RESUMO
Prokaryotic evolution is influenced by the exchange of genetic information between species through a process referred to as recombination. The rate of recombination is a useful measure for the adaptive capacity of a prokaryotic population. We introduce Rhometa (https://github.com/sid-krish/Rhometa), a new software package to determine recombination rates from shotgun sequencing reads of metagenomes. It extends the composite likelihood approach for population recombination rate estimation and enables the analysis of modern short-read datasets. We evaluated Rhometa over a broad range of sequencing depths and complexities, using simulated and real experimental short-read data aligned to external reference genomes. Rhometa offers a comprehensive solution for determining population recombination rates from contemporary metagenomic read datasets. Rhometa extends the capabilities of conventional sequence-based composite likelihood population recombination rate estimators to include modern aligned metagenomic read datasets with diverse sequencing depths, thereby enabling the effective application of these techniques and their high accuracy rates to the field of metagenomics. Using simulated datasets, we show that our method performs well, with its accuracy improving with increasing numbers of genomes. Rhometa was validated on a real S. pneumoniae transformation experiment, where we show that it obtains plausible estimates of the rate of recombination. Finally, the program was also run on ocean surface water metagenomic datasets, through which we demonstrate that the program works on uncultured metagenomic datasets.
Assuntos
Metagenoma , Metagenômica , Metagenômica/métodos , Metagenoma/genética , Análise de Sequência de DNA/métodos , Funções Verossimilhança , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Software , Recombinação Genética/genética , AlgoritmosRESUMO
Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Células-Tronco Hematopoéticas , Humanos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Cromatina/metabolismoRESUMO
Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Epigênese Genética , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/fisiopatologia , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other's, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis.
Assuntos
Fator de Transcrição GATA2/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Células Eritroides/metabolismo , Células Eritroides/patologia , Redes Reguladoras de Genes , Hematopoese , Humanos , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Regulador Transcricional ERG/genéticaRESUMO
MYCN amplification occurs in approximately 20-30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.
Assuntos
Antimitóticos , Neuroblastoma , Humanos , Camundongos , Animais , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Linhagem Celular Tumoral , Camundongos Transgênicos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.
Assuntos
Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/genética , Histona Desmetilases/metabolismo , Leucemia Mieloide Aguda/patologia , Fatores de Transcrição da Família Snail/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células HEK293 , Células HL-60 , Histona Desmetilases/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Ligação Proteica , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismoRESUMO
Promoters are DNA sequences that have an essential role in controlling gene expression. While recent whole cancer genome analyses have identified numerous hotspots of somatic point mutations within promoters, many have not yet been shown to perturb gene expression or drive cancer development. As such, positive selection alone may not adequately explain the frequency of promoter point mutations in cancer genomes. Here we show that increased mutation density at gene promoters can be linked to promoter activity and differential nucleotide excision repair (NER). By analysing 1,161 human cancer genomes across 14 cancer types, we find evidence for increased local density of somatic point mutations within the centres of DNase I-hypersensitive sites (DHSs) in gene promoters. Mutated DHSs were strongly associated with transcription initiation activity, in which active promoters but not enhancers of equal DNase I hypersensitivity were most mutated relative to their flanking regions. Notably, analysis of genome-wide maps of NER shows that NER is impaired within the DHS centre of active gene promoters, while XPC-deficient skin cancers do not show increased promoter mutation density, pinpointing differential NER as the underlying cause of these mutation hotspots. Consistent with this finding, we observe that melanomas with an ultraviolet-induced DNA damage mutation signature show greatest enrichment of promoter mutations, whereas cancers that are not highly dependent on NER, such as colon cancer, show no sign of such enrichment. Taken together, our analysis has uncovered the presence of a previously unknown mechanism linking transcription initiation and NER as a major contributor of somatic point mutation hotspots at active gene promoters in cancer genomes.
