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Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells [ATAC-seq]

ABSTRACT: When human cord blood derived CD34+ cells are induced to differentiate in vitro, they undergo rapid and dynamic morphological and molecular transformation that are critical for the fate commitment. Using ATAC-seq and single-cell RNA sequencing we detected two phases of this process. We show that a rapid and widespread chromatin opening - that makes most of the gene promoters in the genome accessible - precedes the high and unrestrained transcription of a large variety of genes. This phase results in a quasi-random gene expression profile unique to each cell. During the second phase, slow chromatin closing precedes the downregulation of transcription and the emergence of coherent expression profiles. Transcription factors may play a role in the maintenance of the transcription activity of their target genes rather than in their initial activation. These observations are consistent with a model based on the spontaneous probabilistic organization of the cellular process of fate commitment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156733 | GEO | 2020/08/25

REPOSITORIES: GEO

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Project description:When human cord blood derived CD34+ cells are induced to differentiate, they undergo rapid and dynamic morphological and molecular transformations that are critical for fate commitment. In particular, the cells passe through a transitory phase known as “multilineage primed” state. These cells are characterized by a mixed gene expression profile, different in each cell, with the co-expression of many genes characteristic for concurrent cell lineages. The aim of our study is to understand the mechanisms of the establishment and the exit from this transitory state. We investigated this issue using single-cell RNA sequencing and ATAC-seq. Two phases were detected. The first phase is a rapid and global chromatin decompaction that makes most of the gene promoters in the genome accessible for transcription. It results 24h later in enhanced and pervasive transcription of the genome leading to the concomitant increase in the cell-to-cell variability of transcriptional profiles. The second phase is the exit from the multilineage-primed phase marked by a slow chromatin closure and a subsequent overall downregulation of gene transcription. This process is selective and results in the emergence of coherent expression profiles corresponding to distinct cell subpopulations. The typical time scale of these events spans 48 to 72 hours. These observations suggest that the non-specificity of genome decompaction is the condition for the generation of a highly variable multilineage expression profile. The non-specific phase is followed by specific regulatory actions that stabilize and maintain the activity of key genes, while the rest of the genome becomes repressed again by the chromatin re-compaction. Thus, the initiation of differentiation is reminiscent of a trial-and-error process that associates the spontaneous generation of gene expression diversity to subsequent regulatory actions that maintain the activity of some genes while the rest of the genome sinks back to the repressive closed chromatin state.

Project description:ABSTRACT Rationale Premature senescence is conducive to aging and cardiovascular diseases. Nrf2 transcription factor, the master orchestrator of adoptive response to cellular stress, has been implicated in regulation of premature senescence in fibroblasts, neural and mesenchymal stem cells by transactivation of antioxidant gene expression. However, as we show here, human primary endothelial cells (ECs) devoid of Nrf2 and murine Nrf2 transcriptional knockout (tKO) aortas are senescent but do not encounter oxidative stress and damage, what contradicts this mechanism. Moreover, a molecular switch between normal, senescent and apoptotic fate remains unknown. Objective To elucidate the mechanism of Nrf2-related premature senescence of vascular system, to understand why Nrf2 deregulation does not cause oxidative stress exclusionary in ECs and to indicate a molecular switch determining ECs fate. Methods and Results Herein we evidence that ECs deficient in Nrf2 protein, or with limited Nrf2 activity in shear stress conditions, exhibit excessive S-nitrosylation of proteins. It is also a characteristic of Nrf2 tKO murine aortas, as determined by biotin switch assay in situ. Mass spectrometry analysis reveals that NOX4 is S-nitrosylated exclusively in ECs devoid of Nrf2. A functional role of S-nitrosylation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage. As a result Nrf2-deficient ECs preserve oxidative balance but are redirected to premature senescence. The same phenotype is seen in Nrf2 tKO aortas. These effects are mediated by Keap1, a direct binding partner of Nrf2 and repressor of its transcriptional activity, remaining in cytoplasm unrestrained by Nrf2. S-nitrosylation, followed by senescence, can also be triggered in smooth muscle cells (SMCs) by EC-derived paracrine induction of iNOS. Conclusions Collectively, Keap1-dependent S-nitrosylation of NOX4 hampers oxidative detriment in ECs with disturbed Nrf2 signaling and may provide defence in the adjacent aortic cells. Overabundance of unrestrained Keap1 in the cytoplasm determines fate of ECs.

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Global genome decompaction leads to stochastic activation of gene expression as a first step toward fate commitment in human hematopoietic cells [ATAC-seq]

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