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1.
Cell ; 185(7): 1157-1171.e22, 2022 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-35259335

RESUMO

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.


Assuntos
Toxinas Bacterianas/metabolismo , Enterococcus , Leucócitos Mononucleares , Fatores de Virulência/metabolismo , Animais , Bovinos , Enterococcus/metabolismo , Enterococcus/patogenicidade , Cavalos , Camundongos , Testes de Sensibilidade Microbiana , Suínos
2.
Nat Methods ; 19(11): 1393-1402, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36216958

RESUMO

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.


Assuntos
DNA , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Complementar , DNA/genética
3.
Nature ; 544(7649): 250-254, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28371798

RESUMO

Blockade of angiogenesis can retard tumour growth, but may also paradoxically increase metastasis. This paradox may be resolved by vessel normalization, which involves increased pericyte coverage, improved tumour vessel perfusion, reduced vascular permeability, and consequently mitigated hypoxia. Although these processes alter tumour progression, their regulation is poorly understood. Here we show that type 1 T helper (TH1) cells play a crucial role in vessel normalization. Bioinformatic analyses revealed that gene expression features related to vessel normalization correlate with immunostimulatory pathways, especially T lymphocyte infiltration or activity. To delineate the causal relationship, we used various mouse models with vessel normalization or T lymphocyte deficiencies. Although disruption of vessel normalization reduced T lymphocyte infiltration as expected, reciprocal depletion or inactivation of CD4+ T lymphocytes decreased vessel normalization, indicating a mutually regulatory loop. In addition, activation of CD4+ T lymphocytes by immune checkpoint blockade increased vessel normalization. TH1 cells that secrete interferon-γ are a major population of cells associated with vessel normalization. Patient-derived xenograft tumours growing in immunodeficient mice exhibited enhanced hypoxia compared to the original tumours in immunocompetent humans, and hypoxia was reduced by adoptive TH1 transfer. Our findings elucidate an unexpected role of TH1 cells in vasculature and immune reprogramming. TH1 cells may be a marker and a determinant of both immune checkpoint blockade and anti-angiogenesis efficacy.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Neovascularização Fisiológica/imunologia , Neovascularização Fisiológica/fisiologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/transplante , Permeabilidade Capilar , Hipóxia Celular/fisiologia , Células Endoteliais/imunologia , Células Endoteliais/fisiologia , Feminino , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Neovascularização Patológica/patologia , Pericitos/citologia , Pericitos/fisiologia , Prognóstico , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Methods ; 14(3): 267-270, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28092691

RESUMO

The quantification of transcriptional variation in single cells, particularly within the same cell population, is currently limited by the low sensitivity and high technical noise of single-cell RNA-seq assays. We report multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq), a highly sensitive and quantitative method for single-cell sequencing of total RNA. By systematically determining technical noise, we show that MATQ-seq captures genuine biological variation between whole transcriptomes of single cells.


Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Linhagem Celular , Células HEK293 , Humanos , Análise de Célula Única/métodos , Transcriptoma/genética
5.
Nat Comput Sci ; 4(6): 423-428, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38849559

RESUMO

Orthogonal DNA barcode library design is an essential task in bioengineering. Here we present seqwalk, an efficient method for designing barcode libraries that satisfy a sequence symmetry minimization (SSM) heuristic for orthogonality, with theoretical guarantees of maximal or near-maximal library size under certain design constraints. Seqwalk encodes SSM constraints in a de Bruijn graph representation of sequence space, enabling the application of recent advances in discrete mathematics1 to the problem of orthogonal sequence design. We demonstrate the scalability of seqwalk by designing a library of >106 SSM-satisfying barcode sequences in less than 20 s on a standard laptop.


