Roles for Histone Acetylation in Regulation of Telomere Elongation and Two‐cell State in Mouse ES Cells

Mammalian telomeres contain repetitive G-rich sequences and associated proteins at the ends of linear chromosomes (Blackburn, 2001). Telomeres protect chromosome ends and maintain chromosomal stability (Palm and de Lange, 2008). Telomere length maintenance is primarily achieved by telomerase that adds telomere repeats de novo during each cell division, counteracting telomere erosion (Chan and Blackburn, 2002). Telomere length also can be maintained by telomerase-independent mechanisms, including an alternative lengthening of telomeres (ALT) mechanism, based on homologous recombination between telomere repeats (Muntoni and Reddel, 2005). Telomeres and subtelomeres are densely compacted with repressive DNA methylation and histone modifications, forming condensed heterochromatin structures (Blasco, 2007). Differential abundance of those epigenetic modifications at telomeres and subtelomeres contributes to the formation of a “closed” or “open” chromatin state, regulating telomere length, possibly through regulating the access of telomerase to telomeres or the ALT mechanism (Blasco, 2007). Mouse embryonic stem (ES) cells deficient for DNA methyltransferases Dnmt1 or Dnmt3a/3b exhibit reduced DNA methylation at subtelomere regions, increased telomere recombination as indicated by telomere sister-chromatid exchange (T-SCE), and elongated telomeres (Gonzalo et al., 2006). Repressive histones H3K9me3 and H4K20me3, as well as heterochromatin protein 1 isoforms, are also enriched at condensed heterochromatin regions (Blasco, 2007). H3K9me3 and H4K20me3 are detected at satellite, telomeres, and active long-terminal repeats, and can spread to proximal unique sequences (Mikkelsen et al., 2007). Mouse embryonic fibroblast (MEF) cells lacking Suv39h1 and Suv39h2 histone methyltransferases (HMTs), which govern methylation of heterochromatic H3K9me3, show abnormal telomere lengthening and increased T-SCE (Garcia-Cao et al., 2004), suggesting an essential role ofH3K9me3 in suppression of telomere length. Similarly, mouse ES and MEF cells deficient for Suv4-20h2 HMTs that is responsible for trimethylating H4K20 display abnormally elongated telomeres and increased T-SCE (Benetti et al., 2007). Furthermore, mouse MEF cells deficient for all three members of retinoblastoma gene family (RB1, RBL1 and RBL2) also exhibit decreased levels of H4K20me3 at telomeres and global reduction of DNA methylation, accompanied by aberrantly elongated telomeres (Gonzalo and Blasco, 2005). In addition, mammalian telomeres and subtelomeres are bound by low levels of acetylated H3 (AcH3) and H4 (AcH4) (Blasco, 2007; Wong, 2010). However, whether histone acetylation also participates in telomere length regulation in ES cells remains elusive. ES cell cultures are a heterogeneous mixture of metastable cells with fluctuating activation of 2-cell embryo specific genes (2C-genes) and endogenous transposable element (TE) activities (Macfarlan et al., 2012; Torres-Padilla and Chambers, 2014), suggesting that ES cells in the 2C-state might resemble the totipotent zygotes/2C-stage embryos. In this regard, the 2C-state was postulated as a “super” state of ES cells (Surani and Tischler, 2012). Zscan4, specifically expressed in ES cells and 2-cell embryos (Falco et al., 2007; Zalzman et al., 2010), and highly enriched within 2C::tdTomato+ mouse ES cells (Macfarlan et al., 2012), can also faithfully represent the 2C-state of mouse ES cells. Zscan4 is only expressed in about 3–5% of ES cells at any given time, and Zscan4+ and Zscan4− ES cells can interconvert to each other and nearly all ES cells activate Zscan4 at least once during nine passages (Zalzman et al., 2010). Without intermittent activation of Zscan4, embryos delay preimlantation development and ES cells lose their ability to proliferate indefinitely (Falco et al., 2007; Zalzman et al., 2010), suggesting that the equilibrium between the 2C-state and canonical ES state is essential for properembryonic development. The major function of transient Zscan4 expression in ES cells is telomere lengthening by recombination involving T-SCE (Zalzman et al., 2010). We find that histone acetylation positively regulates telomere length by Zscan4/2C-mediated telomere recombination as well as up-regulation of Terc.