RESUMO
Genome-wide association studies have identified multiple novel genomic loci associated with vascular diseases. Many of these loci are common non-coding variants that affect the expression of disease-relevant genes within coronary vascular cells. To identify such genes on a genome-wide level, we performed deep transcriptomic analysis of genotyped primary human coronary artery smooth muscle cells (HCASMCs) and coronary endothelial cells (HCAECs) from the same subjects, including splicing Quantitative Trait Loci (sQTL), allele-specific expression (ASE), and colocalization analyses. We identified sQTLs for TARS2, YAP1, CFDP1, and STAT6 in HCASMCs and HCAECs, and 233 ASE genes, a subset of which are also GTEx eGenes in arterial tissues. Colocalization of GWAS association signals for coronary artery disease (CAD), migraine, stroke and abdominal aortic aneurysm with GTEx eGenes in aorta, coronary artery and tibial artery discovered novel candidate risk genes for these diseases. At the CAD and stroke locus tagged by rs2107595 we demonstrate colocalization with expression of the proximal gene TWIST1. We show that disrupting the rs2107595 locus alters TWIST1 expression and that the risk allele has increased binding of the NOTCH signaling protein RBPJ. Finally, we provide data that TWIST1 expression influences vascular SMC phenotypes, including proliferation and calcification, as a potential mechanism supporting a role for TWIST1 in CAD.
Assuntos
Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genética , Doenças Vasculares/genética , Células Cultivadas , Vasos Coronários/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Transcriptoma , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Coronary artery disease (CAD) is the leading cause of death globally. Genome-wide association studies (GWASs) have identified more than 95 independent loci that influence CAD risk, most of which reside in non-coding regions of the genome. To interpret these loci, we generated transcriptome and whole-genome datasets using human coronary artery smooth muscle cells (HCASMCs) from 52 unrelated donors, as well as epigenomic datasets using ATAC-seq on a subset of 8 donors. Through systematic comparison with publicly available datasets from GTEx and ENCODE projects, we identified transcriptomic, epigenetic, and genetic regulatory mechanisms specific to HCASMCs. We assessed the relevance of HCASMCs to CAD risk using transcriptomic and epigenomic level analyses. By jointly modeling eQTL and GWAS datasets, we identified five genes (SIPA1, TCF21, SMAD3, FES, and PDGFRA) that may modulate CAD risk through HCASMCs, all of which have relevant functional roles in vascular remodeling. Comparison with GTEx data suggests that SIPA1 and PDGFRA influence CAD risk predominantly through HCASMCs, while other annotated genes may have multiple cell and tissue targets. Together, these results provide tissue-specific and mechanistic insights into the regulation of a critical vascular cell type associated with CAD in human populations.
Assuntos
Doença da Artéria Coronariana/genética , Vasos Coronários/fisiologia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Miócitos de Músculo Liso/fisiologia , Locos de Características Quantitativas/genética , Linhagem Celular , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Polimorfismo de Nucleotídeo Único/genética , RiscoRESUMO
Recent advances in sequencing and biotechnological methodologies have led to the generation of large volumes of molecular data of different omics layers, such as genomics, transcriptomics, proteomics and metabolomics. Integration of these data with clinical information provides new opportunities to discover how perturbations in biological processes lead to disease. Using data-driven approaches for the integration and interpretation of multi-omics data could stably identify links between structural and functional information and propose causal molecular networks with potential impact on cancer pathophysiology. This knowledge can then be used to improve disease diagnosis, prognosis, prevention, and therapy. This review will summarize and categorize the most current computational methodologies and tools for integration of distinct molecular layers in the context of translational cancer research and personalized therapy. Additionally, the bioinformatics tools Multi-Omics Factor Analysis (MOFA) and netDX will be tested using omics data from public cancer resources, to assess their overall robustness, provide reproducible workflows for gaining biological knowledge from multi-omics data, and to comprehensively understand the significantly perturbed biological entities in distinct cancer types. We show that the performed supervised and unsupervised analyses result in meaningful and novel findings.
