CRISPR-Cas9 Screens Reveal Genes Regulating a G0-like State in Human Neural Progenitors [scRNA-seq]
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ABSTRACT: The coordination of developmental potential and proliferation in stem and progenitor cells is essential for mammalian development and tissue homeostasis. To better understand this coordination in human neural progenitor cells (hNPCs), we performed CRISPR-Cas9 screens and identified genes that limit their expansion. These screens revealed that knockout of growth-limiting genes, including CREBBP, NF2, PTPN14, TAOK1, or TP53, caused increased hNPC expansion via skipping of a transient G0-like state, accompanied by transcriptional reprogramming of G1 subpopulations. Hallmarks of the G0-like state included expression of genes associated with quiescent neural stem cells and neural development and molecular features found in quiescent cells (e.g., hypo-phosphorylated Rb, CDK2low activity, and p27high). Further, G0-skip genes act through both distinct and convergent downstream effectors, including cell cycle, Hippo-YAP, and novel targets. The results suggest that hNPC expansion is constrained by a transient G0-like state, regulated by multiple pathways, that facilitates retention of neurodevelopmental identity.
Project description:The coordination of developmental potential and proliferation in stem and progenitor cells is essential for mammalian development and tissue homeostasis. To better understand this coordination in human neural progenitor cells (hNPCs), we performed CRISPR-Cas9 screens and identified genes that limit their expansion. These screens revealed that knockout of growth-limiting genes, including CREBBP, NF2, PTPN14, TAOK1, or TP53, caused increased hNPC expansion via skipping of a transient G0-like state, accompanied by transcriptional reprogramming of G1 subpopulations. Hallmarks of the G0-like state included expression of genes associated with quiescent neural stem cells and neural development and molecular features found in quiescent cells (e.g., hypo-phosphorylated Rb, CDK2low activity, and p27high). Further, G0-skip genes act through both distinct and convergent downstream effectors, including cell cycle, Hippo-YAP, and novel targets. The results suggest that hNPC expansion is constrained by a transient G0-like state, regulated by multiple pathways, that facilitates retention of neurodevelopmental identity.
Project description:The coordination of developmental potential and proliferation in stem and progenitor cells is essential for mammalian development and tissue homeostasis. To better understand this coordination in human neural progenitor cells (hNPCs), we performed CRISPR-Cas9 screens and identified genes that limit their expansion. These screens revealed that knockout of growth-limiting genes, including CREBBP, NF2, PTPN14, TAOK1, or TP53, caused increased hNPC expansion via skipping of a transient G0-like state, accompanied by transcriptional reprogramming of G1 subpopulations. Hallmarks of the G0-like state included expression of genes associated with quiescent neural stem cells and neural development and molecular features found in quiescent cells (e.g., hypo-phosphorylated Rb, CDK2low activity, and p27high). Further, G0-skip genes act through both distinct and convergent downstream effectors, including cell cycle, Hippo-YAP, and novel targets. The results suggest that hNPC expansion is constrained by a transient G0-like state, regulated by multiple pathways, that facilitates retention of neurodevelopmental identity.
Project description:Fission yeast Schizosaccharomyces pombe is a model for studying cellular quiescence. Shifting to medium without a nitrogen-source induces proliferative cells to enter long-term G0 quiescence. Klf1 is a Kruppel-like transcription factor with a 7-amino acid-spaced C2H2-type zinc finger motif. The deletion mutant M-bM-^HM-^Fklf1 normally divides in vegetative medium, but proliferation is not restored after long-term G0 quiescence. Cell biologic, transcriptomic, and metabolomic analyses revealed a unique phenotype of the M-bM-^HM-^Fklf1 mutant in quiescence. Mutant cells had diminished transcripts related to signaling molecules for switching to differentiation. In contrast, proliferative metabolites for cell-wall assembly and antioxidants significantly increased. Further, the size of the M-bM-^HM-^Fklf1 cells is markedly increased during quiescence due to the aberrant accumulation of calcofluor-positive chitin-like materials beneath the cell wall. After 4 weeks quiescence, the ability for reversible proliferation is lost, but energy metabolism is maintained. Klf1 thus plays a role in G0 phase longevity through enhancing the differentiation signal and suppressing metabolism for growth. If Klf1 is lost, S. pombe fails to maintain a constant cell size during quiescence. Gene expression profile of fission yeast wild type cells and klf1-gene disruptant cells in proliferating state and in quiescent state. Type of experiment: Comparing between wild type cells and klf1-gene disruptant cells in proliferating condition and in quiescent condition. Experimental factor: Exponentially proliferating state in synthetic medium, EMM2, and quiescent G0 state in nitrogen-depleted synthetic medium, EMM2-N. Quality control steps taken: All experiments were repeated twice in each condition.
Project description:Hematopoietic stem cells (HSCs) are maintained in the quiescent state for protection from exhaustion by a number of self-renewal divisions. To understand the in vivo kinetics of quiescent HSCs, we analyzed the cell cycle of CD201+150+48-Lin-Kit+Sca-1+ cells after transplantation and during development and aging using fluorescent ubiquitination-based cell cycle indicator (Fucci) mice to distinguish HSCs at the G0, G1, and S/G2/M phases. The quiescent HSC population, representing a functional HSC pool, rapidly expanded by three weeks after transplantation and by three weeks of age in development and gradually accumulated in bone marrow with aging. Single-cell RNA-sequencing with flow cytometric index sorting suggested that high CD201 and Sca-1 expression levels and a low level of mitochondrial activity were associated with quiescent HSCs. A novel set of candidate quiescent genes in HSCs was also provided. This study implied that controlling quiescent HSCs is important for the in vivo expansion and maintenance of functional HSCs.
Project description:Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. We induced chemoresistant and quiescent (G0) leukemic cells by serum-starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the up-regulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα—prior to or along with chemotherapy—substantially reduced chemoresistance in primary leukemic cells ex vivo and in vivo. These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE bearing mRNAs that promote chemoresistance. By disrupting this pathway, we developed an effective combination therapy against chemosurvival.
Project description:Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. We induced chemoresistant and quiescent (G0) leukemic cells by serum-starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the up-regulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα—prior to or along with chemotherapy—substantially reduced chemoresistance in primary leukemic cells ex vivo and in vivo. These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE bearing mRNAs that promote chemoresistance. By disrupting this pathway, we developed an effective combination therapy against chemosurvival.