RESUMO
Chronic innate immune signaling in hematopoietic cells is widely described in myelodysplastic syndromes (MDS), and innate immune pathway activation, predominantly via pattern recognition receptors, increases the risk of developing MDS. An inflammatory component to MDS has been reported for many years, but only recently has evidence supported a more direct role of chronic innate immune signaling and associated inflammatory pathways in the pathogenesis of MDS. Here we review recent findings and discuss relevant questions related to chronic immune response dysregulation in MDS.
Assuntos
Imunidade Inata , Síndromes Mielodisplásicas/imunologia , Animais , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Síndromes Mielodisplásicas/patologiaRESUMO
GATA-1 and its cofactor FOG-1 are required for the differentiation of erythrocytes and megakaryocytes. In contrast, mast cell development requires GATA-1 and the absence of FOG-1. Through genome-wide comparison of the chromatin occupancy of GATA-1 and a naturally occurring mutant that cannot bind FOG-1 (GATA-1(V205G)), we reveal that FOG-1 intricately regulates the chromatin occupancy of GATA-1. We identified GATA1-selective and GATA-1(V205G)-selective binding sites and show that GATA-1, in the absence of FOG-1, occupies GATA-1(V205G)-selective sites, but not GATA1-selective sites. By integrating ChIP-seq and gene expression data, we discovered that GATA-1(V205G) binds and activates mast cell-specific genes via GATA-1(V205G)-selective sites. We further show that exogenous expression of FOG-1 in mast cells leads to displacement of GATA-1 from mast cell-specific genes and causes their downregulation. Together these findings establish a mechanism of gene regulation whereby a non-DNA binding cofactor directly modulates the occupancy of a transcription factor to control lineage specification.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Regulação para Baixo , Regulação da Expressão Gênica/genética , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Ligação Proteica , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The development of complex organisms requires the formation of diverse cell types from common stem and progenitor cells. GATA family transcriptional regulators and their dedicated co-factors, termed Friend of GATA (FOG) proteins, control cell fate and differentiation in multiple tissue types from Drosophila to man. FOGs can both facilitate and antagonize GATA factor transcriptional regulation depending on the factor, cell, and even the specific gene target. In this review, we highlight recent studies that have elucidated mechanisms by which FOGs regulate GATA factor function and discuss how these factors use these diverse modes of gene regulation to control cell lineage specification throughout metazoans.
Assuntos
Fatores de Transcrição GATA/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição GATA/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Nucleares/genética , Fatores de Transcrição/genéticaRESUMO
UNLABELLED: DNA repair plays a crucial role in embryonic and somatic stem cell biology and cell reprogramming. The Fanconi anemia (FA) pathway, which promotes error-free repair of DNA double-strand breaks, is required for somatic cell reprogramming to induced pluripotent stem cells (iPSC). Thus, cells from Fanconi anemia patients, which lack this critical pathway, fail to be reprogrammed to iPSC under standard conditions unless the defective FA gene is complemented. In this study, we utilized the oncogenes of high-risk human papillomavirus 16 (HPV16) to overcome the resistance of FA patient cells to reprogramming. We found that E6, but not E7, recovers FA iPSC colony formation and, furthermore, that p53 inhibition is necessary and sufficient for this activity. The iPSC colonies resulting from each of these approaches stained positive for alkaline phosphatase, NANOG, and Tra-1-60, indicating that they were fully reprogrammed into pluripotent cells. However, FA iPSC were incapable of outgrowth into stable iPSC lines regardless of p53 suppression, whereas their FA-complemented counterparts grew efficiently. Thus, we conclude that the FA pathway is required for the growth of iPSC beyond reprogramming and that p53-independent mechanisms are involved. IMPORTANCE: A novel approach is described whereby HPV oncogenes are used as tools to uncover DNA repair-related molecular mechanisms affecting somatic cell reprogramming. The findings indicate that p53-dependent mechanisms block FA cells from reprogramming but also uncover a previously unrecognized defect in FA iPSC proliferation independent of p53.
