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Genome imprinting in stem cells: A mini-review
Gene Expression Patterns 34 (2019) 119063
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Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Genome imprinting in stem cells: A mini-review a
b
Rasoul Godini , Keyvan Karami , Hossein Fallahi
T
c,*
a
Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, 3800, Australia Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran c Department of Biology, School of Sciences, Razi University, Kermanshah, Iran b
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Keywords: Genomic imprinting Embryonic stem cell iPS cells Mono-allelic expression Precision medicine
Genomic imprinting is an epigenetic process result in silencing of one of the two alleles (maternal or paternal) based on the parent of origin. Dysregulation of imprinted genes results in detectable developmental and differential abnormalities. Epigenetics erasure is required for resetting the cell identity to a ground state during the production of induced pluripotent stem (iPS) cells from somatic cells. There are some contradictory reports regarding the status of the imprinting marks in the genome of iPS cells. Additionally, many studies highlighted the existence of subtle differences in the imprinting loci between different types of iPS cells and embryonic stem (ES) cells. These observations could ultimately undermine the use of patient-derived iPS cells for regenerative medicine.
1. Introduction In diploid organisms, two copies of each gene are inherited from each parent. For most of the loci, both alleles are active and equally expressed. However, only one allele in the imprinting loci is active, either maternal or paternal allele (Butler, 2009). Imprinting is an epigenetic event, causing silencing in one of the alleles inherited from one of the parents. Therefore, if the paternal allele of the gene is imprinted, the other allele from mother would be expressed and vice versa (Fig. 1). Imprinting generally violates Mendelian inheritance and affect the segregation pattern of imprinting alleles (Úbeda, 2008). In human, genomic imprinting determines the fate of a few genes, which are crucial for the correct development (Lawson et al., 2013). A majority of imprinted genes play main roles in the control of embryonic and placental growth and development. Expectedly, dysregulation of the imprinted genes has been associated with abnormal growth and development. For example, abnormal imprinting in the DIK1-Dio3 locus results in developmental syndromes and specific diseases (Swanzey and Stadtfeld, 2016). Besides, Angelman syndrome and Prader-Willi syndrome are the typical known human diseases linked to abnormal genomic imprinting. The exact mechanisms by which some genes undergo imprinting is not fully understood (Babak et al., 2015), but the role of epigenetics in the imprinting is evident. The imprinting process involves known epigenetic modifications of the genome including DNA methylation (Li
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et al., 1993; Stelzer et al., 2016), histone modifications (Xin et al., 2001) and non-coding RNAs (Sleutels et al., 2002). Differentially Methylated Regions (DMRs) are essential for parental imprinting of several genes. Imprinted genes are located in clusters (in approximate 80% of imprinted genes in mammals) and in the CpG rich regions of the genomes, called Imprinting Control Region (ICR) (Barlow, 2011). The genes in clusters are coordinately regulated through ICR. Other characteristics of imprinted loci include having fewer and smaller introns than non-imprinted genes, containing repetitive sequences and a high number of retro transposable elements (McVean and Moore, 1996; Ono et al., 2001). In diploid animals, it has been suggested that the presence of both paternal and maternal genomes is required for normal growth. Kono and colleagues showed that parthenogenesis, a natural form of asexual reproduction, would be affected in presence of two copies of mice female genomes, confirming the need for both paternal and maternal chromosomes for proper development (Kono et al., 2004). This could be an evidence for imprinting in several loci. Using uniparental diploid models, the map of these imprinted genes in the mouse has been created (Williamson et al., 2013). Many types of genomic imprinting have been documented in different organisms including animals, fungi, plants (Martienssen and Colot, 2001; Feil and Berger, 2007). It appears that, there an almost equal number of maternally imprinted genes as paternally imprinted genes. In mammalian, it was first described by Lyon and Glenister by results obtained from the reciprocal
Corresponding author. E-mail addresses: [email protected] (R. Godini), [email protected] (K. Karami), [email protected] (H. Fallahi).
https://doi.org/10.1016/j.gep.2019.119063 Received 18 February 2019; Received in revised form 21 May 2019; Accepted 30 June 2019 Available online 04 July 2019 1567-133X/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. General model for imprinting. For non-imprinted genes, expression occurs at both alleles. However, depending on the imprinting status the gene expression at the imprinted locus occurs either at the maternal or paternal allele. If the paternal allele of the gene is imprinted, the other allele from mother would be expressed and vice versa.
