Eternally young cells and zinc fingers: details
Scientists investigated the role of the Zscan4 gene clusterAlexey Skobkin, LibyMax
Telomere elongation and genomic stability of embryonic stem cells are regulated by the Zscan4 protein.
This was proved by scientists from the National Institute on Aging in Baltimore, Maryland, USA.
As previously known, mouse embryonic stem cells (ESCs) originate from the internal cell mass (ECM) of the blastocyst and are characterized by a gene expression pattern similar to ECM cells. One of the distinguishing features of mouse ESCs is the exceptional stability of the genome. It was noted that the frequency of mutations in ESCs is more than 100 times lower than in mouse embryonic fibroblasts and other somatic cells [1]. Mouse ESCs also have a significantly reduced frequency of chromosomal abnormalities compared to cells with similar characteristics, such as embryonic carcinoma cells and even some lines of human ESCs [2]. However, the mechanism is still unknown, and it is not clear which specific genes are responsible for maintaining such genomic stability.
Other distinctive features of ESCs are pluripotency and the ability to self-renew. Both of these properties have been the focus of intensive research for many years [3]. Finally, another important feature of ESC is the ignoring of all signals of differentiation and the ability to pass more than 250 cell cycles without undergoing transformation [4].
In 2007, a new cluster of Zscan4 genes was described, which was characterized as a potential regulator of genome stability at the level of a two-cell embryo and ESC [5]. The name of the genes suggests that the structure of their proteins includes the domains "zinc finger" (zinc finger) and SCAN. The Zscan4 cluster consists of six transcribed paralog genes with high primary sequence similarity (Zscan4a-f), collectively called Zscan4. Stable expression of Zscan4 is a marker of the late two-cell mouse embryo stage and is necessary for embryo implantation into the uterus in vivo and blastocyst formation in tissue culture. The Zscan4d gene is transcribed mainly at the stage of the two-cell embryo, while the Zscan4c protein is found in the ESC and is associated with self-renewal processes.
In an original paper published in the journal Nature, a team of authors led by Sung-Lim Lee and the discoverer of Zscan4, Geppino Falco (National Institute of Aging, Baltimore), investigated the role of the Zscan4 gene cluster and showed that their expression is necessary for the long-term cultivation of ESCs and maintenance karyotype integrity associated with regulated telomere recombination in normal undifferentiated ESCs.
(We recently reported on this work, not in so much detail, but it is much clearer for non–specialists - VM.)
The main conclusions from the presented study:
- Zscan4 Knockout Leads to Cultivation Crisis;
- The Zscan4 protein is involved in the telomere elongation process in ESCs;
- Zscan4 inhibits spontaneous exchange of sister chromatids (OSX);
- The Zscan4 protein is localized in the telomere region.
Fig. 1. Model describing the action of Zscan4 genes in ES cells
In a single period of time, Zscan4 is expressed in about 5% of ESCs and characterizes an unstable state of pluripotency peculiar to ESCs, while other genes heterogeneously expressed in ESCs often mark specific cell lines of differentiation. Moreover, almost all ESC during long-term cultivation express Zscan4 at least once in nine passages. The authors note that further research is needed to determine the signals leading to the activation of Zscan4. Nevertheless, it is noted that the unique nature of Zscan4 expression implies the action of a clear regulatory mechanism. In favor of the existence of a complex regulatory mechanism, observations indicate that the knockdown of Zscan4 does not affect cells immediately, but over time leads to karyotype disorders, a decrease in the rate of proliferation and causes apoptosis by the 7-8 passage. Thus, without the abrupt activation of Zscan4, ESC lose their most important ability – unlimited proliferation.
The researchers note that, apparently, karyotype disorders in ESCs with suppressed Zscan4 expression are associated with telomere shortening and simultaneous increase in spontaneous OCC not associated with telomeres. Zscan4 acts as an activator of spontaneous telomeric exchange of sister chromatids (T-OCC) [6], which leads to telomere elongation in undifferentiated ESC. Thus, a new model of telomere length regulation in mouse ESCs involving Zscan4 is described, which differs from the previously described mechanisms. The main characteristics of the presented new model are:
- Zscan4+ cells undergo rapid telomere elongation, most likely due to telomere recombination or T-OSX. Apparently, the main function of the Zscan4 protein is to induce the expression of mediators of meiotic homologous recombination with the formation of a configuration interaction of these molecules with telomere sites;
- The researchers demonstrated that telomerase is active in undifferentiated ESCs expressing Zscan4. This pattern is not observed in cells in which T-OCC usually occurs, such as ESCs that survive telomerase Terc-/-knockout [7], and tumor cells with unrecoverable or insufficient telomerase activity [8]. Zscan4-mediated telomere elongation does not require telomerase, however, it remains unclear to what extent Zscan4-mediated telomere elongation is able to compensate for the loss of telomeres in Terc-/-ESCs, which stop proliferating after 450 doublings [7];
- Most of the genes previously identified as telomere length regulators, such as DNA methyltransferases DNMT1 and DNMT3 [9], as well as the Werner syndrome gene WRN [10], are inhibitors of T-OSX. Suppression of the expression of these genes leads to an increase in the frequency of T-OCC and/or telomere length [11]. The exception is the Rtel1 gene. In Rtel1-/-ESC, telomere shortening occurs after differentiation induction [12]. Zscan4 demonstrates a similar phenotype on undifferentiated ESC;
- in contrast to the usual T-OCC, which occurs as a result of general chromosomal instability and is accompanied by an increased frequency of OCC of non-telomeric sequences, Zscan4-mediated T-OCC is not associated with an increase in total OCC, and the normal karyotype remains stable against the background of a low level of spontaneous OCC.
Fig. 2. Confocal microscopy of ESCs at the stage of mitosis demonstrates that:
a) the Zscan4 protein (red, immuno-staining) is localized at the ends of chromosomes (blue, DAPI staining) and is associated with telomeres (green, T-FISH using a DNA sample conjugated with Alexa488);
b) the marker of homologous recombination SPO11 (red, immuno-staining) is localized on telomeres 3 days after induction of Zscan4c.
In conclusion, it can be noted that the expression of Zscan4 varies among different types of pluripotent cells, which may correlate with their differences in genome stability [13]. By selecting cells capable of activating Zscan4, it is quite possible to advance towards obtaining cell cultures suitable for therapeutic use. Moreover, the ability to control the expression of Zscan4 can increase the genome stability of other cell types, such as stem cells or tumor cells.
Based on the materials: www.grc.nia.nih.gov , Zalzman M., Falco G., Sharova L.V. et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature 2010; advance online publication: doi: 10.1038/nature08882, as well as NIAZH Cell Transplantology and tissue Engineering – www.celltranspl.ru .
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Portal "Eternal youth" http://vechnayamolodost.ru17.05.2010