Assuntos
Reprogramação Celular/genética , Anemia de Fanconi/genética , Células-Tronco Pluripotentes Induzidas/virologia , Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Proteína Homeobox Nanog , Proteínas Oncogênicas Virais/metabolismo , Cultura Primária de Células , Proteoglicanas/genética , Proteoglicanas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Transdução Genética , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Children with Down syndrome develop a unique congenital clonal megakaryocytic proliferation disorder (transient myeloproliferative disorder [TMD]). It is caused by an expansion of fetal megakaryocyte-erythroid progenitors (MEPs) triggered by trisomy of chromosome 21 and is further enhanced by the somatic acquisition of a mutation in GATA1. These mutations result in the expression of a short-isoform GATA1s lacking the N-terminal domain. To examine the hypothesis that the Hsa21 ETS transcription factor ERG cooperates with GATA1s in this process, we generated double-transgenic mice expressing hERG and Gata1s. We show that increased expression of ERG by itself is sufficient to induce expansion of MEPs in fetal livers. Gata1s expression synergizes with ERG in enhancing the expansion of fetal MEPs and megakaryocytic precursors, resulting in hepatic fibrosis, transient postnatal thrombocytosis, anemia, a gene expression profile that is similar to that of human TMD and progression to progenitor myeloid leukemia by 3 months of age. This ERG/Gata1s transgenic mouse model also uncovers an essential role for the N terminus of Gata1 in erythropoiesis and the antagonistic role of ERG in fetal erythroid differentiation and survival. The human relevance of this finding is underscored by the recent discovery of similar mutations in GATA1 in patients with Diamond-Blackfan anemia.
Assuntos
Síndrome de Down/sangue , Síndrome de Down/complicações , Hematopoese , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/complicações , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Fígado/embriologia , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Oncogênicas/metabolismo , Células-Tronco/citologia , Fatores de Transcrição , Regulador Transcricional ERGRESUMO
The transcription factor Ikaros regulates the development of hematopoietic cells. Ikaros-deficient animals fail to develop B cells and display a T-cell malignancy, which is correlated with altered Notch signaling. Recently, loss of Ikaros was associated with progression of myeloproliferative neoplasms to acute myeloid leukemia and increasing evidence shows that Ikaros is also critical for the regulation of myeloid development. Previous studies showed that Ikaros-deficient mice have increased megakaryopoiesis, but the molecular mechanism of this phenomenon remains unknown. Here, we show that Ikaros overexpression decreases NOTCH-induced megakaryocytic specification, and represses expression of several megakaryocytic genes including GATA-1 to block differentiation and terminal maturation. We also demonstrate that Ikaros expression is differentially regulated by GATA-2 and GATA-1 during megakaryocytic differentiation and reveal that the combined loss of Ikzf1 and Gata1 leads to synthetic lethality in vivo associated with prominent defects in erythroid cells and an expansion of megakaryocyte progenitors. Taken together, our observations demonstrate an important functional interplay between Ikaros, GATA factors, and the NOTCH signaling pathway in specification and homeostasis of the megakaryocyte lineage.
Assuntos
Fator de Transcrição GATA1/metabolismo , Fator de Transcrição Ikaros/fisiologia , Receptores Notch/metabolismo , Trombopoese/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Regulação para Baixo/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Megacariócitos/metabolismo , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
GATA1 is a master transcriptional regulator of the differentiation of several related myeloid blood cell types, including erythrocytes and megakaryocytes. Germ-line mutations that cause loss of full length GATA1, but allow for expression of the short isoform (GATA1s), are associated with defective erythropoiesis in a subset of patients with Diamond Blackfan Anemia. Despite extensive studies of GATA1s in megakaryopoiesis, the mechanism by which GATA1s fails to support normal erythropoiesis is not understood. In this study, we used global gene expression and chromatin occupancy analysis to compare the transcriptional activity of GATA1s to GATA1. We discovered that compared to GATA1, GATA1s is less able to activate the erythroid gene expression program and terminal differentiation in cells with dual erythroid-megakaryocytic differentiation potential. Moreover, we found that GATA1s bound to many of its erythroid-specific target genes less efficiently than full length GATA1. These results suggest that the impaired ability of GATA1s to promote erythropoiesis in DBA may be caused by failure to occupy erythroid-specific gene regulatory elements.