Assuntos
Código de Barras de DNA Taxonômico , Biblioteca Gênica , Código de Barras de DNA Taxonômico/métodos , Algoritmos , DNA/genética , DNA/química
6.
Nat Biotechnol ; 2024 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-39075149

RESUMO

Mass cytometry uses metal-isotope-tagged antibodies to label targets of interest, which enables simultaneous measurements of ~50 proteins or protein modifications in millions of single cells, but its sensitivity is limited. Here, we present a signal amplification technology, termed Amplification by Cyclic Extension (ACE), implementing thermal-cycling-based DNA in situ concatenation in combination with 3-cyanovinylcarbazole phosphoramidite-based DNA crosslinking to enable signal amplification simultaneously on >30 protein epitopes. We demonstrate the utility of ACE in low-abundance protein quantification with suspension mass cytometry to characterize molecular reprogramming during the epithelial-to-mesenchymal transition as well as the mesenchymal-to-epithelial transition. We show the capability of ACE to quantify the dynamics of signaling network responses in human T lymphocytes. We further present the application of ACE in imaging mass cytometry-based multiparametric tissue imaging to identify tissue compartments and profile spatial aspects related to pathological states in polycystic kidney tissues.

7.
Nat Commun ; 14(1): 5130, 2023 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-37612289

RESUMO

Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.


Assuntos
Escherichia coli , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Escherichia coli/genética , RNA-Seq , Antibacterianos/farmacologia , Primers do DNA , RNA Ribossômico/genética
8.
Nat Commun ; 13(1): 6786, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36351897

RESUMO

Toxin B (TcdB) is a major exotoxin responsible for diseases associated with Clostridioides difficile infection. Its sequence variations among clinical isolates may contribute to the difficulty in developing effective therapeutics. Here, we investigate receptor-binding specificity of major TcdB subtypes (TcdB1 to TcdB12). We find that representative members of subtypes 2, 4, 7, 10, 11, and 12 do not recognize the established host receptor, frizzled proteins (FZDs). Using a genome-wide CRISPR-Cas9-mediated screen, we identify tissue factor pathway inhibitor (TFPI) as a host receptor for TcdB4. TFPI is recognized by a region in TcdB4 that is homologous to the FZD-binding site in TcdB1. Analysis of 206 TcdB variant sequences reveals a set of six residues within this receptor-binding site that defines a TFPI binding-associated haplotype (designated B4/B7) that is present in all TcdB4 members, a subset of TcdB7, and one member of TcdB2. Intragenic micro-recombination (IR) events have occurred around this receptor-binding region in TcdB7 and TcdB2 members, resulting in either TFPI- or FZD-binding capabilities. Introduction of B4/B7-haplotype residues into TcdB1 enables dual recognition of TFPI and FZDs. Finally, TcdB10 also recognizes TFPI, although it does not belong to the B4/B7 haplotype, and shows species selectivity: it recognizes TFPI of chicken and to a lesser degree mouse, but not human, dog, or cattle versions. These findings identify TFPI as a TcdB receptor and reveal IR-driven changes on receptor-specificity among TcdB variants.


Assuntos
Toxinas Bacterianas , Clostridioides difficile , Animais , Bovinos , Cães , Camundongos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Clostridioides difficile/genética , Recombinação Genética , Humanos
9.
ACS Nano ; 15(3): 5631-5638, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33687214

RESUMO

Circulating extracellular vesicles (EVs)-biological nanomaterials shed from most mammalian cells-have emerged as promising biomarkers, drug delivery vesicles, and treatment modulators. While different types of vesicles are being explored for these applications, it is becoming clear that human EVs are quite heterogeneous even in homogeneous or monoclonal cell populations. Since it is the surface EV protein composition that will largely dictate their biological behavior, high-throughput single EV profiling methods are needed to better define EV subpopulations. Here, we present an antibody-based immunosequencing method that allows multiplexed measurement of protein molecules from individual nanometer-sized EVs. We use droplet microfluidics to compartmentalize and barcode individual EVs. The barcodes/antibody-DNA are then sequenced to determine protein composition. Using this highly sensitive technology, we detected specific proteins at the single EV level. We expect that this technology can be further adapted for multiplexed protein analysis of any nanoparticle.


