Maintaining genomic stability in pluripotent stem cells
Review Article
Ping Zheng Genome Instability & Disease 1, 92–97(2020)
Abstract
Pluripotent stem cells (PSCs) are capable of generating all types of cells in the body and have promising applications in basic research and cell-based regenerative medicine. Compared to the differentiated cells, PSCs are superior in maintaining genomic stability. However, the underlying molecular mechanisms are far from clear. Here, we summarized the understandings on the molecules and pathways that PSCs specifically utilize to cope with DNA replication-associated stress, to repair DNA damages and to determine cell fates.
Maintaining genomic stability in pluripotent stem cells
Pluripotent stem cells (PSCs) are capable of generating all cell types in the body and hold great promise for disease modeling and cell-based regenerative medicine (Rowe and Daley 2019). Pluripotent cells exist in vivo as epiblast cells in the inner cell mass of a blastocyst. Under suitable culture conditions, the pluripotency of epiblast cells can be captured and indefinitely maintained in vitro. These cells are named as embryonic stem cells (ESCs) (Nichols and Smith 2012). Pluripotent stem cells can also be obtained by reprogramming differentiated cells via Yamanaka factors or small molecules (induced pluripotent stem cells, iPSCs) (Takahashi and Yamanaka 2006; Zhang et al. 2012). These in vivo pluripotent epiblast cells and in vitro ESCs or iPSCs possess similar molecular signatures and biological characteristics including pluripotency, fast self-renewal proliferation and unique cell cycle composition (Boroviak et al. 2015; Boheler 2009; Savatier et al. 2002).
Specifically, PSCs have higher capacities than the differentiated cells to preserve genomic stability (Vitale et al. 2017; Oliveira et al. 2014) due to their unique functional purposes. For instance, cultured mouse ESCs display 100-times lower mutation rate than their isogenic mouse embryonic fibroblasts (Tichy and Stambrook 2008). However, it remains largely unknown how PSCs efficiently secure the genomic integrity. Limited studies suggested that PSCs employed specific regulators or pathways to improve the efficiency. Elucidating the involved key players and their functional mechanisms would improve the PSCs’ genome stability, which is crucial for their applications in basic research and regenerative medicine (Tapia and Scholer 2016). Moreover, the PSC-specific strategies can be hijacked by cancer stem cells (CSCs) to initiate the tumorigenesis and to survive the chemotherapy or radiotherapy (Vitale et al. 2017). For example, CSCs have robust DNA damage response, increased DNA damage repair efficiency, and elevated PARP1 activity than the differentiated malignant cells (Lim et al. 2012; Yuan et al. 2014; Venere et al. 2014). Thus, the knowledge obtained from PSCs could help to fight against cancer. Since many excellent papers have reviewed the molecules and pathways that operate in differentiated cells to maintain genomic stability, in this mini-review, we will focus on the unique characteristics and players in ensuring genomic stability of PSCs.
DNA replication-associated stress response in PSCs
Cells face enormous endogenous and exogenous genotoxic insults. The major sources of endogenous genotoxic insults come from the cell metabolism and metabolites. For instance, during DNA replication in eukaryotic cells, replication fork progression can be frequently impeded by physical obstacles including high-order DNA structure, DNA–RNA complex, DNA–protein complex, or DNA damages, leading to fork slowing or stalling. This phenomenon is called DNA replication stress (Zeman and Cimprich 2014). Stalled replication forks are vulnerable to be converted into DNA double-strand breaks (DSBs), which are the most deleterious form of DNA damages in the cells. Other types of genomic instability, including chromosome translocation and nonrecurrent copy number variations (CNVs), can be derived from DSBs (Arlt et al. 2012). Thus, DNA replication stress represents a major endogenous source of DNA damage and genome instability (Techer et al. 2017).