Assuntos
Reparo do DNA/genética , Genoma Humano/genética , Mutagênese/genética , Taxa de Mutação , Neoplasias/genética , Regiões Promotoras Genéticas/genética , Iniciação da Transcrição Genética , Neoplasias do Colo/genética , Dano ao DNA/genética , Reparo do DNA/efeitos da radiação , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/genética , Melanoma/genética , Mutação Puntual/genética , Raios UltravioletaRESUMO
The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Plaquetas/fisiologia , Proteínas de Neoplasias/fisiologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/fisiologia , Trombopoese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica , Megacariócitos/patologia , Megacariócitos/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Trombocitopenia/sangue , Trombocitopenia/genéticaRESUMO
Acute myeloid leukemia (AML) is a complex disease characterized by a diverse range of recurrent molecular aberrations that occur in many different combinations. Components of transcriptional networks are a common target of these aberrations, leading to network-wide changes and deployment of novel or developmentally inappropriate transcriptional programs. Genome-wide techniques are beginning to reveal the full complexity of normal hematopoietic stem cell transcriptional networks and the extent to which they are deregulated in AML, and new understandings of the mechanisms by which AML cells maintain self-renewal and block differentiation are starting to emerge. The hope is that increased understanding of the network architecture in AML will lead to identification of key oncogenic dependencies that are downstream of multiple network aberrations, and that this knowledge will be translated into new therapies that target these dependencies. Here, we review the current state of knowledge of network perturbation in AML with a focus on major mechanisms of transcription factor dysregulation, including mutation, translocation, and transcriptional dysregulation, and discuss how these perturbations propagate across transcriptional networks. We will also review emerging mechanisms of network disruption, and briefly discuss how increased knowledge of network disruption is already being used to develop new therapies.
Assuntos
Redes Reguladoras de Genes/genética , Leucemia Mieloide Aguda/genética , Transcrição Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Mutação/genética , Fatores de Transcrição/genética , Translocação Genética/genéticaRESUMO
Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.
Assuntos
Exossomos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas rab27 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Meios de Cultivo Condicionados , Exossomos/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanossomas/genética , Melanossomas/metabolismo , Camundongos , Invasividade Neoplásica , Nevo/genética , Nevo/metabolismo , Proteômica , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Esferoides Celulares , Proteínas rab27 de Ligação ao GTP/biossíntese , Proteínas rab27 de Ligação ao GTP/genéticaRESUMO
Inference of ancestry from biological evidence can provide investigative information, especially for unknown DNA donors. Although tools for predicting ancestry have been developing, ancestry research focusing on populations relevant for South Korea is not common and markers are seldom chosen specifically to differentiate Koreans from other East Asian and South East Asian populations. Here, we report ancestry informative markers (AIMs) for distinguishing six East/South East Asian regional populations: China, Japan, Indonesia, Philippines, South Korea and Thailand. Individual genotypes from these six populations were available in PanSNPdb: The HUGO Pan-Asian SNP Database. To select AIMs, we calculated four population divergence metrics for each SNP: Nei's FST, Rosenberg's Informativeness (In), the average absolute allele frequency difference between populations (δFmean) and the maximum allele frequency difference between populations (δFmax). Based on these values, we selected 100 single nucleotide polymorphisms (SNPs) for distinguishing the six populations, 13 of which exhibited large allele frequency differences between Koreans and non-Koreans. To assess the performance of the AIMs, we performed principal coordinates analysis (PCoA) on the individuals from all six populations and inferred ancestral population clusters using the STRUCTURE program. In conclusion, we found that the selected AIMs can be applied to distinguish the six East/South East Asian groups and we suggest the markers in this study will be helpful to establish ancestry panels for Korea and neighbouring populations.
Assuntos
Povo Asiático/genética , Marcadores Genéticos , Genética Populacional , Polimorfismo de Nucleotídeo Único , Ásia , Impressões Digitais de DNA , Bases de Dados Genéticas , Frequência do Gene , Genótipo , Humanos , Análise de Componente PrincipalRESUMO
Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.