Assuntos
Vesículas Extracelulares , Nanoestruturas , Animais , Biomarcadores , Humanos
10.
Cell Rep ; 35(3): 109009, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33882319

RESUMO

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.


Assuntos
Adenocarcinoma de Pulmão/genética , Fibroblastos Associados a Câncer/metabolismo , Células Epiteliais/metabolismo , Neoplasias Renais/genética , Neoplasias Pulmonares/genética , Células-Tronco Mesenquimais/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/secundário , alfa-Globulinas/genética , alfa-Globulinas/metabolismo , Animais , Fibroblastos Associados a Câncer/patologia , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Receptor com Domínio Discoidina 2/genética , Receptor com Domínio Discoidina 2/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/secundário , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Microambiente Tumoral/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
11.
medRxiv ; 2020 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-32839783

RESUMO

We report the single-strand Recombinase Polymerase Amplification (ssRPA) method, which merges the fast, isothermal amplification of RPA with subsequent rapid conversion of the double-strand DNA amplicon to single strands, and hence enables facile hybridization-based, high-specificity readout. We demonstrate the utility of ssRPA for sensitive and rapid (4 copies per 50 µL reaction within 10 min, or 8 copies within 8 min) visual detection of SARS-CoV-2 RNA spiked samples, as well as clinical saliva and nasopharyngeal swabs in VTM or water, on lateral flow devices. The ssRPA method promises rapid, sensitive, and accessible RNA detection to facilitate mass testing in the COVID-19 pandemic.

12.
Adv Sci (Weinh) ; 7(8): 1903463, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32328429

RESUMO

Droplet-based single cell sequencing technologies, such as inDrop, Drop-seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.

13.
Methods Mol Biol ; 1979: 57-71, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028632

RESUMO

Single-cell technologies have emerged as advanced tools to study various biological processes that demand the single cell resolution. To detect subtle heterogeneity in the transcriptome, high accuracy and sensitivity are still desired for single-cell RNA-seq. We describe here multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq) with ~90% capture efficiency. In addition, MATQ-seq is a total RNA assay allowing for detection of nonpolyadenylated transcripts.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Animais , DNA Complementar/genética , Biblioteca Gênica , Humanos , Transcrição Reversa
14.
Elife ; 82019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31033441

RESUMO

The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Células Ciliadas Auditivas/metabolismo , Regeneração/fisiologia , Sáculo e Utrículo/metabolismo , Transcriptoma , Animais , Ciclo Celular , Morte Celular , Cóclea , Feminino , Masculino , Camundongos , Fatores de Transcrição , Transdução Genética
15.
Nat Cell Biol ; 21(9): 1113-1126, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31451770

RESUMO

Cancer-induced immune responses affect tumour progression and therapeutic response. In multiple murine models and clinical datasets, we identified large variations of neutrophils and macrophages that define 'immune subtypes' of triple-negative breast cancer (TNBC), including neutrophil-enriched (NES) and macrophage-enriched subtypes (MES). Different tumour-intrinsic pathways and mutual regulation between macrophages (or monocytes) and neutrophils contribute to the development of a dichotomous myeloid compartment. MES contains predominantly macrophages that are CCR2-dependent and exhibit variable responses to immune checkpoint blockade (ICB). NES exhibits systemic and local accumulation of immunosuppressive neutrophils (or granulocytic myeloid-derived suppressor cells), is resistant to ICB, and contains a minority of macrophages that seem to be unaffected by CCR2 knockout. A MES-to-NES conversion mediated acquired ICB resistance of initially sensitive MES models. Our results demonstrate diverse myeloid cell frequencies, functionality and potential roles in immunotherapies, and highlight the need to better understand the inter-patient heterogeneity of the myeloid compartment.


