Epigenetic Regulation Of Telomere Length In Mouse ES Cells And Human ALT Cancer Cells

Mouse embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts and thought to be functionally equivalent to ICM, harboring similar gene expression patterns compared to ICM cells. Mouse ES cells are featured with pluripotency and self-renewal, which are maintained by complex intrinsic and extrinsic regulation. It is also to note that mouse ES cell cultures are a heterogeneous mixture of metastable cells with fluctuating expression of pluripotent genes, including Zscan4, a very unique gene, expressed specifically in2-cell embryos and transiently in sporadic ES cells (3-5%) at any given time, marks a transient2C-state of mouse ES cells. Zscan4+and Zscan4-ES cells also can interconvert to each other and nearly all ES cells activate Zscan4at least once during nine passages. Zscan4is critical required for preimplantation embryonic development and lengthening telomeres promptly by recombination-based mechanisms and maintaining genomic stability. Without intermittent activation of Zscan4, embryos delay preimlantation development, blastocysts die for defect in implantation, and ES cells lose their ability to maintain normal karyotype, telomere length and proliferate indefinitely. This unique mode of expression suggests a tightly regulated mechanism, but the factors that control Zscan4expression remain a mystery.Herein, we investigated why Zscan4was limited to only3-5%ES cells, and the underlined molecular mechanisms, and also the biological significance of this unique expression pattern. Our results demonstrated that Zscan4was negatively regulated by Rif1, which was highly expressed in mouse ES cells and testis.Rif1, therefore, plays a critical role in regulating ES cell heterogeneity, and maintaining telomere length homeostasis and genomic stability for ES cell self-renewal and pluripotency. Rifl depleted ES cells exhibit remarkably high expression levels of Zscan4, and abnormal telomere elongation and heterogeneity by Zscan4mediated hyper telomere recombination or named telomere sister chromatid exchange (T-SCE), and also pronounced telomere loss and end-to-end chromosomal fusions. This data is substantiated by the experiments showing that double depletion of Rifl and Zscan4 rescues the telomere recombination phenotypes, suggesting that Rif1maintains telomere length homeostasis via regulation of Zscan4. Elevated terminal recombination also may contribute to telomere attrition and genomic instability. Therefore, it is not surprising that Rif1depleted ES cells exhibit greatly reduced pluripotency both in vitro and in vivo, and that simultaneous depletion of Rifl and Zscan4significantly rescues the defective embryogenesis caused by Rif1deficiency. These results indicate that Zscan4must be maintained at a stable level, aberrantly high and low levels of Zscan4both impair ES cell self-renewal, pluripotency and embryogenesis. Interestingly, Zscan4activated its own promoter activity through SCAN domain, forming a positive feedback loop for auto-activation. Without the inhibition of Rif1highly expressed in ES cells, positive regulation of Zscan4expression by Zscan4itself could cause Zscan4+in more ES cells to more than11%from3-5%, leading to hyper telomere recombination and genomic instability, reduced self-renewal and loss of pluripotency of ES cells.Furthermore, our transcriptome analysis suggests that Rif1is a transcriptional repressor. More genes are up-regulated than down-regulated following stable or transient Rifl depletion. Notably, in Rif1depleted ES cells, a significant portion of up-regulated genes (about13%) are located at sub-telomeric regions, including Zscan4and Tcstv1/3.Telomeres and subtelomeres are densely compacted with repressive DNA methylation and histone modifications (e.g. H3K9me3and H4K20me3) that are important negative regulators of mammalian telomere lengths. These repressive epigenetic marks at condensed sub-telomeric heterochromatin regions form an inhibitory effect to the genes nearby, known as Telomere Position Effect (TPE). Mechanistically, we found Rifl is a new component of H3K9methylation complex. Rifl selectively interacted with H3K9me3and other core components of H3K9methylation complex, and stabilized the H3K9methylation machinery in ES cells. In Rif1depleted ES cells, the bridge and interaction among these core components were both greatly disrupted, leading to destabilization of H3K9methylation complex, degradation of the core components, in line with reduced overall protein levels of core components G9a, Setdbl, Kap1, HP1α and Glp, and reduced protein level of H3K9me3and occupancy in Rifl-repressed genes mostly located at peri-centromeres and subtelomeres.Besides the epigenetic regulation by Rifl, we also found Zscan4was influenced by Tbx3mediated epigenetic regulation. Our results proved that Tbx3is new regulator of Zscan4+/2C state and telomere elongation in mouse ES cells. Tbx3over-expression (OE) led to up-regulation of Tet2and down-regulation of Dnmt3b protein level, respectively, which synergistically reduced global DNA methylation, leading to de-repression of Zscan4and elongation of telomeres. Moreover, besides the negative epigenetic regulation by repressive epigenetic marks condensed in telomeric and sub-telomeric heterochromatin regions, the active epigenetic mark histone H3acetylation with less aboundant in telomeric and sub-telomeric heterochromatin regions was first indentified to participate in positive regulation of Zscan4and telomere elongation in mouse ES cells.Proper telomere length is critically required for mammalian cell proliferation, and critically short telomeres will trigger replicative cell senescence and aging. The telomere length of late generation G4Terc-/-ES cells had reduced to very short level with reduced cell proliferation rate, while could undergo more than450cell divisions. In the absence of telomerase activity involvement, the dynamic of telomere length of G4Terc-/-ES cells during passaging and how the G4Terc-/-ES cells compensate for telomere loss are both unclear. Our results demonstrated that the telomere length in G4Terc-/-ES cells maintained at a stable level during passaging. The repressive epigenetic marks DNA methylation and histone methylation H3K9me3were reduced, while active epigenetic mark AcH3increased in G4Terc-/-ES, leading to less condensed telomere heterochromatin structure and reduced TPE, activation of Zscan4and telomere recombination and compensation of telomere loss. These mechanisms used during evolution by G4Terc-/-ES faithfully solve the end-replication problems in the absence of telomerase involvement. About10to15%of human cancers lack detectable telomerase activity, and many of these use an alternative lengthening of telomeres (ALT) mechanism to maintain telomeres in cancer growth, including U2OS cells. We found that H3K9me3and AcH3were also reduced and increased in ALT U2OS cells compared to telomerase positive HeLa cells, respectively, leading to highly up-regulation of ZSCAN4in U2OS cells, suggesting ZSCAN4was also regulated by epigenetic marks. We also indicated that ZSCAN4might also take place in activation of ALT in human ALT cancers. ZSCAN4KD in U2OS cells led to significantly telomere shortening and partially cell proliferation reduction, suggesting ZSCAN4also compensates for telomere loss in ALT cells similar to that in G4Terc-/-ES cells, and also is an promising target for cancer therapy. Anti-telomerase therapy provokes ALT and other adaptive mechanisms in telomerase positive cancers, limiting its potential therapeutic usage in clinical medicine. Therefore, combination of anti-telomerase and anti-ALT therapies may shed promising light to anti-cancer battle.