RESUMO
Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.
RESUMO
The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.
Assuntos
Neoplasias Encefálicas/genética , Mapeamento Cromossômico/métodos , Glioblastoma/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Cromatina/genética , Cromatina/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Genoma Humano , Glioblastoma/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Células Jurkat , Desequilíbrio de Ligação , Camundongos , Camundongos Nus , Elongação da Transcrição GenéticaRESUMO
Most studies of responses to transcriptional stimuli measure changes in cellular mRNA concentrations. By sequencing nascent RNA instead, it is possible to detect changes in transcription in minutes rather than hours and thereby distinguish primary from secondary responses to regulatory signals. Here, we describe the use of PRO-seq to characterize the immediate transcriptional response in human cells to celastrol, a compound derived from traditional Chinese medicine that has potent anti-inflammatory, tumor-inhibitory, and obesity-controlling effects. Celastrol is known to elicit a cellular stress response resembling the response to heat shock, but the transcriptional basis of this response remains unclear. Our analysis of PRO-seq data for K562 cells reveals dramatic transcriptional effects soon after celastrol treatment at a broad collection of both coding and noncoding transcription units. This transcriptional response occurred in two major waves, one within 10 min, and a second 40-60 min after treatment. Transcriptional activity was generally repressed by celastrol, but one distinct group of genes, enriched for roles in the heat shock response, displayed strong activation. Using a regression approach, we identified key transcription factors that appear to drive these transcriptional responses, including members of the E2F and RFX families. We also found sequence-based evidence that particular transcription factors drive the activation of enhancers. We observed increased polymerase pausing at both genes and enhancers, suggesting that pause release may be widely inhibited during the celastrol response. Our study demonstrates that a careful analysis of PRO-seq time-course data can disentangle key aspects of a complex transcriptional response, and it provides new insights into the activity of a powerful pharmacological agent.