Assuntos
Imunoprecipitação da Cromatina , Células Eritroides/metabolismo , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Isoformas de RNA , Transcriptoma , Sítios de Ligação , Diferenciação Celular/genética , Linhagem Celular , Análise por Conglomerados , Células Eritroides/citologia , Eritropoese/genética , Fator de Transcrição GATA1/metabolismo , Perfilação da Expressão Gênica , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Trombopoese/genéticaRESUMO
There are many examples of transcription factor families whose members control gene expression profiles of diverse cell types. However, the mechanism by which closely related factors occupy distinct regulatory elements and impart lineage specificity is largely undefined. Here we demonstrate on a genome wide scale that the hematopoietic GATA factors GATA-1 and GATA-2 bind overlapping sets of genes, often at distinct sites, as a means to differentially regulate target gene expression and to regulate the balance between proliferation and differentiation. We also reveal that the GATA switch, which entails a chromatin occupancy exchange between GATA2 and GATA1 in the course of differentiation, operates on more than one-third of GATA1 bound genes. The switch is equally likely to lead to transcriptional activation or repression; and in general, GATA1 and GATA2 act oppositely on switch target genes. In addition, we show that genomic regions co-occupied by GATA2 and the ETS factor ETS1 are strongly enriched for regions marked by H3K4me3 and occupied by Pol II. Finally, by comparing GATA1 occupancy in erythroid cells and megakaryocytes, we find that the presence of ETS factor motifs is a major discriminator of megakaryocyte versus red cell specification.
Assuntos
Cromatina/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA2/genética , Genes de Troca/genética , Estudo de Associação Genômica Ampla , Hematopoese/genética , Animais , Linhagem da Célula/fisiologia , Cromatina/metabolismo , Células Eritroides/citologia , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Histonas/metabolismo , Megacariócitos/citologia , Megacariócitos/fisiologia , Metilação , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismoRESUMO
Germline heterozygous mutations in DDX41 predispose individuals to hematologic malignancies in adulthood. Most of these DDX41 mutations result in a truncated protein, leading to loss of protein function. To investigate the impact of these mutations on hematopoiesis, we generated mice with hematopoietic-specific knockout of one Ddx41 allele. Under normal steady-state conditions, there was minimal effect on lifelong hematopoiesis, resulting in a mild yet persistent reduction in red blood cell counts. However, stress induced by transplantation of the Ddx41+/- BM resulted in hematopoietic stem/progenitor cell (HSPC) defects and onset of hematopoietic failure upon aging. Transcriptomic analysis of HSPC subsets from the transplanted BM revealed activation of cellular stress responses, including upregulation of p53 target genes in erythroid progenitors. To understand how the loss of p53 affects the phenotype of Ddx41+/- HSPCs, we generated mice with combined Ddx41 and Trp53 heterozygous deletions. The reduction in p53 expression rescued the fitness defects in HSPC caused by Ddx41 heterozygosity. However, the combined Ddx41 and Trp53 mutant mice were prone to developing hematologic malignancies that resemble human myelodysplastic syndrome and acute myeloid leukemia. In conclusion, DDX41 heterozygosity causes dysregulation of the response to hematopoietic stress, which increases the risk of transformation with a p53 mutation.
Assuntos
RNA Helicases DEAD-box , Haploinsuficiência , Neoplasias Hematológicas , Hematopoese , Mutação , Proteína Supressora de Tumor p53 , Animais , Proteína Supressora de Tumor p53/genética , RNA Helicases DEAD-box/genética , Camundongos , Hematopoese/genética , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/etiologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Camundongos Knockout , Humanos , Estresse Fisiológico/genética , Camundongos Endogâmicos C57BLRESUMO
DDX41 mutations are the most common germline alterations in adult myelodysplastic syndromes (MDSs). The majority of affected individuals harbor germline monoallelic frameshift DDX41 mutations and subsequently acquire somatic mutations in their other DDX41 allele, typically missense R525H. Hematopoietic progenitor cells (HPCs) with biallelic frameshift and R525H mutations undergo cell cycle arrest and apoptosis, causing bone marrow failure in mice. Mechanistically, DDX41 is essential for small nucleolar RNA (snoRNA) processing, ribosome assembly, and protein synthesis. Although monoallelic DDX41 mutations do not affect hematopoiesis in young mice, a subset of aged mice develops features of MDS. Biallelic mutations in DDX41 are observed at a low frequency in non-dominant hematopoietic stem cell clones in bone marrow (BM) from individuals with MDS. Mice chimeric for monoallelic DDX41 mutant BM cells and a minor population of biallelic mutant BM cells develop hematopoietic defects at a younger age, suggesting that biallelic DDX41 mutant cells are disease modifying in the context of monoallelic DDX41 mutant BM.
