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FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells

View ORCID ProfileDavid C. Klein, Santana M. Lardo, View ORCID ProfileSarah J. Hainer
doi: https://doi.org/10.1101/2021.07.30.454509
David C. Klein
1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15213
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Santana M. Lardo
1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15213
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Sarah J. Hainer
1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15213
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  • For correspondence: sarah.hainer@pitt.edu
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Abstract

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.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted July 31, 2021.
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FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells
David C. Klein, Santana M. Lardo, Sarah J. Hainer
bioRxiv 2021.07.30.454509; doi: https://doi.org/10.1101/2021.07.30.454509
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FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells
David C. Klein, Santana M. Lardo, Sarah J. Hainer
bioRxiv 2021.07.30.454509; doi: https://doi.org/10.1101/2021.07.30.454509

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