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1.
Cell ; 182(4): 992-1008.e21, 2020 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-32710817

RESUMEN

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.


Asunto(s)
Elementos Transponibles de ADN/genética , Análisis de la Célula Individual/métodos , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Corteza Cerebral/metabolismo , Inmunoprecipitación de Cromatina , Expresión Génica , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Ratones , Unión Proteica , Análisis de Secuencia de ARN , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/genética
2.
Nature ; 564(7735): 219-224, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30518857

RESUMEN

Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here we present 'CellTagging', a combinatorial cell-indexing methodology that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a 'dead-end' state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming.


Asunto(s)
Linaje de la Célula , Rastreo Celular/métodos , Reprogramación Celular , Células Clonales/citología , Análisis de la Célula Individual/métodos , Animales , Linaje de la Célula/efectos de los fármacos , Separación Celular , Reprogramación Celular/efectos de los fármacos , Células Clonales/efectos de los fármacos , Endodermo/citología , Endodermo/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HEK293 , Humanos , Metiltransferasas/metabolismo , Ratones , Células Madre/citología , Células Madre/efectos de los fármacos , Factores de Tiempo
3.
J Cell Mol Med ; 19(1): 103-12, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25352422

RESUMEN

Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell-derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non-coding RNA, microRNA-24 (miR-24), as a pro-survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR-24 via viral transduction or chemical transfection, where the observed protective role of miR-24 in cardiomyocytes might have partially resulted from its effect on non-cardiomyocyte cells. To elucidate the cardiomyocyte-specific effects of miR-24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR-24 expression is driven by the cardiac-specific Myh6 promoter. The Myh6-miR-24 transgenic mice did not exhibit apparent difference from their wild-type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post-MI compared to their wild-type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non-genetic approach, raising the possibility that non-genetic approaches of introducing miR-24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte-specific role of miR-24 in the adult heart, and may provide insight to strategy design when considering miRNA-based therapies for cardiovascular diseases.


Asunto(s)
Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Biomarcadores/metabolismo , Supervivencia Celular , Pruebas de Función Cardíaca , Proteínas de la Membrana/metabolismo , Ratones Transgénicos , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Cadenas Pesadas de Miosina/metabolismo , Especificidad de Órganos , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal
4.
Genome Biol ; 20(1): 90, 2019 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-31072405

RESUMEN

High-throughput single-cell assays increasingly require special consideration in experimental design, sample multiplexing, batch effect removal, and data interpretation. Here, we describe a lentiviral barcode-based multiplexing approach, CellTag Indexing, which uses predefined genetic barcodes that are heritable, enabling cell populations to be tagged, pooled, and tracked over time in the same experimental replicate. We demonstrate the utility of CellTag Indexing by sequencing transcriptomes using a variety of cell types, including long-term tracking of cell engraftment and differentiation in vivo. Together, this presents CellTag Indexing as a broadly applicable genetic multiplexing tool that is complementary with existing single-cell technologies.


Asunto(s)
Rastreo Celular/métodos , Genómica/métodos , Análisis de la Célula Individual , Lentivirus , Transcriptoma
5.
Curr Opin Genet Dev ; 46: 50-57, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28667865

RESUMEN

Cellular reprogramming can be achieved by ectopically expressing transcription factors that directly convert one differentiated cell type into another, bypassing embryonic states. A number of different cell types have been generated by such 'direct lineage reprogramming' methods, but their practical utility has been limited because, in most protocols, the resulting populations are often partially differentiated or incompletely specified. Here, we review mechanisms of lineage reprogramming by pioneer transcription factors, a unique class of transcriptional regulators that has the capacity to engage with silent chromatin to activate target gene regulatory networks. We assess the possible barriers to successful reprogramming in the context of higher-order chromatin landscape, considering how the mechanistic relationship between nuclear organization and cell identity will be crucial to unlocking the full potential of cell fate engineering.


Asunto(s)
Diferenciación Celular/genética , Ingeniería Celular , Cromatina/genética , Epigénesis Genética , Animales , Linaje de la Célula/genética , Reprogramación Celular/genética , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Factores de Transcripción/genética
6.
Curr Gene Ther ; 13(2): 133-8, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23320478

RESUMEN

Cardiac diseases are the major causes of morbidity and mortality in the world. Cardiomyocyte death is a common consequence of many types of heart diseases and is usually irreversible. Scar tissues formed by cardiac fibroblasts serve compensatory roles for the injured heart but eventually weaken cardiac function and result in life-threatening heart failures. Unfortunately, adult human hearts have limited regenerative capacities. In the past decades, many interventional approaches have been taken in an attempt to restore functional cardiomyocytes in an injured heart. Promising advances have been made in directly reprogramming mouse fibroblasts into cardiomyocyte-like cells both in vitro and in vivo. Recently, several different methods have been reported, including the use of transcription factors and microRNAs. In addition, two in vivo studies showed heart function improvements with delivery of reprogramming factors in mouse infarcted hearts. Although many of these studies are at early preliminary stages, the plausibility of applying cardiac reprogramming on patients for regenerative purposes is exciting, and may lead to numerous novel research directions in the field. This review will discuss the history, recent advances and challenges of cellular reprogramming, specifically in the field of cardiac regeneration.


Asunto(s)
Reprogramación Celular/genética , Cardiopatías/terapia , Miocardio/patología , Miocitos Cardíacos/trasplante , Animales , Diferenciación Celular , Tratamiento Basado en Trasplante de Células y Tejidos , Cardiopatías/patología , Humanos , Ratones , Medicina Regenerativa , Trasplante de Células Madre
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