythroid disorders.
Asunto(s)
Células Madre Hematopoyéticas , Proteómica , Humanos , Diferenciación Celular , Ciclo Celular , Eritropoyesis , Redes y Vías Metabólicas , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Precursoras EritroidesRESUMEN
ABSTRACT: Regulation of RNA polymerase II (RNAPII) activity is an essential process that governs gene expression; however, its contribution to the fundamental process of erythropoiesis remains unclear. hexamethylene bis-acetamide inducible 1 (HEXIM1) regulates RNAPII activity by controlling the location and activity of positive transcription factor ß. We identified a key role for HEXIM1 in controlling erythroid gene expression and function, with overexpression of HEXIM1 promoting erythroid proliferation and fetal globin expression. HEXIM1 regulated erythroid proliferation by enforcing RNAPII pausing at cell cycle check point genes and increasing RNAPII occupancy at genes that promote cycle progression. Genome-wide profiling of HEXIM1 revealed that it was increased at both repressed and activated genes. Surprisingly, there were also genome-wide changes in the distribution of GATA-binding factor 1 (GATA1) and RNAPII. The most dramatic changes occurred at the ß-globin loci, where there was loss of RNAPII and GATA1 at ß-globin and gain of these factors at γ-globin. This resulted in increased expression of fetal globin, and BGLT3, a long noncoding RNA in the ß-globin locus that regulates fetal globin expression. GATA1 was a key determinant of the ability of HEXIM1 to repress or activate gene expression. Genes that gained both HEXIM1 and GATA1 had increased RNAPII and increased gene expression, whereas genes that gained HEXIM1 but lost GATA1 had an increase in RNAPII pausing and decreased expression. Together, our findings reveal a central role for universal transcription machinery in regulating key aspects of erythropoiesis, including cell cycle progression and fetal gene expression, which could be exploited for therapeutic benefit.
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Eritropoyesis , Factores de Transcripción , Humanos , Eritropoyesis/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Globinas beta/genética , Globinas beta/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Arthrogryposis multiplex congenita (AMC) [also known as multiple joints contracture or Fetal Akinesia Deformation Sequence (FADS)] is etiologically a heterogeneous condition with an estimated incidence of approximately 1 in 3000 live births and much higher incidence when prenatally diagnosed cases are included. The condition can be acquired or secondary to fetal exposures and can also be caused by a variety of single-gene disorders affecting the brain, spinal cord, peripheral nerves, neuromuscular junction, muscle, and a variety of disorders affecting the connective tissues (Niles et al., Prenatal Diagnosis, 2019; 39:720-731). The introduction of next-generation gene sequencing uncovered many genes and causative variants of AMC but also identified genes that cause both dominant and recessive inherited conditions with the variability of clinical manifestations depending on the genes and variants. Molecular diagnosis in these cases is not only important for prognostication but also for the determination of recurrence risk and for providing reproductive options including preimplantation and prenatal diagnosis. TTN, the largest known gene in the human genome, has been known to be associated with autosomal dominant dilated cardiomyopathy. However, homozygote and compound heterozygote pathogenic variants with recessive inheritance have rarely been reported. We report the effect of recessive variants located within the fetal IC and/or N2BA isoforms in association with severe FADS in three families. All parents were healthy obligate carriers and none of them had cardiac or skeletal muscle abnormalities. This report solidifies FADS as an alternative phenotypic presentation associated with homozygote/compound heterozygous pathogenic variants in the TTN.