model, which is also involved in gene expression of many other nonimprinted genes, methylation or any other modifications at the promoter region prevent assemblies of the transcription machinery and the target gene fails to express. As a result, differential methylation state could be detected for the promoter regions of the imprinted genes on the maternal and paternal chromosomes (Cui et al., 2002; Yagi et al., 2017). In the second model, two or more genes are competing over a single enhancer or a set of common enhancers (Fig. 2B). Activation or silencing of a given gene depends on the activity of the enhancer activity. Such a scenario is observed for the known reciprocals imprinted genes IGF2 and H19 (Lyko et al., 1997). IGF2 is expressed from the paternal allele and H19 is expressed from the maternal allele. The ICR in the maternal allele is not methylated. Therefore, an enhancer blocking factor (CTCF) binds to the ICR and prevent IGF2 promoter activation by a downstream enhancer. In the paternal allele, ICR is methylated and prevent CTCF binding, results in the availability of enhancer for transcription of IGF2 (Ishihara et al., 2006). Thus, in this case, the DNA methylation regulates CTCF-insulation activity. Chromatin structure could affect gene expression via isolating a gene from the regulatory the enhancers (Fig. 2C). Silencer elements are present in the H19 ICR sequence (Lyko et al., 1997). Maternal H19 and Igf2 in mice would be repressed by binding of PEG3, a paternal DNAbinding protein, to the silencer sequences (Ye et al., 2016). Non-coding RNAs regulate expression of protein-coding genes at both transcriptional and post-transcriptional levels. These molecules could also silence a gene by interfering with the gene promoter or directly binding to the mRNA (Kornienko et al., 2013). The transcriptional interference mechanism is involved in the expression of IGF2R and Air (Fig. 2D). Here, differential methylation between paternal and maternal chromosomes determine which gene gets expressed. These non-coding RNAs often transcribed in opposite direction to the proteincoding sequences in one of the imprinted genes in a cluster. For example, the receptor for IGF2 (IGF2R) is transcribed exclusively from the maternal allele due to the repression of paternal transcription by the non-coding Air transcript in the opposite direction to the imprinted IGF2R gene (Kohama et al., 2011).
chromosomal translocation in mouse (Lyon and Glenister, 1977). In human, the studies concerning the effects of imprinting on different aspects of gene regulation are lagging behind, due to the absence of a suitable model. While different studies put the number of imprinting genes in the human genome between 100 and 2000, only a few have been experimentally validated (Daelemans et al., 2010). For example, one of the most studied imprinted genes in human is insulin-like growth factor 2 (IGF2) (a maternally imprinted gene), which is expressed only from the paternal allele (Giannoukakis et al., 1993). Interestingly, imprinting processed seems to be dynamic; some of the imprinted genes remain imprinted constantly, while others exhibit tissue-specific and temporal or time specific pattern of expression. As noted, the imprinted genes could affect the development of mammalian organisms. Imprinting poses serious concerns regarding the use of stem cells in regenerative medicine (Passier and Mummery, 2003). Stem cells are classified into two main classes; ES and iPS cells. Genome imprinting affects all aspects of stem cells from establishment to differentiation. Accordingly, here we have collected recently available information on the genome imprinting in stem cells and highlighted the need for assessing genome imprinting during iPS cells study and applications. 2. Regulation of gene expression through genome imprinting Genomic imprinting is a cis-acting event, affecting that expression of an allele located on only one of the two chromosomes based on a specific pattern (Barlow and Bartolomei, 2014). Although genomic imprinting works through repression of gene expression, it is not necessary a silencing process. Accordingly, any regulatory elements including promoters (Vu and Hoffman, 1994), enhancers (Webber et al., 1998), splicing junctions (Croteau et al., 2003) or polyadenylation sequences could be targeted by imprinting machinery (Wood et al., 2008). Although several different mechanisms for the impacts of imprinting on the gene expression have been proposed in different studies, the most common scheme is presented in Fig. 2. Repression of gene expression via direct methylation of the promoter region is a widespread mechanism of imprinting (Fig. 2A). In this 2
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Fig. 2. At least four models for the imprinting mechanism have been proposed. A) direct methylation/demethylation of the regulatory regions for many imprinted genes. B) Inhibition of gene expression with a competition between methylation and binding activity of a protein (CTCF, CCCTC-binding factor) to the ICR (Imprinting Control Region) sequence. Binding of CTCF prevents methylation and allow expression of the gene 2 and the downstream enhancer would be occupied. This blocks the enhancer activity on gene 1. In contrast, methylation on the ICR region prevent expression from gene 2 and the enhancer is free to activate the gene 1. C) Direct repression by PEG3, a newly identified transcriptional repressor. PEG3 occupies the binding site of the CTCF in the ICR, it would prevent gene expression either with a mechanism similar to part b or a new mechanism shown here. D) The role of non-coding RNAs in the imprinting mechanism; expression from a gene in opposite direction produces an antisense transcript that in turns silences other genes in the cluster. While, methylation of the promoter region of the ncRNA gene would prevent suppression, allowing expression of the imprinted genes from the counterpart locus.