PSCs proliferate fast (around 8–12 h in a cell cycle), and have a long S-phase occupying about 65% of the cell cycle (Savatier et al. 2002). Moreover, PSCs lack G1 cell cycle checkpoint (Desmarais et al. 2012; Hong and Stambrook 2004), which surveillances the genome and prevents cell cycle progression into S phase before the damages are repaired. Due to the frequent DNA replication and, in particular, the lack of G1 checkpoint, PSCs encounter high level of replication stress (Ahuja et al. 2016; Vallabhaneni et al. 2018). Resolving the replication stress with high efficiency is, therefore, extremely critical for PSCs to prevent the endogenous DNA damage and to ensure the completion of DNA replication. Indeed, ours and other’s works showed that compared to differentiated cells, PSCs are superior in protecting nascent DNA from enzymatic degradation, in promoting stalled fork restart, and in initiating dormant origins firing within the active replication cluster to ensure the completion of DNA replication (Zhao et al. 2018; Ge et al. 2015). To understand the molecular basis, we utilized iPOND (isolate proteins on nascent DNA) (Sirbu et al. 2012) combined with mass-spectrum analysis to search for unique players regulating these events. We identified two PSC-specific proteins Filia (official symbol Khdc3, also known as Ecat1) (Zhao et al. 2015) and Floped (official symbol Ooep, also known as Sddr) (Miura et al. 2010) on replication forks. Filia and Floped are abundantly expressed in pluripotent but not differentiated cells. They physically interact with each other and form protein complex. The Filia–Floped protein complexes can reside on replication forks under normal condition and their accumulation on replication forks are robustly enhanced by fork stalling. They form functional scaffolds once the serine 151 residue of Filia is phosphorylated in ATR-dependent manner. The scaffolds promote the stalled fork restart through two independent pathways. On one hand, the scaffolds recruit E3 ubiquitin ligase Trim25 to the stalled replication forks. Trim25 in turn ubiquitinizes the substrate Blm (Bloom syndrome), thereby enhancing its stabilization and retention on damaged replication forks. The helicase Blm plays critical roles in fork reversal and restart (Davies et al. 2007); its amount on stalled replication forks is closely relevant to the fork restart ability (Zhao et al. 2018). Meanwhile, the Filia–Floped scaffolds can stimulate ATR activation in unknown mechanism. Therefore, the ATR-Chk1 signaling is amplified through the positive feedback loop between Filia–Floped scaffolds and ATR. In differentiated cells, the recruitment of the Trim25-Blm to replication forks and the activation of ATR signaling are much less robust due to the lack of Filia–Floped scaffolds. Thus, by adding the scaffold as an additional regulatory layer, PSCs acquire the higher competence to protect the stalled forks from collapse (Zhao et al. 2018). Of note, ectopic expression of Filia–Floped proteins in differentiated cells can promote stalled fork restart, but the efficiency is still lower than that in ESCs depleted of Filia–Floped proteins (Zhao et al. 2018). This implicates that other unknown PSC-specific factors are involved in replication stress response.
During each round of DNA replication, telomeres of the chromosomes face the crisis of attrition due to its specific structure and the end replication problem (Lundblad 2012). Differentiated cells and PSCs utilize distinct mechanisms to maintain telomere length. Telomere in differentiated cells is maintained mainly through telomerase, which adds telomere at a slow pace (50–150 bp per cell cycle) (Pfeiffer and Lingner 2013). Unlike in the differentiated cells, the telomerase plays minor roles in telomere maintenance in PSCs. Instead, PSCs preferentially utilize a recombination-based mechanism to lengthen the telomere (Zalzman et al. 2010). Compared to the telomerase pathway, telomere recombination is more robust and requires the participation of PSC-specific protein Zscan4. Concordantly, mouse ESCs without telomerase activity can proliferate for > 450 population doublings (Niida et al. 2000), whereas depletion of Zscan4 in mouse ESCs causes rapid telomere attrition, genomic instability and culture crisis by passage eight (around 31 population doublings) (Zalzman et al. 2010). Zscan4 is transiently expressed by PSCs and regulates telomere recombination through two distinctive pathways. On one hand, Zscan4 can localize at telomere and regulate the event of telomere sister chromatid exchange (T-SCE) (Zalzman et al. 2010). Alternatively, Zscan4 promotes the DNA demethylation to facilitate telomere recombination and elongation (Dan et al. 2017). Owing to its critical role in maintaining telomere integrity in PSCs, inclusion of Zscan4 during somatic cell reprogramming with Yamanaka factors can not only promote the efficiency of iPSC generation, but also improve the quality of iPSCs (Jiang et al. 2013). Intriguingly, the expression of Zscan4 in cultured PSCs is stimulated by feeder cells (Guo et al. 2018).