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Artrogriposis , Embarazo , Femenino , Humanos , Artrogriposis/diagnóstico , Artrogriposis/genética , Diagnóstico Prenatal , Homocigoto , Atención Prenatal , Síndrome , Conectina/genéticaRESUMEN
Activating KRAS mutations and functional loss of members of the SWI/SNF complex, including ARID1A, are found together in the primary liver tumor cholangiocarcinoma (CC). How these mutations cooperate to promote CC has not been established. Using murine models of hepatocyte and biliary-specific lineage tracing, we show that Kras and Arid1a mutations drive the formation of CC and tumor precursors from the biliary compartment, which are accelerated by liver inflammation. Using cultured cells, we find that Arid1a loss causes cellular proliferation, escape from cell-cycle control, senescence, and widespread changes in chromatin structure. Notably, we show that the biliary proliferative response elicited by Kras/Arid1a cooperation and tissue injury in CC is caused by failed engagement of the TGF-ß-Smad4 tumor suppressor pathway. We thus identify an ARID1A-TGF-ß-Smad4 axis as essential in limiting the biliary epithelial response to oncogenic insults, while its loss leads to biliary pre-neoplasia and CC.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Animales , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/patología , Colangiocarcinoma/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Mutación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Anemia of inflammation, also known as anemia of chronic disease, is refractory to erythropoietin (EPO) treatment, but the mechanisms underlying the EPO refractory state are unclear. Here, we demonstrate that high mobility group box-1 protein (HMGB1), a damage-associated molecular pattern molecule recently implicated in anemia development during sepsis, leads to reduced expansion and increased death of EPO-sensitive erythroid precursors in human models of erythropoiesis. HMGB1 significantly attenuates EPO-mediated phosphorylation of the Janus kinase 2/STAT5 and mTOR signaling pathways. Genetic ablation of receptor for advanced glycation end products, the only known HMGB1 receptor expressed by erythroid precursors, does not rescue the deleterious effects of HMGB1 on EPO signaling, either in human or murine precursors. Furthermore, surface plasmon resonance studies highlight the ability of HMGB1 to interfere with the binding between EPO and the EPOR. Administration of a monoclonal anti-HMGB1 antibody after sepsis onset in mice partially restores EPO signaling in vivo. Thus, HMGB1-mediated restriction of EPO signaling contributes to the chronic phase of anemia of inflammation.
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Anemia , Eritropoyetina , Proteína HMGB1 , Sepsis , Anemia/genética , Animales , Eritropoyesis/genética , Eritropoyetina/metabolismo , Inflamación , Ratones , Receptores de Eritropoyetina/metabolismo , Sepsis/complicacionesRESUMEN
The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.
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Eritroblastos/metabolismo , Células Eritroides/metabolismo , ARN Polimerasa II/metabolismo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Eritroblastos/citología , Células Eritroides/citología , Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , ARN Polimerasa II/genética , Transcripción GenéticaRESUMEN
The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during hemoglobin accumulation, rapid cell division, and nuclear condensation. Congenital dyserythropoietic anemia type I (CDA-I) is an autosomal recessive disease that presents with erythroid hyperplasia in the bone marrow. Erythroblasts in patients with CDA-I are frequently binucleate and have chromatin bridging and defective chromatin condensation. CDA-1 is most commonly caused by mutations in Codanin-1 (CDAN1). The function of CDAN1 is poorly understood but it is thought to regulate histone incorporation into nascent DNA during cellular replication. The study of CDA-1 has been limited by the lack of in vitro models that recapitulate key features of the disease, and most studies on CDAN1 function have been done in nonerythroid cells. To model CDA-I we generated HUDEP2 mutant lines with deletion or mutation of R1042 of CDAN1, mirroring mutations found in CDA-1 patients. CDAN1 mutant cell lines had decreased viability and increased intercellular bridges and binucleate cells. Further, they had alterations in histone acetylation associated with prematurely elevated erythroid gene expression, including gamma globin. Together, these data imply a specific functional role for CDAN1, specifically R1042 on exon 24, in the regulation of DNA replication and organization during erythroid maturation. Most importantly, generation of models with specific patient mutations, such as R1042, will provide further mechanistic insights into CDA-I pathology.
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Anemia Diseritropoyética Congénita/genética , Células Eritroides/citología , Eritropoyesis/genética , Glicoproteínas/genética , Proteínas Nucleares/genética , Acetilación , Anemia Diseritropoyética Congénita/sangre , Sistemas CRISPR-Cas , Línea Celular , Núcleo Celular/ultraestructura , Supervivencia Celular , Cromatina/ultraestructura , Células Eritroides/metabolismo , Eritropoyesis/fisiología , Exones/genética , Edición Génica , Glicoproteínas/deficiencia , Glicoproteínas/fisiología , Código de Histonas , Humanos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/fisiología , Fenotipo , Procesamiento Proteico-PostraduccionalRESUMEN
BACKGROUND: SETD8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. SETD8 and H4K20me1 play a role in a number of essential biologic processes, including cell cycle progression, establishment of higher order chromatin structure, and transcriptional regulation. SETD8 is highly expressed in erythroid cells and erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia, suggesting that it has an erythroid-specific function. The function of SETD8 in the hemopoietic system is poorly understood. The goal of our study was to gain insights into the function of SETD8 during erythroid differentiation. RESULTS: We performed ATAC-seq (assay for transposase-accessible chromatin) on sorted populations of E10.5 Setd8 mutant and control erythroblasts. Accessibility profiles were integrated with expression changes and a mark of heterochromatin (H3K27me3) performed in wild-type E10.5 erythroblasts to further understand the role of SETD8 in erythropoiesis. Data integration identified regions of greater chromatin accessibility in Setd8 mutant cells that co-located with H3K27me3 in wild-type E10.5 erythroblasts suggesting that these regions, and their associated genes, are repressed during normal erythropoiesis. The majority of these more accessible regions were located in promoters and they frequently co-located with the NFY complex. Pathway analysis of genes identified through data integration revealed stemness-related pathways. Among those genes were multiple transcriptional regulators active in multipotent progenitors, but repressed during erythroid differentiation including Hhex, Hlx, and Gata2. Consistent with a role for SETD8 in erythroid specification, SETD8 expression is up-regulated upon erythroid commitment, and Setd8 disruption impairs erythroid colony forming ability. CONCLUSION: Taken together, our results suggest that SETD8 is an important regulator of the chromatin landscape during erythroid differentiation, particularly at promoters. Our results also identify a novel role for Setd8 in the establishment of appropriate patterns of lineage-restricted gene expression during erythroid differentiation.
