RESUMO
Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.
Assuntos
Sistemas CRISPR-Cas/genética , Linhagem da Célula/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Transcriptoma/genética , Animais , Linhagem Celular , Feminino , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/fisiologia , Masculino , Camundongos , Transdução Genética/métodosRESUMO
X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP) are uncommon diseases caused by loss-of-function and gain-of-function mutations, respectively, in the erythroid form of 5-aminolevulinic acid synthetase, ALAS2, which encodes the first enzyme in heme biosynthesis. A related sideroblastic anemia is due to mutations in SLC25A38, which supplies mitochondrial glycine for ALAS2 (SLC25A38-CSA). The lack of viable animal models has limited studies on the pathophysiology and development of therapies for these conditions. Here, using CRISPR-CAS9 gene editing technology, we have generated knock-in mouse models that recapitulate the main features of XLSA and XLPP, and, using conventional conditional gene targeting in embryonic stem cells, we also developed a faithful model of the SLC25A38-CSA. In addition to examining the phenotypes and natural history of each disease, we determine the effect of restriction or supplementation of dietary pyridoxine (vitamin B6), the essential cofactor of ALAS2, on the anemia and porphyria. In addition to the well-documented response of XLSA mutations to pyridoxine supplementation, we also demonstrate the relative insensitivity of the XLPP porphyria, severe sensitivity of the XLSA models, and an extreme hypersensitivity of the SLC25A38-CSA model to pyridoxine deficiency, a phenotype that is not shared with another mouse hereditary anemia model, Hbbth3/+ ï¢-thalassemia intermedia. Thus, in addition to generating animal models useful for examining the pathophysiology and treatment of these diseases, we have uncovered an unsuspected conditional synthetic lethality between the heme synthesis-related CSAs and pyridoxine deficiency. These findings have the potential to inform novel therapeutic paradigms for the treatment of these diseases.
RESUMO
c-Myc (Myc) is an important transcriptional regulator in embryonic stem (ES) cells, somatic cell reprogramming, and cancer. Here, we identify a Myc-centered regulatory network in ES cells by combining protein-protein and protein-DNA interaction studies and show that Myc interacts with the NuA4 complex, a regulator of ES cell identity. In combination with regulatory network information, we define three ES cell modules (Core, Polycomb, and Myc) and show that the modules are functionally separable, illustrating that the overall ES cell transcription program is composed of distinct units. With these modules as an analytical tool, we have reassessed the hypothesis linking an ES cell signature with cancer or cancer stem cells. We find that the Myc module, independent of the Core module, is active in various cancers and predicts cancer outcome. The apparent similarity of cancer and ES cell signatures reflects, in large part, the pervasive nature of Myc regulatory networks.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Acetilação , Animais , Linhagem Celular , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Trimethylation on histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) regulates the balance between self-renewal and differentiation of embryonic stem cells (ESCs). The mechanisms controlling the activity and recruitment of PRC2 are largely unknown. Here we demonstrate that the founding member of the Jumonji family, JMJ (JUMONJI or JARID2), is associated with PRC2, colocalizes with PRC2 and H3K27me3 on chromatin, and modulates PRC2 function. In vitro JMJ inhibits PRC2 methyltransferase activity, consistent with increased H3K27me3 marks at PRC2 targets in Jmj(-/-) ESCs. Paradoxically, JMJ is required for efficient binding of PRC2, indicating that the interplay of PRC2 and JMJ fine-tunes deposition of the H3K27me3 mark. During differentiation, activation of genes marked by H3K27me3 and lineage commitments are delayed in Jmj(-/-) ESCs. Our results demonstrate that dynamic regulation of Polycomb complex activity orchestrated by JMJ balances self-renewal and differentiation, highlighting the involvement of chromatin dynamics in cell-fate transitions.
Assuntos
Células-Tronco Embrionárias/citologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular , Montagem e Desmontagem da Cromatina , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Complexo Repressor Polycomb 2 , Proteínas do Grupo PolycombRESUMO
How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.