Assuntos
Biomarcadores Tumorais , Biologia Computacional , Genômica , Metabolômica , Neoplasias , Proteômica , Pesquisa Translacional Biomédica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapiaRESUMO
Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase ε or δ genes (POLE and POLD1, respectively), and an ultrahigh tumor mutational burden (TMB). These tumors may be associated with enhanced antitumor immunity and preferentially afflict younger patients, but this notion awaits validation by accrual of further cases for detailed correlative phenotypic and molecular study. We performed POLE and POLD1 exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somatic POLE p.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. A POLE p.V411L was found in another two microsatellite-stable tumors with ultrahigh TMBs. Two of these four tumors were from young patients (<50 years old, nonsyndromic), and there was seen a prominent T-cell infiltration in three of them. Furthermore, a somatic POLE p.A465T was found in a Lynch-associated tumor, which, hypothetically, might have enhanced TMB (which was the highest of all). In two tumors, a somatic POLE p.V411L and a POLD1 p.E279K, respectively, were found only focally, and TMBs were low. It is commonly assumed that compromise of one allele is sufficient, but this has not been specifically addressed. Therefore, resequencing of the POLE or POLD1 mutations was done with DNA from tumor cells isolated by laser-capture microdissection. This demonstrated that the mutations were monoallelic, and there was no evidence of a "second hit", neither by allelic loss (allelotyping with polymorphic microsatellite markers), nor by promoter methylation (Pyromark CpG assays). Taken together, including at least the more common DNA polymerase mutations in NGS panels allows for straightforward identification of hypermutator-type colorectal carcinomas which often may be "immunoreactive". This is important at least in young patients or when a metastasizing stage of disease has been reached and immune-checkpoint therapy enters deliberation.
Assuntos
Neoplasias Colorretais/genética , DNA Polimerase III/genética , DNA Polimerase II/genética , Perda de Heterozigosidade , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Neoplasias Colorretais/patologia , Metilação de DNA , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk. METHODS: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0×10-3 (Bonferroni correction for 50 tests). RESULTS: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers (P=1.3×10-16) in comparison with 5% in ever-smokers (P=2.5×10-4), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value=8.7×10-5). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. CONCLUSIONS: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers.
Assuntos
Doença das Coronárias/genética , Doença das Coronárias/prevenção & controle , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Fumar/genética , Proteína ADAMTS7/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Doença das Coronárias/epidemiologia , Vasos Coronários/patologia , Vasos Coronários/fisiologia , Feminino , Interação Gene-Ambiente , Predisposição Genética para Doença/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Fumar/efeitos adversos , Fumar/epidemiologiaRESUMO
Genome-wide association studies have provided a rich collection of ≈ 58 coronary artery disease (CAD) loci that suggest the existence of previously unsuspected new biology relevant to atherosclerosis. However, these studies only identify genomic loci associated with CAD, and many questions remain even after a genomic locus is definitively implicated, including the nature of the causal variant(s) and the causal gene(s), as well as the directionality of effect. There are several tools that can be used for investigation of the functional genomics of these loci, and progress has been made on a limited number of novel CAD loci. New biology regarding atherosclerosis and CAD will be learned through the functional genomics of these loci, and the hope is that at least some of these new pathways relevant to CAD pathogenesis will yield new therapeutic targets for the prevention and treatment of CAD.
Assuntos
Doença da Artéria Coronariana/genética , Loci Gênicos , Animais , Doença da Artéria Coronariana/diagnóstico , Doença da Artéria Coronariana/epidemiologia , Doença da Artéria Coronariana/terapia , Marcadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Fenótipo , Prognóstico , Medição de Risco , Fatores de RiscoRESUMO
To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including "growth factor binding," "matrix interaction," and "smooth muscle contraction." We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.