3. The status of imprinted genes in somatic cells
hES cells, variable expression for the imprinted loci has been observed (Park et al., 2014), which might be due to disrupted DNA methylation pattern at these loci (Kim et al., 2007). Indeed, the imprinted gene expression patterns vary between mice cloned by nuclear transfer and the ES cell as a result of instability in the epigenetic marks at the imprinted loci (Humpherys et al., 2001). Besides, cloned mice derived from ES cells of the same subclone showed significant differences in the imprinted gene expression (Humpherys et al., 2001). However, some studies suggest that epigenetic instability is not a common phenomenon and is apparently a cell-line dependent event (Rugg-Gunn et al., 2007). Moreover, in some stem cells, the mono-allelic expression has been detected even in the absence of DNA methylation (Umlauf et al., 2004). These conflicting reports might indicate the existence of some other mechanisms rather than a generally accepted DNA methylation model for maintaining imprinting loci. Some explanatory models proposed for the widespread loss of imprinting (LOI) in hES cells (Rugg-Gunn et al., 2007). First, global reshuffling of the transcription programs could result in differential gene expression of many genes including imprinted genes. Second, differences in expression show differential methylation patterns in the maternal and paternal genome. Third, differential genes expression could be due to the growth advantage that the affected gene provide to hES cells. A similar mechanism could explain the variation observed in the imprinted gene expression in the iPS cells, where growth advantages count most in the cell culture. Notably, the extent of epigenetic stability of hES cells could be experimentally examined by measuring the expression level of the imprinted genes. While some imprinted genes such as Snrpn, Ipw, and Kcnq1ot1 shown to be highly stable in the hES cells, others including H19, Igf2 and Meg3 show significant variations in the degree of imprinting from cells to cells. Such measures could indicate the general status of epigenetic modifications in the ES cells (Rugg-Gunn et al., 2007).
In somatic cells, the allele-specific expression has been described for many of the imprinted genes in a tissue-depended pattern (Kohama et al., 2011). In the brain, for example, an interesting pattern of imprinting has been detected. Maternal gene expression occurs in the developing brain, while mostly paternal gene expression found in the adult brain (Gregg et al., 2010). The crucial role of epigenetic modifications, specifically genome imprinting, is the subject of many studies during gametogenesis, pre- and post-fertilization and early development of embryos. Thanks to these studies acceptable level of success achieved in the assisted reproduction technology (ART) (Fraser and Lin, 2016). It is known that global epigenetic remodeling in the primordial germ cell (PGC) is required to establish an epigenetic ground state. Therefore, incomplete epigenetic remodeling might be one of the reasons for inefficiencies in overall germ cells production, inefficient meiotic progression and the incomplete imprinting events observed for in vitro mediated differentiation (Durruthy et al., 2014). Hypo-methylated in the ICRs of imprinted genes such as SNRPN, H19/IGF2, and IGF2R has been observed at early stages of development in the embryos derived by somatic cell nuclear transfer (SCNT) and IVF techniques. Therefore, epigenetic erasure might occur at the imprinted loci during in vitro reprogramming and culture of oocytes or embryos (Smith et al., 2015). However, epigenetic anomalies appear to be less evident at the imprinted ICRs later during development; suggesting either proper re-methylation or survival of less affected embryos to the later stages of development (Smith et al., 2015). In somatic cells, the imprinted gene H19, its new antisense transcript 91H, and its miRNA are involved in the regulation the function of IGF receptor and in cell cycle progression (Vennin et al., 2013). Furthermore, it has been shown that Peg10 and Peg 4 imprinted genes are involved in PGC reprogramming and their epigenetic status linked to Tet1 function (Yamaguchi et al., 2013). Therefore, it has been suggested that imprinting has a vital role in somatic cells both at regulation and maintenance levels. Besides, an imprinted gene network (IGN) has been detected in adult somatic stem cells when compared to their differentiated progeny (Berg et al., 2011), suggesting the role of imprinting in general cell growth and tissue homeostasis.
