RESUMEN
Transcriptional regulation controls cellular functions through interactions between transcription factors (TFs) and their chromosomal targets. However, understanding the fate conversion potential of multiple TFs in an inducible manner remains limited. Here, we introduce iTF-seq as a method for identifying individual TFs that can alter cell fate toward specific lineages at a single-cell level. iTF-seq enables time course monitoring of transcriptome changes, and with biotinylated individual TFs, it provides a multi-omics approach to understanding the mechanisms behind TF-mediated cell fate changes. Our iTF-seq study in mouse embryonic stem cells identified multiple TFs that trigger rapid transcriptome changes indicative of differentiation within a day of induction. Moreover, cells expressing these potent TFs often show a slower cell cycle and increased cell death. Further analysis using bioChIP-seq revealed that GCM1 and OTX2 act as pioneer factors and activators by increasing gene accessibility and activating the expression of lineage specification genes during cell fate conversion. iTF-seq has utility in both mapping cell fate conversion and understanding cell fate conversion mechanisms.
Asunto(s)
Diferenciación Celular , Factores de Transcripción , Animales , Ratones , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Diferenciación Celular/genética , Análisis de la Célula Individual/métodos , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , Linaje de la Célula/genética , Transcriptoma , Análisis de Secuencia de ARN/métodos , RNA-Seq/métodos , Perfilación de la Expresión Génica/métodos , ARN Citoplasmático Pequeño/genética , ARN Citoplasmático Pequeño/metabolismo , Multiómica , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Translation regulation is critical for early mammalian embryonic development1. However, previous studies had been restricted to bulk measurements2, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.