FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

Abstract

Background The FACT complex is a conserved histone chaperone with critical roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear. Results To explore the mechanism for FACT function, we generated AID-tagged murine embryonic cell lines for FACT subunit SPT16 and paired depletion with nascent transcription and chromatin accessibility analyses. We also analyzed SPT16 occupancy using CUT&RUN and found that SPT16 localizes to both promoter and enhancer elements, with a strong overlap in binding with OCT4, SOX2, and NANOG. Over a timecourse of SPT16 depletion, nucleosomes invade new loci, including promoters, regions bound by SPT16, OCT4, SOX2, and NANOG, and TSS-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (encoding OCT4), Sox2, Nanog, and enhancer RNAs produced from these genes’ associated enhancers are downregulated. Conclusions We propose that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.

Chromatin remodeling with combined FACT and BET inhibition disrupts oncogenic transcription in Diffuse Midline Glioma

“…Our findings indicate that FACT maintains open chromatin at promoters and enhancers, supporting transcription driven by developmental TFs (TCF12, OLIG2, SNAI1) in DMG. This parallels a recent study showing FACT's role in maintaining open chromatin at regulatory elements to promote transcription by core pluripotency TFs in embryonic stem cells [83].…”

Widespread impact of nucleosome remodelers on transcription at cis-regulatory elements

“…CUT&RUN experiments were performed as previously described 132,134,135 , with the following modifications. The following antibodies were used in CUT&RUN experiments performed on 100,000 lightly crosslinked wildtype ES cells using a low salt elution method for fragment release: SRCAP (Kerafast ESL103, 1:50) SMARCAL (Invitrogen PA5-28980, lot YF3945097B, 1:50), and IgG (Sigma-Aldrich, 06-371,1:250).…”

“…TT-seq was conducted as previously described 132,133 . 48 h post transfection, media was aspirated from transfected plates and replaced with 10 mL of 500 nM 4-thio-uridine (4sU) containing ESC media and the plates incubated at 37°C with 5% CO 2 for 5 min.…”