5. The status of imprinted genes in the iPS cells Currently, the impacts of the reprogramming procedures on the status of epigenetic marks, including imprinted genes, have not been fully understood in the iPS cells. Additionally, conflicting reports make it difficult to determine if cell type of origin would affect the dynamics of epigenetic and imprinting reprogramming. However, it has been suggested that the self-renewal of the stem cells derived from different types of adult tissues depends on the status of imprinted genes (Zacharek et al., 2011). On the other hand, imprinted genes play an important role during development and involved in some types of diseases. It is, therefore, crucial to uncover the consequences of cellular reprogramming on these loci in the iPS cells (Tomizawa and Sasaki, 2012). Similar imprinting pattern exists between iPS cells and their somatic cell of origin (Rouhani et al., 2014). Accordingly, some studies suggest that loss of imprinting during reprogramming is mediated by TETs at the H19 locus, similar to ES cells, and depends on the epigenetic marks of the precursor cells (Bermejo-Álvarez et al., 2015). Surprisingly, imprinting errors are observed in iPS clones, indicating the presence of epigenetic anomalies that are related to the reprogramming process.
4. The status of imprinted genes in ES cells The status of imprinting has not been fully established in the ES cells because of these cells affected by global epigenetic remodeling during embryonic developmental stages (Rugg-Gunn et al., 2007). Apparently, maintaining self-renewal properties of adult stem cells require epigenetic modification in the imprinted genes (Kelsey, 2011). Additionally, knockdown of some imprinting genes related long non-coding RNAs affects the stem cell function and self-renewal (Luo et al., 2015), where stem cell differentiation is shown to be under direct control of the noncoding RNAs (Luo et al., 2015). Furthermore, a direct role of maternally-imprinted locus Dlk1-Gtl2 is well documented in the self-renewal of hematopoietic stem cell (HSC) (Qian et al., 2016). On the other hand, several defects have been observed in the genomic imprinting loci in the mouse and human embryonic stem (hES) cells (John and Helen, 2008). Unexpectedly, during differentiation of 4
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Fig. 3. The impacts of imprinting on different aspects of stem cell biology as indicated in the literature. Embryonic stem cells (A), somatic cells (B) and iPS cells (C) were compared. Self-renewal, cell growth, differentiation, homeostasis, and development are affected by imprinting status in ESCs, iPS and their somatic derivatives. The most updated reference for each step is given in the brackets.