Assuntos
Aterosclerose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doença da Artéria Coronariana/genética , Proteínas de Ligação a DNA/genética , Redes Reguladoras de Genes , Aterosclerose/patologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Sítios de Ligação , Doença da Artéria Coronariana/patologia , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including "vascular disease," "disorder of artery," and "occlusion of artery," as well as disease-related cellular functions including "cellular movement" and "cellular growth and proliferation." In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Doença da Artéria Coronariana/genética , Miócitos de Músculo Liso/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Linhagem da Célula/genética , Doença da Artéria Coronariana/patologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Mioblastos/metabolismo , Mioblastos/patologia , Miócitos de Músculo Liso/metabolismo , Células-TroncoRESUMO
Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor TCF21 gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a cis-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered TCF21 expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-ß) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at TCF21.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Doença das Coronárias/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Fator de Transcrição AP-1/genética , Alelos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Doença das Coronárias/patologia , Vasos Coronários/citologia , Vasos Coronários/crescimento & desenvolvimento , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Miócitos de Músculo Liso/citologia , Polimorfismo de Nucleotídeo Único , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Tumor de Wilms/genética , Tumor de Wilms/metabolismoRESUMO
We recently identified 68 genomic loci where common sequence variants are associated with platelet count and volume. Platelets are formed in the bone marrow by megakaryocytes, which are derived from hematopoietic stem cells by a process mainly controlled by transcription factors. The homeobox transcription factor MEIS1 is uniquely transcribed in megakaryocytes and not in the other lineage-committed blood cells. By ChIP-seq, we show that 5 of the 68 loci pinpoint a MEIS1 binding event within a group of 252 MK-overexpressed genes. In one such locus in DNM3, regulating platelet volume, the MEIS1 binding site falls within a region acting as an alternative promoter that is solely used in megakaryocytes, where allelic variation dictates different levels of a shorter transcript. The importance of dynamin activity to the latter stages of thrombopoiesis was confirmed by the observation that the inhibitor Dynasore reduced murine proplatelet for-mation in vitro.
Assuntos
Plaquetas/metabolismo , Dinamina III/genética , Genoma Humano/genética , Proteínas de Homeodomínio/genética , Megacariócitos/metabolismo , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação/genética , Plaquetas/efeitos dos fármacos , Linhagem Celular Tumoral , Linhagem da Célula/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Expressão Gênica , Variação Genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hidrazonas/farmacologia , Camundongos , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Contagem de Plaquetas , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
3D printing offers the possibility to prepare personalized tablets on demand, making it an intriguing technology for hospital pharmacies. For the implementation of 3D-printed tablets into the digital Closed Loop Medication Management system, the required tablet formulation and development of the manufacturing process as well as the pharmaceutical validation were conducted. The goal of the formulation development was to enable an optimal printing process and rapid dissolution of the printed tablets for the selected model drugs Levodopa/Carbidopa. The 3D printed tablets were prepared by direct powder extrusion. Printability, thermal properties, disintegration, dissolution, physical properties and storage stability were investigated by employing analytical methods such as HPLC-UV, DSC and TGA. The developed formulation shows a high dose accuracy and an immediate drug release for Levodopa. In addition, the tablets exhibit high crushing strength and very low friability. Unfortunately, Carbidopa did not tolerate the printing process. This is the first study to develop an immediate release excipient composition via direct powder extrusion in a hospital pharmacy setting. The developed process is suitable for the implementation in Closed-Loop Medication Management systems in hospital pharmacies and could therefore contribute to medication safety.