Assuntos
Imunoterapia , Células Mieloides/imunologia , Neoplasias de Mama Triplo Negativas/terapia , Microambiente Tumoral/imunologia , Animais , Modelos Animais de Doenças , Feminino , Granulócitos/imunologia , Imunoterapia/métodos , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Neoplasias de Mama Triplo Negativas/patologia
16.
Nat Commun ; 9(1): 3356, 2018 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30135482

RESUMO

Decoding the molecular composition of individual Ngn3 + endocrine progenitors (EPs) during pancreatic morphogenesis could provide insight into the mechanisms regulating hormonal cell fate. Here, we identify population markers and extensive cellular diversity including four EP subtypes reflecting EP maturation using high-resolution single-cell RNA-sequencing of the e14.5 and e16.5 mouse pancreas. While e14.5 and e16.5 EPs are constantly born and share select genes, these EPs are overall transcriptionally distinct concomitant with changes in the underlying epithelium. As a consequence, e16.5 EPs are not the same as e14.5 EPs: e16.5 EPs have a higher propensity to form beta cells. Analysis of e14.5 and e16.5 EP chromatin states reveals temporal shifts, with enrichment of beta cell motifs in accessible regions at later stages. Finally, we provide transcriptional maps outlining the route progenitors take as they make cell fate decisions, which can be applied to advance the in vitro generation of beta cells.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/citologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Masculino , Camundongos Endogâmicos ICR , Morfogênese/genética , Gravidez , Células-Tronco/citologia , Células-Tronco/metabolismo
17.
Nat Commun ; 8: 15045, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28429794

RESUMO

The majority of breast cancer models for drug discovery are based on orthotopic or subcutaneous tumours. Therapeutic responses of metastases, especially microscopic metastases, are likely to differ from these tumours due to distinct cancer-microenvironment crosstalk in distant organs. Here, to recapitulate such differences, we established an ex vivo bone metastasis model, termed bone-in-culture array or BICA, by fragmenting mouse bones preloaded with breast cancer cells via intra-iliac artery injection. Cancer cells in BICA maintain features of in vivo bone micrometastases regarding the microenvironmental niche, gene expression profile, metastatic growth kinetics and therapeutic responses. Through a proof-of-principle drug screening using BICA, we found that danusertib, an inhibitor of the Aurora kinase family, preferentially inhibits bone micrometastases. In contrast, certain histone methyltransferase inhibitors stimulate metastatic outgrowth of indolent cancer cells, specifically in the bone. Thus, BICA can be used to investigate mechanisms involved in bone colonization and to rapidly test drug efficacies on bone micrometastases.


Assuntos
Antineoplásicos/farmacologia , Aurora Quinases/antagonistas & inibidores , Benzamidas/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Osso e Ossos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Animais , Aurora Quinases/genética , Aurora Quinases/metabolismo , Benzamidas/efeitos adversos , Compostos de Bifenilo , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Osso e Ossos/enzimologia , Osso e Ossos/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Morfolinas , Piridonas/efeitos adversos , Técnicas de Cultura de Tecidos , Microambiente Tumoral
18.
Cell Stem Cell ; 16(4): 426-38, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25772072

RESUMO

Hematopoietic stem cells (HSCs) possess unique gene expression programs that enforce their identity and regulate lineage commitment. Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cell fate decisions, although their functions in HSCs are unclear. Here we profiled the transcriptome of purified HSCs by deep sequencing and identified 323 unannotated lncRNAs. Comparing their expression in differentiated lineages revealed 159 lncRNAs enriched in HSCs, some of which are likely HSC specific (LncHSCs). These lncRNA genes share epigenetic features with protein-coding genes, including regulated expression via DNA methylation, and knocking down two LncHSCs revealed distinct effects on HSC self-renewal and lineage commitment. We mapped the genomic binding sites of one of these candidates and found enrichment for key hematopoietic transcription factor binding sites, especially E2A. Together, these results demonstrate that lncRNAs play important roles in regulating HSCs, providing an additional layer to the genetic circuitry controlling HSC function.


Assuntos
Células da Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/fisiologia , RNA Longo não Codificante/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Autorrenovação Celular/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Metiltransferase 3A , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética
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