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Ensamble y Desensamble de Cromatina , Eritropoyesis , N-Metiltransferasa de Histona-Lisina/metabolismo , Factores de Transcripción/genética , Animales , Línea Celular , Células Cultivadas , Eritroblastos/citología , Eritroblastos/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Ratones , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a lethal disorder of lung development. ACDMPV is associated with haploinsufficiency of the transcription factor FOXF1, which plays an important role in the development of the lung and intestine. CNVs upstream of the FOXF1 gene have also been associated with an ACDMPV phenotype, but mechanism(s) by which these deletions disrupt lung development are not well understood. The objective of our study is to gain insights into the mechanisms by which CNVs contribute to an ACDMPV phenotype. METHODS: We analysed primary lung tissue from an infant with classic clinical and histological findings of ACDMPV and harboured a 340 kb deletion on chromosome 16q24.1 located 250 kb upstream of FOXF1. RESULTS: In RNA generated from paraffin-fixed lung sections, our patient had lower expression of FOXF1 than age-matched controls. He also had an abnormal pattern of FOXF1 protein expression, with a dramatic loss of FOXF1 expression in the lung. To gain insights into the mechanisms underlying these changes, we assessed the epigenetic landscape using chromatin immunoprecipitation, which demonstrated loss of histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic mark of active enhancers, in the region of the deletion. CONCLUSIONS: Together, these data suggest that the deletion disrupts an enhancer responsible for directing FOXF1 expression in the developing lung and provide novel insights into the mechanisms underlying a fatal developmental lung disorder.
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Factores de Transcripción Forkhead/genética , Predisposición Genética a la Enfermedad , Pulmón/metabolismo , Síndrome de Circulación Fetal Persistente/genética , Cromosomas Humanos Par 16/genética , Elementos de Facilitación Genéticos/genética , Eliminación de Gen , Regulación de la Expresión Génica/genética , Haploinsuficiencia/genética , Humanos , Lactante , Recién Nacido , Pulmón/crecimiento & desarrollo , Pulmón/patología , Síndrome de Circulación Fetal Persistente/patologíaRESUMEN
A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.
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Técnicas de Cultivo de Célula , Diferenciación Celular , Eritroblastos/enzimología , Mutación , Proteínas Proto-Oncogénicas c-kit , Sistemas CRISPR-Cas , Técnicas de Cultivo de Célula/economía , Técnicas de Cultivo de Célula/métodos , Línea Celular Transformada , Edición Génica , Humanos , Células K562 , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismoRESUMEN
OBJECTIVE: Here, we evaluate the contribution of AT-rich interaction domain-containing protein 1A (ARID1A), the most frequently mutated member of the SWItch/sucrose non-fermentable (SWI/SNF) complex, in pancreatic homeostasis and pancreatic ductal adenocarcinoma (PDAC) pathogenesis using mouse models. DESIGN: Mice with a targeted deletion of Arid1a in the pancreas by itself and in the context of two common genetic alterations in PDAC, Kras and p53, were followed longitudinally. Pancreases were examined and analysed for proliferation, response to injury and tumourigenesis. Cancer cell lines derived from these models were analysed for clonogenic, migratory, invasive and transcriptomic changes. RESULTS: Arid1a deletion in the pancreas results in progressive acinar-to-ductal metaplasia (ADM), loss of acinar mass, diminished acinar regeneration in response to injury and ductal cell expansion. Mutant Kras cooperates with homozygous deletion of Arid1a, leading to intraductal papillary mucinous neoplasm (IPMN). Arid1a loss in the context of mutant Kras and p53 leads to shorter tumour latency, with the resulting tumours being poorly differentiated. Cancer cell lines derived from Arid1a-mutant tumours are more mesenchymal, migratory, invasive and capable of anchorage-independent growth; gene expression analysis showed activation of epithelial-mesenchymal transition (EMT) and stem cell identity pathways that are partially dependent on Arid1a loss for dysregulation. CONCLUSIONS: ARID1A plays a key role in pancreatic acinar homeostasis and response to injury. Furthermore, ARID1A restrains oncogenic KRAS-driven formation of premalignant proliferative IPMN. Arid1a-deficient PDACs are poorly differentiated and have mesenchymal features conferring migratory/invasive and stem-like properties.
