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| Status |
Public on Jun 28, 2024 |
| Title |
Stem cells tightly regulate dead cell clearance to maintain tissue fitness [CUT&Run] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Macrophages and dendritic cells have long been appreciated for their ability to migrate to and engulf dying cells and debris, including some of the billions of cells that are naturally eliminated from our body daily. However, a substantial amount of these dying cells are cleared by local tissue cells, so-called ‘non-professional phagocytes’, critical to preserve organismal fitness. How non-professional phagocytes are able to sense and digest nearby apoptotic corpses while still performing their normal tissue functions is unclear. Here, we explore the molecular mechanisms underlying this balancing act. Exploiting the cyclical bouts of tissue regeneration and degeneration during the hair cycle, we show that stem cells can transiently become non-professional phagocytes when confronted with dying cells. Adoption of this phagocytic state requires not only local lipids produced by apoptotic corpses, which are necessary for RXRα activation, but also tissue-specific retinoids able to activate RARg. The dual factor dependency enables tight regulation of the genes requisite to activate phagocytic apoptotic clearance. The tunable phagocytic program we describe here offers an attractive mechanism to offset phagocytic duties against the primary stem cell function of replenishing differentiated cells to preserve tissue integrity during homeostasis. Our findings have broad implications for other non-motile stem or progenitor cells which experience cell death in an immune-privileged niche.
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| Overall design |
500,000 cultured hair follicle stem cells per sample were trypsinized, washed with PBS and pelleted. Cut-&-Run-seq was performed as described before in Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6 (2017). Briefly, HFSCs were cross-linked in cross-linking buffer with rotation for 10 min, quenched with glycine, washed and resuspended in NE1 buffer and rotated for 10min at 4oC. Nuclei were washed with CNR binding buffer and incubated with concanavalin-A beads or 10 min at 4 °C. ConA-bead bound nuclei were incubated overnight at 4 °C in CNR antibody buffer and 1:50 RXRa antibody (Cell Signaling Technologies, clone D6H10, #3085). ConA-bead bound nuclei were washed with CNR Triton wash buffer. then resuspended and incubated at 4 °C for 60 min in CUT&RUN antibody buffer and 2.5 μl pAG-MNase (EpiCypher). Following this, ConA-bead bound nuclei were washed twice with CUT&RUN Triton wash buffer, resuspended in 100 μl of Triton wash buffer and incubated on ice for 5 min before 2ul of 100mM CaCl2 was added per sample. Samples were incubated on ice for 30 min and the reaction was then stopped by adding 100ul of 2× stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM egtazic acid, 0.1% Triton X-100 and 50 μg ml−1 RNaseA) and incubated at 37 °C for 10 min. ConA-bound nuclei were captured on a magnet, and supernatant containing Cut-and-Run DNA fragments was collected. Supernatant was incubated at 70 °C for 4 h with 2 μl 10% sodium dodecyl sulfate and 2.5 μL 20 mg ml−1 proteinase K, prior to DNA purification using PCI reagent (phenol:chloroform:isoamyl alcohol, Millipore). DNA fragments were precipitated overnight with ethanol and glycogen at -20 oC before resuspension in elution buffer (1 mM Tris–HCl pH 8.0 and 0.1 mM EDTA). CNR sequencing libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina. PCR-amplified libraries were purified using 1X ratio of SPRI beads (Beckman) and eluted in 15 μl EB buffer (Qiagen). All CNR libraries were sequenced on Illumina NextSeq using 40 bp paired-end reads.
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| Web link |
https://www.nature.com/articles/s41586-024-07855-6
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| Contributor(s) |
Stewart KS, Fuchs E |
| Citation(s) |
39169186 |
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| Submission date |
Jun 27, 2024 |
| Last update date |
Sep 27, 2024 |
| Contact name |
Katherine Stewart |
| Organization name |
The Rockefeller University
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| Department |
Laboratory of Mammalian Cell Biology and Development
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| Lab |
Elaine Fuchs
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| Street address |
1230 York Avenue
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| City |
New York City |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (8)
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| This SubSeries is part of SuperSeries: |
| GSE271007 |
Stem cells tightly regulate dead cell clearance to maintain tissue fitness |
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| Relations |
| BioProject |
PRJNA1129097 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE270947_BEL_RXR_pool_dedup_RPGCnorm10bin_sub120.bedgraph.gz |
476.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSE270947_BEL_RXR_pool_dedup_RPGCnorm10bin_sub120.bw |
395.4 Mb |
(ftp)(http) |
BW |
| GSE270947_corpse_RXR_pool_dedup_RPGCnorm10bin_sub120.bedgraph.gz |
494.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSE270947_corpse_RXR_pool_dedup_RPGCnorm10bin_sub120.bw |
415.4 Mb |
(ftp)(http) |
BW |
| GSE270947_mlcl_RXR_pool_dedup_RPGCnorm10bin_sub120.bedgraph.gz |
581.8 Mb |
(ftp)(http) |
BEDGRAPH |
| GSE270947_mlcl_RXR_pool_dedup_RPGCnorm10bin_sub120.bw |
479.6 Mb |
(ftp)(http) |
BW |
| GSE270947_un_RXR_pool_dedup_RPGCnorm10bin_sub120.bedgraph.gz |
625.6 Mb |
(ftp)(http) |
BEDGRAPH |
| GSE270947_un_RXR_pool_dedup_RPGCnorm10bin_sub120.bw |
521.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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