Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo
- Yale University, United States
- Michigan State University, United States
- McGill University, Canada
Abstract
Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.
Data availability
All coding scripts and source datasheets for figure quantifications are made accessible through the Dryad data repository: https://doi.org/10.5061/dryad.5hqbzkh94. Representative raw imaging data are accessible through the same link and full datasets available upon request with no restrictions (due to size) by contacting VG. Source datasheets are included in supplemental information.
Article and author information
Author details
Funding
National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R01AR063663-01)
- Valentina Greco
National Institute of Arthritis and Musculoskeletal and Skin Diseases (1R01AR067755-01A1)
- Valentina Greco
National Institute on Aging (1DP1AG066590-01)
- Valentina Greco
National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR072668)
- Valentina Greco
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All procedures involving animal subjects were performed under the approval of the Institutional Animal Care and Use Committee (IACUC) of the Yale School of Medicine (Protocol #2021-11303). All live imaging was performed under 1-2% isoflurane, and ever effort was made to minimize suffering.
Copyright
© 2023, May et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Live imaging reveals chromatin compaction transitions and dynamic transcriptional bursting during stem cell differentiation in vivo
eLife 12:e83444.
https://doi.org/10.7554/eLife.83444
Further reading
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- Biochemistry and Chemical Biology
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Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.
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- Stem Cells and Regenerative Medicine
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