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    • Our observations are consistent with previous findings

    2018-11-08

    Our observations are consistent with previous findings in vivo that Ahr expression is silenced at the 2-cell stage, becoming again detectable in late blastocysts (Peters and Wiley, 1995; Dey and Nebert, 1998; Wu et al., 2002) and in most developing organs during post-implantation development (Abbott and Probst, 1995; Jain et al., 1998). Such an expression pattern suggests that AHR is developmentally bivalent and that its functions during post-implantation must be silenced if pluripotency is maintained. Studies on the OCT3/4-NANOG-SOX2 regulatory network in ES faah inhibitor have shown that pluripotency factors maintain ES cell properties by simultaneously activating expression of pluripotency-related genes, including their own (Kim et al., 2008; Chambers and Tomlinson, 2009) and repressing key differentiation transcription factors (Boyer et al., 2006; Loh et al., 2006). Among the latter, OCT3/4 was previously implicated on Ahr repression (Loh et al., 2006). In good agreement, we find that, not just OCT3/4, but NANOG and SOX2 as well are actively involved in Ahr repression in ES cells through binding to their cognate sites on the Ahr minus-2kb distal promoter region. Ectopic OSKM expression in mouse hepatoma Hepa-1 cells confirmed this conclusion. Endogenous Ahr expression is very high in these cells, but it was significantly repressed by the transfected OSKM proteins both at the protein and mRNA levels. This finding confirms that pluripotency factors are the main agents of Ahr repression in pluripotent cells and suggests that Ahr repression may be required to maintain pluripotency. In this context, it may be significant that AHR regulates the multipotency of hematopoietic stem cells, since its inhibition promotes their greater expansion (Boitano et al., 2010), while deletion of the Ahr gene increases the proliferative capacity of hematopoietic progenitors (Lindsey and Papoutsakis, 2012; Gasiewicz et al., 2010; Singh et al., 2011). Pluripotency factors do not act alone; they share overlapping networks of target genes with PcG proteins that further silence their targets (Bernstein et al., 2006; Boyer et al., 2006; Loh et al., 2006). In ES cells, differentiation associated genes are subject to flexible epigenetic controls that repress and prime their expression but do not fully silence them. Genes encoding transcription factors that play an essential role during differentiation carry bivalent promoter domain marks in ES cells, consisting of active H3K4me3 and repressive H3K27me3 (Mikkelsen et al., 2007). These marks, through their interplay with PcG and TxG proteins, repress transcription but keep the genes in a primed state (Morey and Helin, 2010; Schuettengruber et al., 2011). The Ahr promoter does not show bivalent domains in ES cells; in fact, in our hands and in good agreement with previous studies (Mikkelsen et al., 2007; Ku et al., 2008), H3K27me3 is low in both ES and differentiated cells and H3K4me3 is low in ES cells and reaches high levels in differentiated cells. Conversely, consistent with recent work that has classified Ahr as a PcG-silenced gene (Brookes et al., 2012), our ChIP analyses show that PcG-mediated mechanisms are strongly involved in Ahr repression in ES cells. Several PcG proteins, including EZH2, SUZ12, RING1B, and TxG proteins MLL and KDM6B showed higher binding on the Ahr promoter in ES than in differentiated cells. The E3 ubiquitin ligase activity of the PRC1 component RING1B mediates pausing of unproductive RNAPII through ubiquitination of histone H2A, contributing a key element to gene repression (Endoh et al., 2012; Stock et al., 2007). We conclude that, in addition to the inhibitory effect resulting from direct binding of pluripotency factors, Ahr is also repressed by PcG-mediated mechanisms. Binding of the two TxG proteins, the H3K4 methyltransferase MLL and the H3K27 demethylase KDM6B, suggests that their role is to quickly activate the Ahr upon reception of morphogenetic signals. The main features of the transition of Ahr repression from undifferentiated ES cells to activation in differentiated cells are schematically presented in Fig. 7.