Assuntos
Fatores Etários , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Animais , Cromatina , Elementos Facilitadores Genéticos/genética , Eritroblastos , Eritropoese/fisiologia , Feminino , Expressão Gênica , Genômica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genéticaRESUMO
Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control.
Assuntos
Células-Tronco Embrionárias/enzimologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Pluripotentes/enzimologia , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
We describe a concise and reliable protocol for the precisely controlled tetradeuteration of straight-chain fatty acids (FAs) at the α- and ß-positions that is generally applicable to a variety of FAs, including trans-FAs, polyunsaturated FAs (PUFAs), and their oxidized derivatives. The precisely controlled introduction of four deuterium atoms into the FAs enables their persistent and quantitative tracking by LC-MS/MS analysis based on their molecular structures. In addition, the phosphatidylcholine (PC) species prepared from the tetradeuterated FAs thus obtained give a diagnostic peak, namely, a phosphocholine fragment that contains deuterium, in the LC-MS/MS analysis. With these features, the metabolism of a representative oxidized linoleic acid, that is, hydroxyoctadecadienoic acid (HODE), was investigated, leading to the identification of acyltransferases that transfer the acyl moiety derived from HODE to lysophosphatidylcholine.
Assuntos
Ácidos Graxos , Ácido Linoleico , Cromatografia Líquida , Deutério , Ácidos Linoleicos/química , Espectrometria de Massas em TandemRESUMO
Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.
Assuntos
Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genômica , HumanosRESUMO
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Proteínas de Transporte/genética , Elementos Facilitadores Genéticos/genética , Engenharia Genética , Mutagênese/genética , Proteínas Nucleares/genética , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA , Eritroblastos/metabolismo , Hemoglobina Fetal/genética , Genoma/genética , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Guia de Cinetoplastídeos/genética , Proteínas Repressoras , Reprodutibilidade dos Testes , Especificidade da EspécieRESUMO
BACKGROUND: This study aimed to explore associations between various elements of primary care, patient satisfaction, and loyalty. METHODS: This cross-sectional study used a modified version of the Primary Care Assessment Tool (PCAT), which was adapted for Japan. We distributed the PCAT questionnaire to patients aged 20 years or older at five rural primary care centres in Japan. We confirmed the validity and reliability of the measure for our study. Next, we examined which elements of primary care were related to patient satisfaction and loyalty using Spearman's correlation and structural equation modelling. RESULTS: Of 220 eligible patients, 206 participated in this study. We developed nine component scales: first contact (regular access), first contact (urgent access), longitudinality, coordination, comprehensiveness (variety of care), comprehensiveness (risk prevention), comprehensiveness (health promotion), family-centeredness, and community orientation. Longitudinality and first contact (urgent access) were related with patient satisfaction. Longitudinality, first contact (regular access), and family-centeredness were related to patient loyalty. In the structural equation modelling analysis, two variables were significantly related to loyalty, namely a combined variable including longitudinality and first contact (regular access), along with family-centeredness. CONCLUSIONS: While a patient satisfaction model could not be distilled from the data, longitudinality, first contact (urgent access), and family-centeredness were identified as important elements for the cultivation of patient loyalty. This implies that primary care providers need to develop a deep understanding of patients' contexts and concerns and pay attention to their level of access to cultivate greater patient loyalty.
Assuntos
Satisfação do Paciente , Atenção Primária à Saúde , Adulto , Estudos Transversais , Humanos , Japão , Qualidade da Assistência à Saúde , Reprodutibilidade dos Testes , Inquéritos e Questionários , Adulto JovemRESUMO
Distinguishing aggressive from indolent disease and developing effective therapy for advanced disease are the major challenges in prostate cancer research. Chromosomal rearrangements involving ETS transcription factors, such as ERG and ETV1, occur frequently in prostate cancer. How they contribute to tumorigenesis and whether they play similar or distinct in vivo roles remain elusive. Here we show that in mice with ERG or ETV1 targeted to the endogenous Tmprss2 locus, either factor cooperated with loss of a single copy of Pten, leading to localized cancer, but only ETV1 appeared to support development of invasive adenocarcinoma under the background of full Pten loss. Mechanistic studies demonstrated that ERG and ETV1 control a common transcriptional network but largely in an opposing fashion. In particular, while ERG negatively regulates the androgen receptor (AR) transcriptional program, ETV1 cooperates with AR signaling by favoring activation of the AR transcriptional program. Furthermore, we found that ETV1 expression, but not that of ERG, promotes autonomous testosterone production. Last, we confirmed the association of an ETV1 expression signature with aggressive disease and poorer outcome in patient data. The distinct biology of ETV1-associated prostate cancer suggests that this disease class may require new therapies directed to underlying programs controlled by ETV1.
