RESUMO
Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.
Assuntos
Anemia/imunologia , Doenças dos Peixes/imunologia , Fator de Transcrição GATA1/metabolismo , Inflamassomos/metabolismo , Inflamação/imunologia , Neutrófilos/imunologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Caspase 1/genética , Caspase 1/metabolismo , Diferenciação Celular , Células Eritroides/citologia , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Humanos , Inflamassomos/genética , Células K562 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Proteínas de Peixe-Zebra/genéticaRESUMO
Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.
Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Cromatina , Dinoprostona , Sequências Reguladoras de Ácido Nucleico , Montagem e Desmontagem da CromatinaRESUMO
Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Proteínas de Ligação a DNA , Hematopoese , Animais , Diferenciação Celular , Linhagem Celular , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Células-Tronco Hematopoéticas , Humanos , Masculino , Camundongos , Baço/citologia , Peixe-ZebraRESUMO
Whole-exome sequencing has been incredibly successful in identifying causal genetic variants and has revealed a number of novel genes associated with blood and other diseases. One limitation of this approach is that it overlooks mutations in noncoding regulatory elements. Furthermore, the mechanisms by which mutations in transcriptionalcis-regulatory elements result in disease remain poorly understood. Here we used CRISPR/Cas9 genome editing to interrogate three such elements harboring mutations in human erythroid disorders, which in all cases are predicted to disrupt a canonical binding motif for the hematopoietic transcription factor GATA1. Deletions of as few as two to four nucleotides resulted in a substantial decrease (>80%) in target gene expression. Isolated deletions of the canonical GATA1 binding motif completely abrogated binding of the cofactor TAL1, which binds to a separate motif. Having verified the functionality of these three GATA1 motifs, we demonstrate strong evolutionary conservation of GATA1 motifs in regulatory elements proximal to other genes implicated in erythroid disorders, and show that targeted disruption of such elements results in altered gene expression. By modeling transcription factor binding patterns, we show that multiple transcription factors are associated with erythroid gene expression, and have created predictive maps modeling putative disruptions of their binding sites at key regulatory elements. Our study provides insight into GATA1 transcriptional activity and may prove a useful resource for investigating the pathogenicity of noncoding variants in human erythroid disorders.
Assuntos
Anemia de Diamond-Blackfan/metabolismo , Fator de Transcrição GATA1/metabolismo , Mutação , Elementos de Resposta , Transcrição Gênica , Anemia de Diamond-Blackfan/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistemas CRISPR-Cas , Fator de Transcrição GATA1/genética , Humanos , Células K562 , Motivos de Nucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células TRESUMO
Eukaryotic translation initiation factor 3 (eIF3) plays a central role in translation initiation and consists of five core (conserved) subunits present in both budding yeast and higher eukaryotes. Higher eukaryotic eIF3 contains additional (noncore or nonconserved) subunits of poorly defined function, including sub-unit h (eIF3h), which in zebrafish is encoded by two distinct genes (eif3ha and eif3hb). Previously we showed that eif3ha encodes the predominant isoform during zebrafish embryogenesis and that depletion of this factor causes defects in the development of the brain and eyes. To investigate the molecular mechanism governing this regulation, we developed a genome-wide polysome-profiling strategy using stage-matched WT and eif3ha morphant zebrafish embryos. This strategy identified a large set of predominantly neural-associated translationally regulated mRNAs. A striking finding was a cohort of lens-associated crystallin isoform mRNAs lost from the eif3ha morphant polysomes, revealing a mechanism by which lens development is translationally controlled. We show that both UTR sequences of a targeted crystallin transcript are necessary but not sufficient for translational regulation by eif3ha. Therefore, our study reveals the role of a noncore eIF3 subunit in modulating a specific developmental program by regulating translation of defined transcripts and highlights the potential of the zebrafish system to identify translational regulatory mechanisms controlling vertebrate development.
Assuntos
Embrião não Mamífero/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Western Blotting , Embrião não Mamífero/embriologia , Fator de Iniciação 3 em Eucariotos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Polirribossomos/genética , Biossíntese de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , gama-Cristalinas/genética , gama-Cristalinas/metabolismoRESUMO
A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.
RESUMO
Eukaryotic translation initiation factor eIF3, which plays a central role in translation initiation, consists of five core subunits that are present in both the budding yeast and higher eukaryotes. However, higher eukaryotic eIF3 contains additional (non-core) subunits that are absent in the budding yeast. We investigated the role of one such non-core eIF3 subunit eIF3h, encoded by two distinct genes-eif3ha and eif3hb, as a regulator of embryonic development in zebrafish. Both eif3h genes are expressed during early embryogenesis, and display overlapping yet distinct and highly dynamic spatial expression patterns. Loss of function analysis using specific morpholino oligomers indicates that each isoform has specific as well as redundant functions during early development. The morphant phenotypes correlate with their spatial expression patterns, indicating that eif3h regulates development of the brain, heart, vasculature, and lateral line. These results indicate that the non-core subunits of eIF3 regulate specific developmental programs during vertebrate embryogenesis.
Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação 3 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Animais , Citoplasma/genética , Citoplasma/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Eucariotos , Fator de Iniciação 3 em Eucariotos/biossíntese , Fatores de Iniciação em Eucariotos/metabolismo , Feminino , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The National Heart, Lung, and Blood Institute Progenitor Cell Translational Consortium Blood Progenitor Meeting was hosted virtually on November 5, 2020, with 93 attendees across 20 research groups. The purpose of this meeting was to exchange recent findings, discuss current efforts, and identify prospective opportunities in the field of hematopoietic stem and progenitor cell research and therapeutic discovery.
Assuntos
Células Sanguíneas/citologia , Comportamento Cooperativo , Células-Tronco/citologia , Microambiente Celular , Hematopoese , Humanos , Nicho de Células-TroncoRESUMO
Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-ß-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.
Assuntos
Eritrócitos/fisiologia , Regulação da Expressão Gênica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Eritrócitos/citologia , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
This perspective describes the usefulness of zebrafish as a model to study interaction of hematopoietic stem cells with the associated niche in vivo, explains how such interactions influence regeneration, migration, and clonality of HSCs, and defines their fate during differentiation.
Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Diferenciação Celular , Movimento Celular , Células-Tronco Hematopoéticas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Regeneração/fisiologia , Transdução de Sinais , Nicho de Células-Tronco/fisiologia , Peixe-ZebraRESUMO
Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.