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Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas de Unión al ADN/genética , Transición Epitelial-Mesenquimal/fisiología , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Células Acinares/patología , Células Acinares/fisiología , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Homeostasis , Ratones , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de TranscripciónRESUMEN
Erythropoiesis is a highly regulated process that generates enucleate red blood cells from committed erythroid progenitors. Chromatin condensation culminating in enucleation is a defining feature of this process. Setd8 is the sole enzyme that can mono-methylate histone H4, lysine 20 and is highly expressed in erythroblasts compared to most other cell types. Erythroid Setd8 deletion results in embryonic lethality from severe anemia due to impaired erythroblast survival and proliferation. Setd8 protein levels are also uniquely regulated in erythroblasts, suggesting a cell-type-specific role for Setd8 during terminal maturation. Consistent with this hypothesis, Setd8 Δ/Δ erythroblasts have profound defects in transcriptional repression, chromatin condensation, and heterochromatin accumulation. Together, these results suggest that Setd8, used by most cells to promote mitotic chromatin condensation, is an essential aspect of the transcriptional repression and chromatin condensation that are hallmarks of terminal erythroid maturation.
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Eritroblastos/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Eritroblastos/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , Femenino , Heterocromatina/genética , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Ratones , EmbarazoRESUMEN
BACKGROUND: CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. RESULTS: Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. CONCLUSION: These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.
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Eritropoyesis , Perfilación de la Expresión Génica/métodos , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Sitios de Unión , Factor de Unión a CCCTC , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Células Eritroides/citología , Células Eritroides/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células K562 , Proteínas Nucleares/genética , Unión Proteica , Mapas de Interacción de Proteínas , Proteínas Represoras/genética , Análisis de Secuencia de ARN/métodosRESUMEN
The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult ß-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation.
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Linaje de la Célula , Ensamble y Desensamble de Cromatina , Eritrocitos/citología , Eritropoyesis , Regulación de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Antígenos Nucleares/metabolismo , Linaje de la Célula/genética , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Eritroblastos/citología , Eritroblastos/metabolismo , Recuento de Eritrocitos , Eritrocitos/metabolismo , Eritropoyesis/genética , Femenino , Hemoglobinas/metabolismo , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Metilación , Ratones , Ratones Noqueados , Nucleasa Microcócica/metabolismo , Complejos Multiproteicos/química , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , Bazo/citología , Factores de Transcripción/metabolismo , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Setd8 is the sole histone methyltransferase in mammals capable of monomethylating histone H4 lysine 20 (H4K20me1). Setd8 is expressed at significantly higher levels in erythroid cells than any other cell or tissue type, suggesting that Setd8 has an erythroid-cell-specific function. To test this hypothesis, stable Setd8 knockdown was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, nontransformed model of erythroid maturation. Knockdown of Setd8 resulted in impaired erythroid maturation characterized by a delay in hemoglobin accumulation, larger mean cell area, persistent ckit expression, incomplete nuclear condensation, and lower rates of enucleation. Setd8 knockdown did not alter ESRE proliferation or viability or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown demonstrated that in erythroid cells, Setd8 functions primarily as a repressor. Most notably, Gata2 expression was significantly higher in knockdown cells than in control cells and Gata2 knockdown rescued some of the maturation impairments associated with Setd8 disruption. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. These results suggest that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2 expression.