DNA damage response and repair in PSCs
Different types of DNA damages require distinct repair pathways. In PSCs, most studies focus on the DNA DSBs, the most deleterious form of damages that threaten the viability of a cell. Upon DSBs, many proteins are recruited to the DSB sites, and the central kinase ATM is activated which then phosphorylates numerous downstream substrates to generate the signaling cascades. In turn, cell cycle is temporarily arrested and the repair proteins are recruited to the damage sites for repair. These integrated cellular reactions are collectively called DNA damage response (DDR) (Jackson and Bartek 2009). Compared to somatic cells, very few studies have been conducted on pluripotent cells regarding their DDR regulations. Limited studies revealed that PSCs displayed many unique properties in DNA damage response and repair (Vitale et al. 2017; Wyles et al. 2014). For example, the DDR is more robust in PSCs than in differentiated cells. PSCs preferentially utilize the high fidelity homologue recombination (HR)-mediated pathway to repair DSBs (Tichy et al. 2010), whereas differentiated cells prominently use error-prone non-homologous end joining (NHEJ) pathway. In addition, ESCs do not have the G1/S cell cycle checkpoint (van der Laan et al. 2013). Instead, intra-S and G2 cell cycle checkpoints play critical functions in ESCs (Momcilovic et al. 2011). The molecular mechanisms underlying these unique characteristics are far from clear. Recently, two PSC-specific proteins have been identified to play unique roles in DDR of PSCs. The first protein is Sall4, which is required for maintaining the stemness of ESCs (Lim et al. 2008; Zhou et al. 2007). Upon DNA DSBs, Sall4 is recruited to DSB sites by interacting with Baf61a, one of the components of chromatin remodeling SWI/SNF complexes which promote the opening of chromatin to facilitate the recruitment of repair proteins to damage sites. At DSB sites, Sall4 interacts with Rad50 and stabilizes the Mre11–Rad50–Nbs1 (MRN) complex, which is essential for the downstream ATM activation (Xiong et al. 2015). Therefore, by expressing Sall4, PSCs can activate ATM signaling in higher efficiency than differentiated cells in response to DNA damages. The second protein is Filia, which is also enriched in undifferentiated PSCs but is not required for the expression of pluripotent markers. Filia promotes the DNA damage response and repair by several distinct pathways. Filia can interact with PARP1 and stimulate PARP1 activation, thereby promoting efficient DNA damage response (Zhao et al. 2015). Filia can also translocate to the DNA DSB sites to facilitate the HR-mediated DNA damage repair (Zhao et al. 2015), but the underlying mechanisms remain elusive. Filia itself participates in the regulation of cell cycle arrest independent of ATM in DDR (Zhao et al. 2015). These roles of Filia in DDR are conserved between mouse and human. In human PSCs, the ortholog KHDC3L binds to PARP1 and stimulates PARP1 activation. It also relocates to DSB sites and promotes the HR repair. Specifically, KHDC3L is a substrate of ATM which phosphorylates two threonine residues to modulate KHDC3L’s functions (unpublished data). Thus, by expressing Filia/KHDC3L, PSCs can have robust DNA damage response and enhanced HR repair.