Assuntos
Adenocarcinoma/patologia , Androgênios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Proteínas Oncogênicas/metabolismo , Próstata/citologia , Próstata/metabolismo , Neoplasias da Próstata/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/genética , Regulador Transcricional ERGRESUMO
Adrenoleukodystrophy (X-ALD) is an X-linked genetic disorder caused by mutation of the ATP-binding cassette subfamily D member 1 gene, which encodes the peroxisomal membrane protein, adrenoleukodystrophy protein (ALDP). ALDP is associated with the transport of very-long-chain fatty acids (VLCFAs; carbon chain length ≥ 24) into peroxisomes. Defective ALDP leads to the accumulation of saturated VLCFAs in plasma and tissues, which results in damage to myelin and the adrenal glands. Here, we profiled the glycosphingolipid (GSL) species in fibroblasts from X-ALD patients. Quantitative analysis was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry with a chiral column in multiple reaction monitoring (MRM) mode. MRM transitions were designed to scan for precursor ions of long-chain bases to detect GSLs, neutral loss of hexose to detect hexosylceramide (HexCer), and precursor ions of phosphorylcholine to detect sphingomyelin (SM). Our results reveal that levels of C25 and C26-containing HexCer, Hex2Cer, NeuAc-Hex2Cer, NeuAc-HexNAc-Hex2Cer, Hex3Cer, HexNAc-Hex3Cer, and SM were elevated in fibroblasts from X-ALD patients. In conclusion, we precisely quantified SM and various GSLs in fibroblasts from X-ALD patients and determined structural information of the elevated VLCFA-containing GSLs.
Assuntos
Adrenoleucodistrofia/metabolismo , Fibroblastos/metabolismo , Glicoesfingolipídeos/metabolismo , Adrenoleucodistrofia/patologia , Biópsia , Estudos de Casos e Controles , Células Cultivadas , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Fibroblastos/patologia , Glicoesfingolipídeos/química , Humanos , Masculino , Pele/metabolismo , Pele/patologiaRESUMO
X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by deleterious mutations in the ABCD1 gene. The ABCD1 protein transports very long-chain FAs (VLCFAs) from the cytosol into the peroxisome where the VLCFAs are degraded through ß-oxidation. ABCD1 dysfunction leads to VLCFA accumulation in individuals with X-ALD. FAs are activated by esterification to CoA before metabolic utilization. However, the intracellular pools and metabolic profiles of individual acyl-CoA esters have not been fully analyzed. In this study, we profiled the acyl-CoA species in fibroblasts from X-ALD patients and in ABCD1-deficient HeLa cells. We found that hexacosenoyl (26:1)-CoA, but not hexacosanoyl (26:0)-CoA, was the most abundantly concentrated among the VLCFA-CoA species in these cells. We also show that 26:1-CoA is mainly synthesized from oleoyl-CoA, and the metabolic turnover rate of 26:1-CoA was almost identical to that of oleoyl-CoA in both WT and ABCD1-deficient HeLa cells. The findings of our study provide precise quantitative and metabolic information of each acyl-CoA species in living cells. Our results suggest that VLCFA is endogenously synthesized as VLCFA-CoA through a FA elongation pathway and is then efficiently converted to other metabolites, such as phospholipids, in the absence of ABCD1.