(He et al., 2014). Of note, in mouse, the expression of Dlk1-Dio3 cluster is shown to be altered in iPS cells (Stadtfeld et al., 2010). Surprisingly, upon reprogramming, culture conditions could affect the epigenetic patterns, imprinting and the properties of the resulting iPS cells. For example, ascorbic acid inhibits the removal of Dlk1-Dio3 imprinting pattern and enhances the generation of iPS cells from B cells (Stadtfeld et al., 2012). Hence, genomic imprinting is known to variably lose during reprogramming of mouse iPS cells (Takikawa et al., 2013). In human induced Pluripotent Stem (hiPS) cells, dysregulation of NNAT, a key imprinted gene, has been observed (Teichroeb et al.,
These epigenetic abnormalities could be responsible for the variable phenotypes and low success rates of iPS differentiation and cloning (Bressan et al., 2014). Furthermore, in mouse primordial germ cells and their cell culture models, resistance to global genome DNA demethylation have been identified, which are linked to imprinting regions (Miyoshi et al., 2016). Despite many similarities between iPS cells and ES cells, there exist significant differences in overall methylation pattern between these two cell types. Clearly, iPS cells are shown to partly retain the methylation pattern of the original somatic cells. In addition, these cells gain unexpected de novo methylation upon or during cellular reprogramming
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cases have been observed during neural lineage differentiation from human somatic iPSCs (Lee et al., 2019). The imprinting genes expression status could also be used as a benchmark for identifications of those cells that would have full developmental potential (Dirks et al., 2019). Some strategies have been suggested for detection of correct imprinting states in hiPSCs (Perrera and Martello, 2019). First and the most applicable approach would be measuring and comparing the expression of a set of imprinted genes in the parental and progeny cell lines to assess the stability of the imprinting processes. This could be easily achieved via quantitative RT-PCR or RNA-sequencing. The difficult parts might be compiling a list of imprinting genes to be used as benchmark for each types of cell lines and tissues. Second types of assay could be detection of mono or biallelic expression for the imprinted genes via SNPs identification (Santoni et al., 2017). This approach relies on RNA-sequencing technology and therefore has some limitations when one produces numerous iPSCs in a single experiment. Additionally, stem cells, derivatives should be also assessed for correct imprinting gene expression prior to therapeutic uses. Therefore, monitoring the status of imprinting should be included in the experimental design before generating iPSCs, during differentiation into other types of cells and in the terminally differentiated progenies before their inclusion in the clinical studies.
2011). Furthermore, in female hiPS cells, many genes located on the Xchromosome show unusual over-expression. These observations suggest that despite a remarkable overall similarity in the transcriptome of the hES cells, even isogenic hiPS cells contain altered gene expression of the imprinted and X-chromosome linked genes (Teichroeb et al., 2011). 6. Knowledge of imprinted genes status is important for successful regenerative medicine Although the epigenetic pattern of freshly developed hiPS cells appears to be different from those of hES cells (Nishino et al., 2011), these differences decline after multiple passages of hiPS cells (Hiura et al., 2013; Pick et al., 2009). In contrast, existing abnormal imprinting would not be removed even after prolonged passaging (Hiura et al., 2013). These observations in addition to the variable expression of imprinted genes among different types of iPS cells suggest that each cell line should be individually assessed before use for regenerative medicine (Pick et al., 2009). Abnormal expression of the imprinted genes would change the dosage of gene products in the stem cells. Consequently, the differentiation and proliferation capacity of the stem cells would be affected. The exact impact of such modification on the fate of the stem cells is unknown. However, a tendency toward specific tissues has been observed, following overexpression of some imprinted genes in ES cells (Prelle et al., 2000). Additionally, as the biallelic expression of several imprinted genes including MEST (Pedersen et al., 1999), MEG3 (Zhang et al., 2003), H19 and IGF2 (Cui et al., 2002), correlate with different types of cancers. These unexpected results raise concerns regarding the use of such cells in cell therapy, where aberrant expression might exist for one or more of these genes (Choi et al., 2017). Surprisingly though, alteration of imprinting status at the Dlk1-Dio3 cluster in ST5 and ST8 iPS cell lines did not result in any considerable changes in the differentiation capacity of these lines to the early hepatic lineage in vitro. However, after further culture to day 19, a statistically significant functional impairment was observed in glycogen storage capacity (Christodoulou et al., 2011). Another example is the expression of the ZFP57, a master regulator in genomic imprinting in the iPS cells derived from MEF cells (Mackay et al., 2008). Similarly, ZFP57 is highly expressed in the undifferentiated ES cells as well (Zuo et al., 2012), albite variation of gene expression from this site in iPS cells might be different.
Abbreviations ART DMRs ES CTCF HSC hES hiPS IGN ICR iPS IGF2 LOI PGC IGF2R SCNT
Assisted reproduction technology Differentially Methylated Regions Embryonic stem Enhancer Blocking Factor Hematopoietic stem cell Human Embryonic Stem Human induced Pluripotent Stem Imprinted gene network Imprinting Control Region Induced pluripotent stem Insulin-like growth factor 2 Loss of imprinting Primordial germ cell Receptor for IGF2 Somatic cell nuclear transfer
Appendix A. Supplementary data 7. Future perspective Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gep.2019.119063.
