RESUMEN
Joint profiling of transcriptome and chromatin accessibility within single cells allows for the deconstruction of the complex relationship between transcriptional states and upstream regulatory programs determining different cell fates. Here, we developed an automated method with high sensitivity, assay for single-cell transcriptome and accessibility regions (ASTAR-seq), for simultaneous measurement of whole-cell transcriptome and chromatin accessibility within the same single cell. To show the utility of ASTAR-seq, we profiled 384 mESCs under naive and primed pluripotent states as well as a two-cell like state, 424 human cells of various lineage origins (BJ, K562, JK1, and Jurkat), and 480 primary cord blood cells undergoing erythroblast differentiation. With the joint profiles, we configured the transcriptional and chromatin accessibility landscapes of discrete cell states, uncovered linked sets of cis-regulatory elements and target genes unique to each state, and constructed interactome and transcription factor (TF)-centered upstream regulatory networks for various cell states.
Asunto(s)
Cromatina/metabolismo , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Células Madre Embrionarias , Epigénesis Genética , Eritroblastos/citología , Eritroblastos/metabolismo , Humanos , Ratones , Elementos Reguladores de la Transcripción , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Incomplete knowledge of the mechanisms at work continues to hamper efforts to maximize reprogramming efficiency. Here, we present a systematic genome-wide RNAi screen to determine the global regulators during the early stages of human reprogramming. Our screen identifies functional repressors and effectors that act to impede or promote the reprogramming process. Repressors and effectors form close interacting networks in pathways, including RNA processing, G protein signaling, protein ubiquitination, and chromatin modification. Combinatorial knockdown of five repressors (SMAD3, ZMYM2, SFRS11, SAE1, and ESET) synergistically resulted in â¼85% TRA-1-60-positive cells. Removal of the novel splicing factor SFRS11 during reprogramming is accompanied by rapid acquisition of pluripotency-specific spliced forms. Mechanistically, SFRS11 regulates exon skipping and mutually exclusive splicing of transcripts in genes involved in cell differentiation, mRNA splicing, and chromatin modification. Our study provides insights into the reprogramming process, which comprises comprehensive and multi-layered transcriptional, splicing, and epigenetic machineries.