Endogenous retroviruses (ERVs) contribute to ∼10 percent of the mouse genome. They are often silenced in differentiated somatic cells but differentially expressed at various embryonic developmental stages. A minority of mouse embryonic stem cells (ESCs), like 2-cell cleavage embryos, highly express ERV MERVL. However, the role of ERVs and mechanism of their activation in these cells are still poorly understood. In this study, we investigated the regulation and function of the stage-specific expressed ERVs, with a particular focus on the totipotency marker MT2/MERVL. We show that the transcription factor Zscan4c functions as an activator of MT2/MERVL and 2-cell/4-cell embryo genes. Zinc finger domains of Zscan4c play an important role in this process. In addition, Zscan4c interacts with MT2 and regulates MT2-nearby 2-cell/4-cell genes through promoting enhancer activity of MT2. Furthermore, MT2 activation is accompanied by enhanced H3K4me1, H3K27ac, and H3K14ac deposition on MT2. Zscan4c also interacts with GBAF chromatin remodelling complex through SCAN domain to further activate MT2 enhancer activity. Taken together, we delineate a previously unrecognized regulatory axis that Zscan4c interacts with and activates MT2/MERVL loci and their nearby genes through epigenetic regulation.

Inhibition of Mek/Erk signaling by pharmacological Mek inhibitors promotes self-renewal and pluripotency of mouse embryonic stem cells (ESCs). Intriguingly, Erk signaling is essential for human ESC selfrenewal. Here we demonstrate that Erk signaling is critical for mouse ESC self-renewal and genomic stability. Erk-depleted ESCs cannot be maintained. Lack of Erk leads to rapid telomere shortening and genomic instability, in association with misregulated expression of pluripotency genes, reduced cell proliferation, G1 cell-cycle arrest, and increased apoptosis. Erk signaling is also required for the activation of differentiation genes but not for the repression of pluripotency genes during ESC differentiation. Furthermore, we find an Erk-independent function of Mek, which may explain the diverse effects of Mek inhibition and Erk knockout on ESC self-renewal. Together, in contrast to the prevailing view, Erk signaling is required for telomere maintenance, genomic stability, and self-renewal of mouse ESCs.Erk | Mek | embryonic stem cells | self-renewal | genomic stability E mbryonic stem cells (ESCs) are pluripotent and, hence, promising donor cell sources for regenerative medicine. Transcriptional regulation plays an essential role in pluripotency maintenance of ESCs, and a transcriptional regulation network for pluripotency has been characterized (1, 2). The core component of the pluripotency transcriptional regulation network is a feed-forward selfregulating circuitry formed by transcription factors Oct4, Sox2, and Nanog (3, 4). ESCs are cultured in media supplemented with growth factors. Through signaling pathways, growth factors affect the pluripotency transcriptional regulation network and regulate the self-renewal and differentiation of ESCs. For example, LIF and BMP signaling controls the transcriptional activities of the downstream effectors Stat3 and Smad1 to promote the self-renewal of mouse ESCs (5-7).The extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) signal-transduction cascade mediates the effect of growth factors by sequential activation of Ras-like GTPase, Raf kinase, serine/threonine protein kinase Mek, and Erk to regulate cell-cycle progression, proliferation, differentiation, and carcinogenesis (8, 9). Erk signaling also plays a pivotal role in pluripotency maintenance. Inhibition of Mek/Erk signaling constrains the differentiation of mouse ESCs (10). Mouse ESCs can be derived and maintained in medium supplemented with inhibitors of Mek and Gsk3 signaling (2i) (11). Moreover, inhibition of Mek facilitates the conversion of mouse epiblast stem cells (epiSCs) to ESC-like cells (12). Similarly, the Mek inhibitor PD0325901 is used in establishing and maintaining human ground-state pluripotent stem cells (13-16). Conversely, activation of Mek/Erk signaling promotes the differentiation of ESCs. Ectopic expression of an activated H-RAS mutant leads to mouse ESC differentiation toward the trophectodermal lineage. Mek/Erk signaling is the downstream effector of Ras medi...

Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3β inhibitors), which also inhibits Dnmt3b, reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4, which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels.

Feeder cells like mouse embryonic fibroblasts (MEFs) have been widely applied for culture of pluripotent stem cells, but their roles remain elusive. Noticeably, ESCs cultured on the feeders display transcriptional heterogeneity. We investigated roles of feeder cells by examining the telomere maintenance. Here we show that telomere is longer in mESCs cultured with than without the feeders. mESC cultures without MEF feeders exhibit telomere loss, chromosomal fusion, and aneuploidy with increasing passages. Notably, feeders facilitate heterogeneous transcription of 2-cell genes including Zscan4 and telomere elongation. Moreover, feeders produce Fstl1 that together with BMP4 periodically activate Zscan4. Interestingly, Zscan4 is repressed in mESCs cultured in 2i (inhibitors of Mek and Gsk3β signaling) media, associated with shorter telomeres and increased chromosome instability. These data suggest the important role of feeders in maintaining telomeres for long-term stable self-renewal and developmental pluripotency of mESCs.

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