nicergoline br Experimental Procedures br Acknowledgments br
Introduction Master transcription regulators control the pluripotent gene expression in embryonic stem nicergoline (ESCs) (Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003; Nichols et al., 1998). X chromosome inactivation (XCI) is a crucial epigenetic process that silences one of the two female X chromosomes to ensure equal X-linked gene expression with XY males (Payer and Lee, 2008; Lee and Bartolomei, 2013). XCI is tightly linked with pluripotency, as this epigenetic silencing occurs upon cellular differentiation and conversion of female somatic cells to the induced stem-ness state is accompanied by a global epigenetic reprogramming and reactivation of the silenced X (Maherali et al., 2007; Navarro et al., 2008). The transcription factor OCT4 lies at the top of the XCI hierarchy regulating the pluripotent-associated long noncoding RNAs (lncRNAs): Xite (the enhancer for Tsix) and Tsix (the anti-sense repressor of Xist) (Donohoe et al., 2009). Together Xite and Tsix mediate X-X homologous pairing and inhibit the silencer Xist prior to X chromosome choice (Xu et al., 2006). In addition to their roles in the study of pluripotency and cellular differentiation, mouse ESCs are established as ex vivo models of XCI, faithfully recapitulating XCI in the embryo (Clerc and Avner, 1998; Lee and Jaenisch, 1997; Lee and Lu, 1999; Penny et al., 1996; Rastan and Robertson, 1985). In undifferentiated ESCs, the single male X and both female X chromosomes are active. The lncRNAs Xite, Tsix, and Xist are all expressed on these active X chromosomes in the pluripotent state. ESCs can be differentiated by suspension culture for 4 days without leukemia inhibitory factor (LIF) and maintained thereafter under adherent conditions (Martin and Evans, 1975). Following differentiation, the male X chromosome loses expression of these lncRNAs to retain activity of the single X, whereas the female ESCs have a choice of active versus inactive X. On the future active X, Xite and Tsix expression persists to keep Xist levels low. In contrast, on the future inactive X, Xite and Tsix are extinguished, and Xist levels are greatly upregulated. OCT4 partners with the chromatin insulator CTCF, specifying the early decisions of XCI (counting, X-X pairing, and choice) (Xu et al., 2006, 2007; Donohoe et al., 2009). During differentiation, ESC chromatin shifts from a transcriptionally permission, euchromatic, to a more heterochromatic state (Azuara et al., 2006; Meshorer and Misteli, 2006; Niwa, 2007). These changes in chromatin packaging are accompanied by alterations in histone post-translational modifications (PTMs) crucial for modulation of chromatin structure and gene expression (Bernstein et al., 2006). Histone PTM writers such as the Polycomb group proteins (Boyer et al., 2006) and erasers such as the demethylases (Adamo et al., 2011; Loh et al., 2007; Mansour et al., 2012; Wang et al., 2011) play important roles in early development. We postulate that histone readers together with OCT4 play a role in the transcriptional control of the XCI lncRNAs as well as pluripotent genes. One candidate is the chromatin reader, BRD4. BRD4 is a member of the BET (bromodomain and extraterminal domain) family of tandem bromodomain-containing proteins that can bind acetylated histones H3 and H4 and influence transcription (Chiang, 2009). BRD4 is an epigenetic reader originally identified as a mitotic chromosome-binding protein that remains associated with acetylated chromatin throughout the entire cell cycle and is thought to provide epigenetic bookmarking after cell division (Dey et al., 2000, 2003). BRD4 has a direct role in transcription as it associates with positive transcription elongation factor b (P-TEFb) to enhance RNA polymerase II (RNAP II) and control productive mRNA synthesis (Yang et al., 2008). At many developmental genes RNAP II stalls or pauses after transcribing a nascent transcript about 20–65 nucleotides in length (Adelman and Lis, 2012). Nearly 30% of the genes in human ESCs commence transcription initiation but do not undergo transcriptional elongation (Guenther et al., 2007). This suggests that transcriptional pausing is an additional checkpoint control during development (Levine, 2011). The release from transcriptional pausing is associated with P-TEFb recruitment, the eviction of pause factors, the phosphorylation at serine 2 of the carboxyl-terminal domain (CTD) in RNAP II, and the production of elongated mRNAs.