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  • Transcriptional elongation is a checkpoint for productive

    2022-07-01

    Transcriptional elongation is a checkpoint for productive transcription. In ESCs, ∼50% of the genes enriched for H3K4me3 are initially transcribed but do not enter productive elongation (Guenther et al., 2007, Rahl et al., 2010). Following promoter escape, the transcriptional elongating protein complex needs to overcome the nucleosome barrier (Kwak and Lis, 2013). During this process, the histone chaperones FACT and Spt6 disassemble and reassemble H2A-H2B and H3-H4 dimers, respectively (Bortvin and Winston, 1996, Belotserkovskaya et al., 2003). Spt6 enhances transcriptional elongation rate (Kaplan et al., 2000, Endoh et al., 2004, Ardehali et al., 2009) and interacts with the elongating RNA polymerase II phosphorylated at Ser2 (Ser2P-PolII) (Yoh et al., 2007), a property also required for Spt6 to promote mRNA processing and nuclear export (Yoh et al., 2008). In addition, Spt6 associates with the H3K27me3 demethylases KDM6A (UTX) and KDM6B (JMJD3) and the H3K36 methyltransferase SETD2 (Wang et al., 2013, Chen et al., 2012). The Tudor domain of the PRC2-associated Polycomb-like proteins PHF1/PLC1 and PHF19/PLC3 recognize H3K36me3 nucleosomes, allowing recruitment and spreading of PRC2 and H3K27me3 into H3K36me3-chromatin regions of ESCs (Cai et al., 2013, Brien et al., 2012). H3K27me3 spreading might be regulated to prevent its intrusion into active Zinc protoporphyrin IX regions.
    Results
    Discussion We found that Spt6 depletion in ESCs resulted in decreased expression of pluripotency TFs, reduced accumulation of the pluripotency marker AP protein, increased transcription of cell-lineage-affiliated master TFs, and morphological changes indicative of early cell differentiation. Transcription of pluripotency TFs, including OSN, was exquisitely sensitive to reduced Spt6 levels, which were, on the other hand, sufficient to sustain transcriptional activation of cell-lineage-affiliated master TFs. Increased expression of developmental regulators in Spt6-depleted ESCs is likely an indirect effect triggered by entrance into differentiation following loss of ESC pluripotency factors and regulators (Boyer et al., 2005, Loh et al., 2006, Masui et al., 2007, Ding et al., 2015, Strikoudis et al., 2016). Expression of pluripotency TFs is controlled by SEs, consisting of cluster of enhancers occupied by high levels of the Mediator complex, RNA polymerase II, and other transcriptional activators (Parker et al., 2013, Whyte et al., 2013). Spt6 was preferentially enriched at ESC-specific SEs, and several lines of evidence supported its role in regulating their activity: Spt6 depletion (1) affected expression of genes associated with ESC-SEs, (2) decreased H3K27ac and increased H3K27me3 at SEs, and (3) reduced expression of SE-transcribed eRNAs. As in the case of other transcriptional regulators (Lovén et al., 2013, Brown et al., 2014, Vahedi et al., 2015, Pelish et al., 2015), Spt6 reduction may selectively affect ESC SE-mediated gene expression by interfering with cooperative and synergistic binding of multiple transcriptional regulators, which is more prominent at SEs than TEs (Whyte et al., 2013, Hnisz et al., 2017). Given the interconnectivity of the autoregulatory loop formed by ESC pluripotency TFs with their respective SEs (Hnisz et al., 2013), reduced expression of ESC pluripotency TFs is expected to negatively affect SE activity. However, it seems unlikely that SE inactivation would be the primary consequence of decreased pluripotency TFs levels, as the TFs themselves would have to be first downregulated to exert a negative effect on SEs. Elongation is a checkpoint in ESC productive transcription (Guenther et al., 2007, Rahl et al., 2010). The PHD-finger protein Phf5a has been recently shown to regulate ESC pluripotency by stabilizing the elongation Paf1 complex. Ph5a silencing resulted in downregulation of ESC pluripotency markers and upregulation of mesoderm development (Strikoudis et al., 2016). A role at ESC enhancers has also been described for Ell3, a member of the Ell family of transcriptional elongation factors and component of the super elongation complex (Miller et al., 2000, Luo et al., 2012). In ESCs, Ell3 occupies enhancer regions that are poised, active, or inactive and is required for priming of developmental regulators for future activation during differentiation. However, despite being recruited at enhancers of pluripotency TFs OSN, Ell3 was dispensable for their expression (Smith et al., 2011). Thus, distinct elongation factors have different roles at ESC enhancers controlling genes involved in maintaining pluripotency or favoring differentiation.