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Identification of dormant cells using a novel fluorescent reporter of RNA-polymerase II activity

ABSTRACT: Identification of dormant cells using a novel fluorescent reporter of RNA-polymerase II activity

PROVIDER: PRJNA612818 | ENA |

REPOSITORIES: ENA

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Identification of dormant cells using a novel fluorescent reporter of RNA-polymerase II activity

Project description:Dormancy is a reversible cellular state in which the cell cycle and metabolic activity of a cell is dramatically decreased, leading to protection from genetic damage and prolonged survival. One example of dormant cells is the somatic stem cell, which is crucial for tissue homeostasis and response to injury or transplantation. However, while dormant cells have been well characterized in some tissues, the ability to identify them irrespective of tissue of origin remains elusive. Moreover, in some tissues (e.g. the intestinal epithelium) dormant stem cells showed heterogeneity in the marker’s profile that makes their identification and isolation challenging. Here, we developed a live cell reporter of dormancy based on our observation that phosphorylation of RNA Polymerase II (RNApII), a hallmark of active mRNA transcription elongation, is largely absent in dormant stem cells from multiple lineages. We show that insertion of a short RNApII kinase target peptide directly into the yellow fluorescent protein Venus generates a live cell Optical Stem Cell Activity Reporter (OSCAR) for detection of dormant cells. We found, using small intestinal crypt cultures, that OSCAR is able to show the dynamics of dormancy induction and cellular differentiation in real time. Generation and analysis of mice ubiquitously expressing OSCAR showed several populations of OSCARhigh and OSCARlow cells. Microscopy, RNA-Seq and single-cell culture confirmed that OSCAR enables direct identification and isolation of cell populations bearing different transcriptional states and provides a useful tool for understanding dormant cell biology.

2021-04-16 | GSE147030 | GEO

Establishment of a fluorescent reporter of RNA-polymerase II activity to identify dormant cells.

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Identifying dormant Nfatc1-marked stem/progenitor cells as an important contributor for mammary lobuloalveoli formation

Project description:The Mammary gland undergoes complicated epithelial remodeling to form lobuloalveoli during pregnancy, in which basal epithelial cells remarkably increase to form a basket-like architecture. However, it remains largely unknown how dormant mammary basal stem/progenitor cells involve in lobuloalveolar development. Here, we show that Nfatc1 expression marks a rare population of mammary epithelial cells with the majority being basal epithelial cells. Nfatc1 reporter-marked basal epithelial cells are relatively dormant mammary stem/progenitor cells. Although Nfatc1 reporter-marked basal epithelial cells show limited contribution to the homeostasis of mammary epithelium, they divide rapidly during pregnancy and contribute to lobuloalveolar development. Furthermore, Nfatc1 reporter-marked basal epithelial cells are preferentially used for multiple pregnancies. Using single-cell RNA-seq analysis, we identify multiple functionally distinct clusters within the Nfatc1 reporter-marked cell-derived progeny cells during pregnancy. Taken together, our findings underscore Nfatc1 reporter-marked basal cells as dormant stem/progenitor cells that contribute to mammary lobuloalveolar development during pregnancy.

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ChIP-chip of MCF7 Sox2 regulatory region 2 transcription activity reporter unresponsive and reporter responsive breast cancer cells

Project description:We found that MCF7 and ZR751 Sox2-expressing breast cancer cell lines comprise of cells with heterogeneous Sox2 transcription activity reporter response. A small subset of Sox2 reporter responsive cells are more tumourigenic than the bulk Sox2 reporter unresponsive cells. We questioned whether Sox2 exhibit differential gene promoter occupancies in the two cell subsets to govern differential gene expression patterns. Sox2 ChIP in reporter unresponsive (RU) and reporter responsive (RR) cells (duplicate samples) were compared. IgG ChIP in RU and RR cells served as the negative controls.

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