Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 15213, USA.
Department of Biology and Epigenetics Institute, University of Pennsylvania, Philadelphia, PA, 19104, USA.
BMC Biol. 2023 Aug 4;21(1):167. doi: 10.1186/s12915-023-01669-0.
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.
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.
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.
FACT 复合物是一种保守的组蛋白伴侣，在转录和组蛋白沉积中具有关键作用。FACT 在多能性和癌细胞中是必不可少的，但在大多数哺乳动物细胞类型中则可有可无。FACT 的缺失或抑制可以阻止多能干细胞的诱导，但 FACT 调节细胞命运决定的机制尚不清楚。
为了探索 FACT 功能的机制，我们生成了带有 AID 标签的小鼠胚胎细胞系，用于 FACT 亚基 SPT16，并与新生转录和染色质可及性分析配对进行耗竭。我们还使用 CUT&RUN 分析了 SPT16 的占据情况，发现 SPT16 定位于启动子和增强子元件，与 OCT4、SOX2 和 NANOG 的结合有很强的重叠。在 SPT16 耗竭的时间过程中，核小体侵入新的位置，包括启动子、SPT16、OCT4、SOX2 和 NANOG 结合的区域以及 TSS 远端的 DNA 酶 I 超敏位点。同时，编码 OCT4 的 Pou5f1（OCT4 基因）、Sox2、Nanog 和从这些基因相关增强子产生的增强子 RNA 的转录下调。
我们提出，FACT 通过一种精确的基于核小体的调控机制，维持细胞的多能性，适当表达与多能性相关的编码和非编码转录本。