Assuntos
Azacitidina/farmacologia , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Células Cultivadas , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/fisiologiaRESUMO
MOTIVATION: Long non-coding RNAs (lncRNAs) have been implicated in the regulation of diverse biological functions. The number of newly identified lncRNAs has increased dramatically in recent years but their expression and function have not yet been described from most diseases. To elucidate lncRNA function in human disease, we have developed a novel network based method (NLCFA) integrating correlations between lncRNA, protein coding genes and noncoding miRNAs. We have also integrated target gene associations and protein-protein interactions and designed our model to provide information on the combined influence of mRNAs, lncRNAs and miRNAs on cellular signal transduction networks. RESULTS: We have generated lncRNA expression profiles from the CD34+ haematopoietic stem and progenitor cells (HSPCs) from patients with Myelodysplastic syndromes (MDS) and healthy donors. We report, for the first time, aberrantly expressed lncRNAs in MDS and further prioritize biologically relevant lncRNAs using the NLCFA. Taken together, our data suggests that aberrant levels of specific lncRNAs are intimately involved in network modules that control multiple cancer-associated signalling pathways and cellular processes. Importantly, our method can be applied to prioritize aberrantly expressed lncRNAs for functional validation in other diseases and biological contexts. AVAILABILITY AND IMPLEMENTATION: The method is implemented in R language and Matlab. CONTACT: xizhou@wakehealth.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Anotação de Sequência Molecular/métodos , Síndromes Mielodisplásicas/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Software , Idoso , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Neoplasias/genética , Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Análise de Sequência de RNA/métodosRESUMO
Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the -8/+7/+9-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/-8-kb enhancer targeted TIE2+/c-KIT+/CD41- endothelial cells that were enriched for hematopoietic potential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters, LRP2, a multiligand receptor, was the only gene that had not previously been associated with hematopoiesis. We show that LRP2 is indeed involved in definitive hematopoiesis and by doing so validate the use of reporter gene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.
Assuntos
Embrião de Mamíferos/metabolismo , Endoglina/metabolismo , Elementos Facilitadores Genéticos/fisiologia , Hematopoese/fisiologia , Sondas Moleculares/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Endoglina/genética , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Sondas Moleculares/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismoRESUMO
Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Elementos Facilitadores Genéticos , Células Mieloides/fisiologia , Mielopoese/genética , Neutrófilos/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Células K562 , Camundongos , Camundongos Knockout , Células U937RESUMO
Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-ALL and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remains poorly understood. In order to discover transcriptional regulators of ERG, we employed a quantitative mass spectrometry-based method to identify factors binding the 321 bp ERG +85 stem cell enhancer region in MOLT-4 T-ALL and KG-1 AML cells. Using this approach, we identified a number of known binders of the +85 enhancer in leukaemic cells along with previously unknown binders, including ETV6 and IKZF1. We confirmed that ETV6 and IKZF1 were also bound at the +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells. Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators not just of ERG, but also of a number of genes regulated by a densely interconnected network of seven transcription factors. At last, we show that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis.
Assuntos
Fator de Transcrição Ikaros/fisiologia , Proteínas Proto-Oncogênicas c-ets/fisiologia , Proteínas Repressoras/fisiologia , Transcrição Gênica , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Consenso , Elementos Facilitadores Genéticos , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Ligação Proteica , Proteoma , Proteômica , Regulador Transcricional ERG/fisiologia , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Glucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed that KLF13 and MYB gene expression changes were significantly greater in dexamethasone-sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB expression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Proteínas de Membrana/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Glucocorticoides/genética , Animais , Antineoplásicos Hormonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteína 11 Semelhante a Bcl-2 , Western Blotting , Imunoprecipitação da Cromatina , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Ensaio de Imunoadsorção Enzimática , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Glucocorticoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The BloodChIP database (http://www.med.unsw.edu.au/CRCWeb.nsf/page/BloodChIP) supports exploration and visualization of combinatorial transcription factor (TF) binding at a particular locus in human CD34-positive and other normal and leukaemic cells or retrieval of target gene sets for user-defined combinations of TFs across one or more cell types. Increasing numbers of genome-wide TF binding profiles are being added to public repositories, and this trend is likely to continue. For the power of these data sets to be fully harnessed by experimental scientists, there is a need for these data to be placed in context and easily accessible for downstream applications. To this end, we have built a user-friendly database that has at its core the genome-wide binding profiles of seven key haematopoietic TFs in human stem/progenitor cells. These binding profiles are compared with binding profiles in normal differentiated and leukaemic cells. We have integrated these TF binding profiles with chromatin marks and expression data in normal and leukaemic cell fractions. All queries can be exported into external sites to construct TF-gene and protein-protein networks and to evaluate the association of genes with cellular processes and tissue expression.