The extent of imprinting loss or gain remains to be fully explored during of reprogramming of somatic cells to iPS cells. What we understand so far is that although imprinting errors may be rare in the iPS cells, they appear to be resistant to reversal epigenetic modifications. Therefore, persistent alteration of imprinting genes expression in the iPS cells is likely to undermine their use in regenerative medicine (Fig. 3). Many concerns raised regarding the use of iPS cells for medical applications (Fraser and Lin, 2016; Durruthy et al., 2014). Based on current knowledge, we suggest rigorous evaluation of the imprinting genes status in the iPS cells prior to their use as primary stem cells for production of other types of cells. As such, any differentiation protocols should include approaches for evaluation of epigenetic changes, including the status of genomic imprinting (Mishra et al., 2018). However, many studies so far suggest that some imprinted loci appear to be more affected by the loss of imprinting. This implies that targeted assays for specific loci should be established, as the first step in the characterization of newly generated iPS cell lines, and also for those that have been passaged repeatedly. This is required to obtain iPS cells resembling ES cells in epigenetic marks. Recent literature highlighted the need for evaluation of imprinting status in the stem cells to minimize the impacts of aberrant imprinting on the pluripotency. Specific
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Contents lists available at ScienceDirect
Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep
Genome imprinting in stem cells: A mini-review a
b
Rasoul Godini , Keyvan Karami , Hossein Fallahi
T
c,*
a
Development and Stem Cells Program, Monash Biomedicine Discovery Institute and Department of Anatomy and Developmental Biology, Monash University, Melbourne, Victoria, 3800, Australia Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran c Department of Biology, School of Sciences, Razi University, Kermanshah, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Genomic imprinting Embryonic stem cell iPS cells Mono-allelic expression Precision medicine
Genomic imprinting is an epigenetic process result in silencing of one of the two alleles (maternal or paternal) based on the parent of origin. Dysregulation of imprinted genes results in detectable developmental and differential abnormalities. Epigenetics erasure is required for resetting the cell identity to a ground state during the production of induced pluripotent stem (iPS) cells from somatic cells. There are some contradictory reports regarding the status of the imprinting marks in the genome of iPS cells. Additionally, many studies highlighted the existence of subtle differences in the imprinting loci between different types of iPS cells and embryonic stem (ES) cells. These observations could ultimately undermine the use of patient-derived iPS cells for regenerative medicine.
1. Introduction In diploid organisms, two copies of each gene are inherited from each parent. For most of the loci, both alleles are active and equally expressed. However, only one allele in the imprinting loci is active, either maternal or paternal allele (Butler, 2009). Imprinting is an epigenetic event, causing silencing in one of the alleles inherited from one of the parents. Therefore, if the paternal allele of the gene is imprinted, the other allele from mother would be expressed and vice versa (Fig. 1). Imprinting generally violates Mendelian inheritance and affect the segregation pattern of imprinting alleles (Úbeda, 2008). In human, genomic imprinting determines the fate of a few genes, which are crucial for the correct development (Lawson et al., 2013). A majority of imprinted genes play main roles in the control of embryonic and placental growth and development. Expectedly, dysregulation of the imprinted genes has been associated with abnormal growth and development. For example, abnormal imprinting in the DIK1-Dio3 locus results in developmental syndromes and specific diseases (Swanzey and Stadtfeld, 2016). Besides, Angelman syndrome and Prader-Willi syndrome are the typical known human diseases linked to abnormal genomic imprinting. The exact mechanisms by which some genes undergo imprinting is not fully understood (Babak et al., 2015), but the role of epigenetics in the imprinting is evident. The imprinting process involves known epigenetic modifications of the genome including DNA methylation (Li
*
et al., 1993; Stelzer et al., 2016), histone modifications (Xin et al., 2001) and non-coding RNAs (Sleutels et al., 2002). Differentially Methylated Regions (DMRs) are essential for parental imprinting of several genes. Imprinted genes are located in clusters (in approximate 80% of imprinted genes in mammals) and in the CpG rich regions of the genomes, called Imprinting Control Region (ICR) (Barlow, 2011). The genes in clusters are coordinately regulated through ICR. Other characteristics of imprinted loci include having fewer and smaller introns than non-imprinted genes, containing repetitive sequences and a high number of retro transposable elements (McVean and Moore, 1996; Ono et al., 2001). In diploid animals, it has been suggested that the presence of both paternal and maternal genomes is required for normal growth. Kono and colleagues showed that parthenogenesis, a natural form of asexual reproduction, would be affected in presence of two copies of mice female genomes, confirming the need for both paternal and maternal chromosomes for proper development (Kono et al., 2004). This could be an evidence for imprinting in several loci. Using uniparental diploid models, the map of these imprinted genes in the mouse has been created (Williamson et al., 2013). Many types of genomic imprinting have been documented in different organisms including animals, fungi, plants (Martienssen and Colot, 2001; Feil and Berger, 2007). It appears that, there an almost equal number of maternally imprinted genes as paternally imprinted genes. In mammalian, it was first described by Lyon and Glenister by results obtained from the reciprocal
Corresponding author. E-mail addresses: [email protected] (R. Godini), [email protected] (K. Karami), [email protected] (H. Fallahi).