Assuntos
Excipientes , Tecnologia Farmacêutica , Pós , Tecnologia Farmacêutica/métodos , Carbidopa , Levodopa , Liberação Controlada de Fármacos , Comprimidos , Impressão Tridimensional , HospitaisRESUMO
INTRODUCTION: The diagnosis and treatment of Parkinson's disease depend on the assessment of motor symptoms. Wearables and machine learning algorithms have emerged to collect large amounts of data and potentially support clinicians in clinical and ambulant settings. STATE OF THE ART: However, a systematical and reusable data architecture for storage, processing, and analysis of inertial sensor data is not available. Consequently, datasets vary significantly between studies and prevent comparability. CONCEPT: To simplify research on the neurodegenerative disorder, we propose an efficient and real-time-optimized architecture compatible with HL7 FHIR backed by a relational database schema. LESSONS LEARNED: We can verify the adequate performance of the system on an experimental benchmark and in a clinical experiment. However, existing standards need to be further optimized to be fully sufficient for data with high temporal resolution.
Assuntos
Doença de Parkinson , Humanos , Doença de Parkinson/diagnóstico , Algoritmos , Benchmarking , Bases de Dados Factuais , Aprendizado de MáquinaRESUMO
In their joint effort against cancer, all involved parties within the German healthcare system are obligated to report diagnostics, treatments, progression, and follow-up information for tumor patients to the respective cancer registries. Given the federal structure of Germany, the oncological basis dataset (oBDS) operates as the legally required national standard for oncological reporting. Unfortunately, the usage of various documentation software solutions leads to semantic and technical heterogeneity of the data, complicating the establishment of research networks and collective data analysis. Within this feasibility study, we evaluated the transferability of all oBDS characteristics to the standardized vocabularies, a metadata repository of the observational medical outcomes partnership (OMOP) common data model (CDM). A total of 17,844 oBDS expressions were mapped automatically or manually to standardized concepts of the OMOP CDM. In a second step, we converted real patient data retrieved from the Hamburg Cancer Registry to the new terminologies. Given our pipeline, we transformed 1773.373 cancer-related data elements to the OMOP CDM. The mapping of the oBDS to the standardized vocabularies of the OMOP CDM promotes the semantic interoperability of oncological data in Germany. Moreover, it allows the participation in network studies of the observational health data sciences and informatics under the usage of federated analysis beyond the level of individual countries.
RESUMO
AIMS: We aim to develop a pragmatic screening tool for heart failure at the general population level. METHODS AND RESULTS: This study was conducted within the Hamburg-City-Health-Study, an ongoing, prospective, observational study enrolling randomly selected inhabitants of the city of Hamburg aged 45-75 years. Heart failure was diagnosed per current guidelines. Using only digital electrocardiograms (ECGs), a convolutional neural network (CNN) was built to discriminate participants with and without heart failure. As comparisons, known risk variables for heart failure were fitted into a logistic regression model and a random forest classifier. Of the 5299 individuals included into this study, 318 individuals (6.0%) had heart failure. Using only the digital ECGs instead of several risk variables as an input, the CNN provided a comparable predictive accuracy for heart failure versus the logistic regression model and the random forest classifier [area under the curve (AUC) of 0.75, a sensitivity of 0.67 and a specificity of 0.69 for the CNN; AUC 0.77, a sensitivity of 0.63 and a specificity of 0.76 for the logistic regression; AUC 0.79, a sensitivity of 0.67 and a specificity of 0.72 for the random forest classifier]. CONCLUSIONS: Using a CNN build on digital ECGs only and requiring no additional input, we derived a screening tool for heart failure in the general population. This could be perfectly embedded into clinical routine of general practitioners, as it builds on an already established diagnostic tool and does not require additional, time-consuming input. This could help to alleviate the underdiagnosis of heart failure.
Assuntos
Insuficiência Cardíaca , Redes Neurais de Computação , Humanos , Estudos Prospectivos , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/epidemiologia , Algoritmo Florestas Aleatórias , EletrocardiografiaRESUMO
Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.
Assuntos
Redes Reguladoras de Genes , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Simulação por Computador , Perfilação da Expressão Gênica , Camundongos , Modelos Teóricos , Análise de Sequência de DNARESUMO
Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.