Cell fate determination after DNA damages in PSCs
In eukaryotic cells, when the extent of DNA damage is beyond repairable, cells undergo apoptosis or senescence to prevent the passage of the mutations to descendent cells. To protect the organism from propagating harmful mutations at the earliest stages of embryonic development, PSCs are exceptionally sensitive to DNA damage and quickly undergo apoptosis rather than attempt to repair a compromised genome in response to stress (Liu et al. 2013). This sensitivity is ensured by several unique mechanisms operated in PSCs. First, compared to differentiated cells, the mitochondria in PSCs are at a high priming state and have a lowered cell-intrinsic threshold for initiating apoptosis (Liu et al. 2013). Mitochondria priming is also called readiness for apoptosis, and is determined by the balance of pro- and anti-apoptotic proteins on the mitochondria membrane (Certo et al. 2006; Vo et al. 2012). PSCs express lower levels of the anti-apoptotic protein Bcl-2 and higher levels of the proapoptotic protein PUMA, which favors the balance shifting toward the proapoptotic end and sensitizes PSCs to apoptosis (Liu et al. 2013). PSCs also utilize alternative way to rapidly initiate apoptosis in response to DNA damage. Pro-apoptotic factor Bax, which makes the life-death decision upon threatening stress, is differentially regulated in differentiated cells and PSCs. In differentiated cells, Bax remains inactive and resides in the cytosol under normal condition. Its activation and translocation to mitochondrial outer membrane must be triggered by the stress stimuli. Intriguingly, Bax remains constitutively active and localizes on the trans Golgi networks in PSCs. Upon DNA damage, active Bax rapidly translocates to the mitochondria in a p53-dependent fashion, thereby yielding a rapid apoptotic response (Dumitru et al. 2012). Except of apoptosis, PSCs can also initiate differentiation to eliminate the damaged cell populations to prevent the spread of mutations (Lin et al. 2005).
One of the factors regulating the cell fate decision is the tumor suppressor protein p53. p53 displays distinct roles in PSCs compared to differentiated cells (Zhao and Xu 2010). It acts as a transcription factor to directly activate and repress gene expressions. A global view of p53 signaling in mouse ESCs revealed that p53 directly activated and suppressed more than 3600 genes (about 55% are activated and 45% are repressed) in response to DNA damage. p53-activated genes and p53-repressed genes are functionally opposite. Many p53-activated genes are associated with differentiation, while most of the PSC core transcription factors (e.g. Oct4, Nanog, Sox2, Zic3, Jmjd1c, Esrrb, Tcfcp2l1, Utf1, n-Myc) are suppressed by p53 (Li et al. 2012). Thus, through the dual functions of p53 on activation of differentiation genes and repression of PSC core regulatory factors, PSCs can undergo fast differentiation in response to DNA damage.
Depletion of p53 does not completely prevent the cell death or differentiation of stem cells, suggesting that other molecules and pathways operate in parallel to determine the cell fate. Our previous work reported that PSC-specific protein Filia regulated the cell fate determination (Zhao et al. 2015). Filia is necessary for PSCs to undergo rapid apoptosis and differentiation upon DNA damage. However, it is not known whether these functions are mediated by p53 or independent of p53. Moreover, Filia can translocate to mitochondria upon DNA damage and this subcellular localization is essential for Filia’s function on apoptosis. It is interesting to understand whether Filia’s role on mitochondria is relevant to mitochondria priming. Answers to these questions would shed more lights on how stem cells utilize different ways to make the life-death decision.
Glucose metabolism in PSCs
Reactive oxygen species (ROS) is the by-product of ATP generation through oxidative phosphorylation, and is detrimental to DNA integrity by causing oxidative lesions (Halliwell 2007). Compared to differentiated cells, PSCs have lower level of ROS and this is achieved by the preferential use of anaerobic glycolysis instead of oxidative phosphorylation. PSCs contain less active mitochondria, which are immature, in a globular shape, have poorly developed cristae and peri-nuclear distribution (Prigione et al. 2010; Ramalho-Santos et al. 2009). The mechanisms that regulate the favor of glycolysis are unclear, but studies hypothesized several basis. For instance, PSCs specifically and abundantly express glucose transporter 3 (Glut3), which has high affinity to glucose and enables the efficient uptake of glucose (Zhang et al. 2017). Glycolysis is very inefficient to generate ATP from glucose; thus, a large amount of glucose is needed to produce necessary ATP through glycolysis. Low expression of Glut3 leads to the inefficient uptake of glucose, which in turn favors the glucose metabolism via oxidative phosphorylation. The expression of Glut3 in PSCs is regulated by PSC-specific transcription factor Zscan10 (Zhang et al. 2017). In addition, PSCs have higher protein levels of Hexokinase II and Pyruvate Dehydrogenase kinases (Varum et al. 2011). Hexokinase II catalyzes the first reaction of glycolysis and is very important for glycolysis. Pyruvate Dehydrogenase kinases phosphorylate Pyruvate Dehydrogenase to inactivate the enzyme complex, thereby reducing the availability of substrate acetyl-CoA, which fuels the TCA cycle and oxidative phosphorylation. Interestingly, hypoxia can induce the expression of Pyruvate Dehydrogenase kinase 1 by HIF-1 transcription factor. Preference of glycolysis in PSCs is also favored by the higher expression of UCP2 (Zhang et al. 2011), which prevents pyruvate from entering into TCA cycle (Zhang et al. 2011).