Assuntos
RNA Helicases DEAD-box , Síndromes Mielodisplásicas , Animais , RNA Helicases DEAD-box/genética , Células Germinativas , Hematopoese/genética , Camundongos , Mutação/genética , Síndromes Mielodisplásicas/genéticaRESUMO
Squamous cell carcinoma (SCC) is a global public health burden originating in epidermal stem and progenitor cells (ESPCs) of the skin and mucosa. To understand how genetic risk factors contribute to SCC, studies of ESPC biology are imperative. Children with Fanconi anemia (FA) are a paradigm for extreme SCC susceptibility caused by germline loss-of-function mutations in FA DNA repair pathway genes. To discover epidermal vulnerabilities, patient-derived pluripotent stem cells (PSCs) conditional for the FA pathway were differentiated into ESPCs and PSC-derived epidermal organotypic rafts (PSC-EORs). FA PSC-EORs harbored diminished cell-cell junctions and increased proliferation in the basal cell compartment. Furthermore, desmosome and hemidesmosome defects were identified in the skin of FA patients, and these translated into accelerated blistering following mechanically induced stress. Together, we demonstrate that a critical DNA repair pathway maintains the structure and function of human skin and provide 3D epidermal models wherein SCC prevention can now be explored.
Assuntos
Carcinoma de Células Escamosas , Anemia de Fanconi , Diferenciação Celular , Criança , Reparo do DNA , Anemia de Fanconi/genética , Humanos , PeleRESUMO
The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mama/efeitos dos fármacos , Mama/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de Superfície Celular/metabolismo , Tretinoína/farmacologia , Proteína de Ligação a Vitamina D/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose , Sequência de Bases , Mama/citologia , Diferenciação Celular , Linhagem Celular , Primers do DNA/genética , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Proteínas Supressoras de Tumor , Proteína de Ligação a Vitamina D/genéticaRESUMO
OBJECTIVE: It has been demonstrated that high concentration of the transcription factor PU.1 (encoded by Sfpi1) promotes macrophage development, whereas low concentration induces B-cell development in vitro. This has led to the hypothesis that lower levels of PU.1 activity are required for B cell than for macrophage development in vivo. We utilized an allele of Sfpi1 (termed BN) with a mutation in the first coding exon, which resulted in a reduction of PU.1 expression in order to test this hypothesis. MATERIALS AND METHODS: Using gene targeting in embryonic stem cells, two ATG-start site codons of PU.1 were mutated, resulting in reduced PU.1 expression originating from a third start codon. Mice were assayed for phenotypic abnormalities using fluorescence-activated cell sorting, microscopy, and colony-forming ability. In addition, isolated cells were tested for their differentiation potential in vitro and in vivo. RESULTS: Lymphoid and myeloid cells derived from cultured Sfpi1(BN/BN) fetal liver cells had reduced levels of PU.1 expression and activity. B-cell development was intrinsically blocked in cells isolated from Sfpi1(BN/BN) mice. In addition, myeloid development was impaired in Sfpi1(BN/BN) fetal liver. However, neonatal Sfpi1(BN/BN) mice had a dramatic expansion and infiltration of immature myeloid cells. CONCLUSION: Contrary to our original hypothesis, high levels of PU.1 activity are required to induce both myeloid and B-cell development. In addition, neonatal mice homozygous for the hypomorphic allele acquire a myeloproliferative disorder and die within 1 month of age.
Assuntos
Linfócitos B/fisiologia , Transtornos Mieloproliferativos/etiologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Alelos , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mielopoese , Proteínas Proto-Oncogênicas/genética , Transativadores/genéticaRESUMO
Pluripotent stem cells (PSCs) maintain a low mutation frequency compared with somatic cell types at least in part by preferentially utilizing error-free homologous recombination (HR) for DNA repair. Many endogenous metabolites cause DNA interstrand crosslinks, which are repaired by the Fanconi anemia (FA) pathway using HR. To determine the effect of failed repair of endogenous DNA lesions on PSC biology, we generated iPSCs harboring a conditional FA pathway. Upon FA pathway loss, iPSCs maintained pluripotency but underwent profound G2 arrest and apoptosis, whereas parental fibroblasts grew normally. Mechanistic studies revealed that G2-phase FA-deficient iPSCs possess large γH2AX-RAD51 foci indicative of accrued DNA damage, which correlated with activated DNA-damage signaling through CHK1. CHK1 inhibition specifically rescued the growth of FA-deficient iPSCs for prolonged culture periods, surprisingly without stimulating excessive karyotypic abnormalities. These studies reveal that PSCs possess hyperactive CHK1 signaling that restricts their self-renewal in the absence of error-free DNA repair.