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| Status |
Public on Mar 15, 2023 |
| Title |
FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells [ATAC-Seq] |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
The mammalian FACT complex is a highly conserved histone chaperone with essential roles in transcription elongation, histone deposition, and maintenance of stem cell state. FACT is essential for viability in pluripotent cells and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block reprogramming of fibroblasts to induced pluripotent stem cells, yet the molecular mechanisms through which FACT regulates cell fate decisions remain unclear. To determine the mechanism by which FACT regulates stem cell identity, we used the auxin-inducible degron systems to deplete murine embryonic stem cells of FACT subunit SPT16 and subjected depleted cells to genome-wide factor localization, nascent transcription analyses, and genome-wide nucleosome profiling. Inducible depletion of SPT16 reveals a critical role in regulating targets of the master regulators of pluripotency: OCT4, KLF4, MYC, NANOG, and SOX2. Depletion of SPT16 leads to increased nucleosome occupancy at genomic loci occupied by these transcription factors, as well as gene-distal regulatory sites defined by DNaseI hypersensitivity. This heightened occupancy suggests a mechanism of nucleosome filling, wherein the sites typically maintained in an accessible state by FACT are occluded through loss of FACT-regulated nucleosome spacing. 20% of transcription arising from gene-distal regions bound by these factors is directly dependent on FACT, and putative gene targets of these non-coding RNAs are highly enriched for pluripotency in pathway analyses. Upon FACT depletion, transcription of Pou5f1 (OCT4), Sox2, and Nanog are downregulated, suggesting that FACT not only co-regulates expression of the encoded proteins’ targets, but also the pluripotency factors themselves. We find that FACT maintains cellular pluripotency through a complex regulatory network of both coding and non-coding transcription.
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| Overall design |
ATAC-seq
8 ATAC-seq libraries, with two replicates per condition in two independently-targeted cell lines (SPT16-V5-AID C-terminal tag). Control samples were treated with EtOH, while experimental samples were treated with IAA.
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| Contributor(s) |
Klein DC, Lardo SM, Hainer SJ |
| Citation |
https://doi.org/10.1101/2021.07.30.454509
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| Submission date |
Apr 28, 2022 |
| Last update date |
Jul 13, 2023 |
| Contact name |
David Charles Klein |
| E-mail(s) |
[email protected]
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| Organization name |
University of Pittsburgh
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| Department |
Biological Sciences
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| Lab |
Hainer Lab
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| Street address |
4249 Fifth Ave
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
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| Platforms (1) |
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| Samples (36)
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| This SubSeries is part of SuperSeries: |
| GSE181624 |
FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells |
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| Relations |
| BioProject |
PRJNA833042 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE201784_RAW.tar |
6.0 Gb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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