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Células Eritroides/citología , Factor de Transcripción GATA2/genética , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Acetilación , Animales , Células Cultivadas , Eritroblastos/citología , Eritroblastos/metabolismo , Células Eritroides/metabolismo , Eritropoyesis , Factor de Transcripción GATA2/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Globin gene regulation occurs in the context of a maturing erythroid cell, which is undergoing significant changes in chromatin structure and gene expression. There are few model systems available that facilitate studies of globin gene regulation in the context of erythroid maturation. Extensively self-renewing erythroblasts (ESREs) are a nontransformed model of erythroid maturation derived from murine fetal liver or yolk sac. Imaging flow cytometry and RNA-seq studies demonstrate that ESREs functionally and molecularly model erythroid maturation. To address the need for a model system that also recapitulates human globin switching, ESREs were derived from mice transgenic for the complete human ß-globin locus (ß-yac ESREs). ß-yac ESREs express ß-globin from the transgenic human locus, with minimal γ-globin expression. When treated with hydroxyurea or inhibitors to histone deacetylases, DNA methyltransferases, or the histone demethylase lysine specific demethylase 1 (LSD1), ß-Yac ESREs significantly increase their γ-globin expression, demonstrating their utility for studying agents that influence maturational globin switching. ß-yac ESREs were further used to characterize the secondary effects of LSD1 inhibition on erythroid maturation, with inhibition of LSD1 resulting in altered cell and nuclear size, prolonged Kit expression, and decreased rates of enucleation consistent with impaired maturation. Taken together, these studies demonstrate that ß-yac ESREs have significant utility for identifying modulators of maturational globin switching as well as for studying the broader role of those modulators in erythroid maturation.
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Eritroblastos/patología , Globinas/metabolismo , Modelos Biológicos , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.
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Elementos de Facilitación Genéticos , Células Eritroides/metabolismo , Regulación de la Expresión Génica , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Secuencia Conservada , Proteína p300 Asociada a E1A/metabolismo , Factor de Transcripción GATA1/metabolismo , Factor de Transcripción GATA1/fisiología , Genes Reporteros , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Anotación de Secuencia Molecular , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Subunidad p45 del Factor de Transcripción NF-E2/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Proteína 1 de la Leucemia Linfocítica T Aguda , TranscriptomaRESUMEN
The human ankyrin-1 gene (ANK1) contains 3 tissue-specific alternative promoters. We have shown previously that the erythroid-specific ankyrin 1 (ANK1E) core promoter contains a 5' DNase I hypersensitive site (HS) with barrier insulator function that prevents gene silencing in vitro and in vivo. Mutations in the ANK1E barrier region lead to decreased ANK1 mRNA levels and hereditary spherocytosis. In this report, we demonstrate a second ANK1E regulatory element located in an adjacent pair of DNase I HS located 5.6 kb 3' of the ANK1E promoter at the 3' boundary of an erythroid-specific DNase I-sensitive chromatin domain. The 3' regulatory element exhibits enhancer activity in vitro and in transgenic mice, and it has the histone modifications associated with an enhancer element. One of the ANK1E 3'HS contains an NF-E2 binding site that is required for enhancer function. We show that a chromatin loop brings the 3' enhancer and NF-E2 into proximity with the 5' barrier region including the ANK1E core promoter. These observations demonstrate a model for the tissue-specific activation of alternative promoters that may be applicable to the â¼ 30% of mammalian genes with alternative promoters that exhibit distinct expression patterns.
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Ancirinas/genética , Cromatina/genética , Elementos de Facilitación Genéticos , Elementos Aisladores , Subunidad p45 del Factor de Transcripción NF-E2/genética , Regiones Promotoras Genéticas , Esferocitosis Hereditaria/genética , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Animales , Ancirinas/metabolismo , Sitios de Unión , Línea Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , Ratones , Ratones Transgénicos , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Especificidad de Órganos , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esferocitosis Hereditaria/metabolismoRESUMEN
Post-translational histone modifications, acting alone or in a context-dependent manner, influence numerous cellular processes via their regulation of gene expression. Monomethylation of histone H3 lysine 27 (K27me1) is a poorly understood histone modification. Some reports describe depletion of K27Me1 at promoters and transcription start sites (TSS), implying its depletion at TSS is necessary for active transcription, while others have associated enrichment of H3K27me1 at TSS with increased levels of mRNA expression. Tissue- and gene-specific patterns of H3K27me1 enrichment and their correlation with gene expression were determined via chromatin immunoprecipitation on chip microarray (ChIP-chip) and human mRNA expression array analyses. Results from erythroid cells were compared with those in neural and muscle cells. H3K27me1 enrichment varied depending on levels of cell-type specific gene expression, with highest enrichment over transcriptionally active genes. Over individual genes, the highest levels of H3K27me1 enrichment were found over the gene bodies of highly expressed genes. In contrast to H3K4me3, which was highly enriched at the TSS of actively transcribing genes, H3K27me1 was selectively depleted at the TSS of actively transcribed genes. There was markedly decreased to no H3K27me1 enrichment in genes with low expression. At some locations, H3K27 monomethylation was also found to be associated with chromatin signatures of gene enhancers.