Although ROS is detrimental to genomic stability, certain level of ROS is also essential to signal cellular stress and to induce DNA damage response (Skamagki et al. 2017). ROS can directly oxidize ATM to activate this kinase and elicit ATM-mediated DNA damage response (Guo et al. 2010). Thus, correct level of ROS is required in PSCs to maintain genomic stability. ROS level is counteracted by antioxidant glutathione, the homeostasis between ROS and glutathione is critical to maintain correct level of ROS. To maintain the homeostasis between ROS and glutathione, PSCs express specific transcription factor Zscan10, which binds to the promoter of glutathione synthetase (GSS) and suppresses its transcription. Inadequate induction of Zscan10 in iPSCs will result in the excessive expression of GSS and the disruption of homeostasis between ROS and glutathione, leading to the genomic instability (Skamagki et al. 2017).
Perspective
Although it has been recognized that PSCs employ unique strategies and mechanisms to efficiently maintain their genomic stability, very limited PSC-specific regulators were discovered so far. In addition to proteins, other molecules including non-coding RNAs (e.g., microRNAs, and long non-coding RNAs) are also involved in regulating genomic stability in differentiated cells (Khanduja et al. 2016). However, PSC-specific non-coding RNAs have not been identified. In the future, more efforts are required to expand the list of PSC-specific factors critical for maintaining genomic stability and to understand the regulatory networks. The knowledge would help us to understand the source of genomic instability in iPSCs to improve the protocol to generate safer iPSCs. In addition, pluripotency and high level of genome stability are two critical properties of PSCs. How the two characteristics are coordinated remains largely unknown. Studying the crosstalks between pluripotency and genomic stability would help to better understand PSCs as a whole.
PSCs exhibit distinct pluripotent states, namely the naïve and primed pluripotent states. These two pluripotent states differ in many cellular and molecular aspects (Weinberger et al. 2016; Theunissen et al. 2016). For instance, they display differential chimeric and differentiation potentials, core pluripotency regulatory circuitry, specific markers, transposon element expression profiles, X chromosome activation state in female cells, and the epigenetic and metabolic profiles. Human ESCs that are commonly used for investigation are at primed pluripotent state (conventional human PSCs). Naïve pluripotent human PSCs were established in recent years. However, compared to the conventional human PSCs, cultured naïve human PSCs suffer from severe genomic instability. It would be interesting to understand if PSCs at different pluripotent states have distinct properties and regulations on maintaining genomic stability. In our previous study aimed to understand the differences between the naïve and primed pluripotent states in monkey in vivo epiblast cells, we found that the genomic stability regulators ZSCAN10, KHDC3L, and OOEP were relatively highly expressed in primed pluripotent cells (Liu et al. 2018). This observation suggested that human naïve and primed PSCs might exhibit distinct characters and regulations on genomic stability.
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State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China
Ping Zheng
Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650223, Yunnan, China
Ping Zheng
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Correspondence to Ping Zheng.
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Zheng, P. Maintaining genomic stability in pluripotent stem cells. GENOME INSTAB. DIS. 1, 92–97 (2020). https://doi.org/10.1007/s42764-019-00008-4
Received05 March 2019
Revised08 October 2019
Accepted07 November 2019
Published20 November 2019
Issue DateMarch 2020
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Pluripotent stem cells
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