Assuntos
Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/deficiência , Acil Coenzima A/metabolismo , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HeLa , HumanosRESUMO
Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Proteínas F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Culina/genética , Ciclina E/genética , Ciclina E/metabolismo , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Tumor Glômico/genética , Tumor Glômico/metabolismo , Células HEK293 , Células HeLa , Humanos , Paraganglioma Extrassuprarrenal/genética , Paraganglioma Extrassuprarrenal/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre-established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage-specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células-Tronco Hematopoéticas/citologia , Mesoderma/embriologia , Mioblastos Cardíacos/citologia , Proteínas Proto-Oncogênicas/metabolismo , Células Cultivadas , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Biblioteca Gênica , Células-Tronco Hematopoéticas/fisiologia , Humanos , Mesoderma/metabolismo , Análise em Microsséries , Modelos Biológicos , Dados de Sequência Molecular , Mioblastos Cardíacos/fisiologia , Análise de Sequência de RNA , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex.
Assuntos
Anemia Hemolítica , Proteínas do Citoesqueleto/genética , Citoesqueleto , Membrana Eritrocítica , Deleção de Genes , Anemia Hemolítica/genética , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patologia , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/patologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Espectrina/genética , Espectrina/metabolismoRESUMO
BCL11A, a repressor of human fetal (γ-)globin expression, is required for immune and hematopoietic stem cell functions and brain development. Regulatory sequences within the gene, which are subject to genetic variation affecting fetal globin expression, display hallmarks of an erythroid enhancer in cell lines and transgenic mice. As such, this enhancer is a novel, attractive target for therapeutic gene editing. To explore the roles of such sequences in vivo, we generated mice in which the orthologous 10-kb intronic sequences were removed. Bcl11a enhancer-deleted mice, Bcl11a(Δenh), phenocopy the BCL11A-null state with respect to alterations of globin expression, yet are viable and exhibit no observable blood, brain, or other abnormalities. These preclinical findings provide strong in vivo support for genetic modification of the enhancer for therapy of hemoglobin disorders.
Assuntos
Proteínas de Transporte/metabolismo , Elementos Facilitadores Genéticos/genética , Células Eritroides/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Compartimento Celular , Proteínas de Ligação a DNA , Hemoglobina Fetal/genética , Hemoglobina Fetal/metabolismo , Inativação Gênica , Humanos , Camundongos , Camundongos Transgênicos , Proteínas RepressorasRESUMO
ERG, an ETS family transcription factor frequently overexpressed in human leukemia, has been implicated as a key regulator of hematopoietic stem cells. However, how ERG controls normal hematopoiesis, particularly at the stem and progenitor cell level, and how it contributes to leukemogenesis remain incompletely understood. Using homologous recombination, we generated an Erg knockdown allele (Ergkd ) in which Erg expression can be conditionally restored by Cre recombinase. Ergkd/kd animals die at E10.5-E11.5 due to defects in endothelial and hematopoietic cells, but can be completely rescued by Tie2-Cre-mediated restoration of Erg in these cells. In Ergkd/+ mice, â¼40% reduction in Erg dosage perturbs both fetal liver and bone marrow hematopoiesis by reducing the numbers of Lin- Sca-1+ c-Kit+ (LSK) hematopoietic stem and progenitor cells (HSPCs) and megakaryocytic progenitors. By genetic mosaic analysis, we find that Erg-restored HSPCs outcompete Ergkd/+ HSPCs for contribution to adult hematopoiesis in vivo. This defect is in part due to increased apoptosis of HSPCs with reduced Erg dosage, a phenotype that becomes more drastic during 5-FU-induced stress hematopoiesis. Expression analysis reveals that reduced Erg expression leads to changes in expression of a subset of ERG target genes involved in regulating survival of HSPCs, including increased expression of a pro-apoptotic regulator Bcl2l11 (Bim) and reduced expression of Jun. Collectively, our data demonstrate that ERG controls survival of HSPCs, a property that may be used by leukemic cells. Stem Cells 2017;35:1773-1785.
Assuntos
Apoptose/genética , Dosagem de Genes , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Oncogênicas/genética , Regulador Transcricional ERG/genética , Animais , Antimetabólitos/farmacologia , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Feminino , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Teste de Complementação Genética , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Integrases/genética , Integrases/metabolismo , Masculino , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas/deficiência , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Regulador Transcricional ERG/deficiênciaRESUMO
Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [14C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [14C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [14C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.