Assuntos
Células Sanguíneas/metabolismo , Bases de Dados Genéticas , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA/química , DNA/metabolismo , Perfilação da Expressão Gênica , Genoma Humano , Células-Tronco Hematopoéticas/metabolismo , Humanos , Internet , Software , Fatores de Transcrição/químicaRESUMO
The progression of benign colorectal adenomas into cancer is associated with the accumulation of chromosomal aberrations. Even though patterns and frequencies of chromosomal aberrations have been well established in colorectal carcinomas, corresponding patterns of aberrations in adenomas are less well documented. The aim of this study was to profile chromosomal aberrations across colorectal adenomas and carcinomas to provide a better insight into key changes during tumor initiation and progression. Single nucleotide polymorphism array analysis was performed on 216 colorectal tumor/normal matched pairs, comprising 60 adenomas and 156 carcinomas. While many chromosomal aberrations were specific to carcinomas, those with the highest frequency in carcinomas (amplification of chromosome 7, 13q, and 20q; deletion of 17p and chromosome 18; LOH of 1p, chromosome 4, 5q, 8p, 17p, chromosome 18, and 20p) were also identified in adenomas. Hierarchical clustering using chromosomal aberrations revealed three distinct subtypes. Interestingly, these subtypes were only partially dependent on tumor staging. A cluster of colorectal cancer patients with frequent chromosomal deletions had the least favorable prognosis, and a number of adenomas (n = 9) were also present in the cluster suggesting that, at least in some tumors, the chromosomal aberration pattern is determined at a very early stage of tumor formation. Finally, analysis of LOH events revealed that copy-neutral/gain LOH (CN/G-LOH) is frequent (>10%) in carcinomas at 5q, 11q, 15q, 17p, chromosome 18, 20p, and 22q. Deletion of the corresponding region is sometimes present in adenomas, suggesting that LOH at these loci may play an important role in tumor initiation.
Assuntos
Adenoma/genética , Carcinoma/genética , Aberrações Cromossômicas , Neoplasias Colorretais/genética , Polimorfismo de Nucleotídeo Único , Adenoma/patologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma/patologia , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Genome-wide combinatorial binding patterns for key transcription factors (TFs) have not been reported for primary human hematopoietic stem and progenitor cells (HSPCs), and have constrained analysis of the global architecture of molecular circuits controlling these cells. Here we provide high-resolution genome-wide binding maps for a heptad of key TFs (FLI1, ERG, GATA2, RUNX1, SCL, LYL1, and LMO2) in human CD34(+) HSPCs, together with quantitative RNA and microRNA expression profiles. We catalog binding of TFs at coding genes and microRNA promoters, and report that combinatorial binding of all 7 TFs is favored and associated with differential expression of genes and microRNA in HSPCs. We also uncover a previously unrecognized association between FLI1 and RUNX1 pairing in HSPCs, we establish a correlation between the density of histone modifications that mark active enhancers and the number of overlapping TFs at a peak, we demonstrate bivalent histone marks at promoters of heptad target genes in CD34(+) cells that are poised for later expression, and we identify complex relationships between specific microRNAs and coding genes regulated by the heptad. Taken together, these data reveal the power of integrating multifactor sequencing of chromatin immunoprecipitates with coding and noncoding gene expression to identify regulatory circuits controlling cell identity.