https://doi.org/10.1016/j.gep.2019.119063 Received 18 February 2019; Received in revised form 21 May 2019; Accepted 30 June 2019 Available online 04 July 2019 1567-133X/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. General model for imprinting. For non-imprinted genes, expression occurs at both alleles. However, depending on the imprinting status the gene expression at the imprinted locus occurs either at the maternal or paternal allele. If the paternal allele of the gene is imprinted, the other allele from mother would be expressed and vice versa.
model, which is also involved in gene expression of many other nonimprinted genes, methylation or any other modifications at the promoter region prevent assemblies of the transcription machinery and the target gene fails to express. As a result, differential methylation state could be detected for the promoter regions of the imprinted genes on the maternal and paternal chromosomes (Cui et al., 2002; Yagi et al., 2017). In the second model, two or more genes are competing over a single enhancer or a set of common enhancers (Fig. 2B). Activation or silencing of a given gene depends on the activity of the enhancer activity. Such a scenario is observed for the known reciprocals imprinted genes IGF2 and H19 (Lyko et al., 1997). IGF2 is expressed from the paternal allele and H19 is expressed from the maternal allele. The ICR in the maternal allele is not methylated. Therefore, an enhancer blocking factor (CTCF) binds to the ICR and prevent IGF2 promoter activation by a downstream enhancer. In the paternal allele, ICR is methylated and prevent CTCF binding, results in the availability of enhancer for transcription of IGF2 (Ishihara et al., 2006). Thus, in this case, the DNA methylation regulates CTCF-insulation activity. Chromatin structure could affect gene expression via isolating a gene from the regulatory the enhancers (Fig. 2C). Silencer elements are present in the H19 ICR sequence (Lyko et al., 1997). Maternal H19 and Igf2 in mice would be repressed by binding of PEG3, a paternal DNAbinding protein, to the silencer sequences (Ye et al., 2016). Non-coding RNAs regulate expression of protein-coding genes at both transcriptional and post-transcriptional levels. These molecules could also silence a gene by interfering with the gene promoter or directly binding to the mRNA (Kornienko et al., 2013). The transcriptional interference mechanism is involved in the expression of IGF2R and Air (Fig. 2D). Here, differential methylation between paternal and maternal chromosomes determine which gene gets expressed. These non-coding RNAs often transcribed in opposite direction to the proteincoding sequences in one of the imprinted genes in a cluster. For example, the receptor for IGF2 (IGF2R) is transcribed exclusively from the maternal allele due to the repression of paternal transcription by the non-coding Air transcript in the opposite direction to the imprinted IGF2R gene (Kohama et al., 2011).
chromosomal translocation in mouse (Lyon and Glenister, 1977). In human, the studies concerning the effects of imprinting on different aspects of gene regulation are lagging behind, due to the absence of a suitable model. While different studies put the number of imprinting genes in the human genome between 100 and 2000, only a few have been experimentally validated (Daelemans et al., 2010). For example, one of the most studied imprinted genes in human is insulin-like growth factor 2 (IGF2) (a maternally imprinted gene), which is expressed only from the paternal allele (Giannoukakis et al., 1993). Interestingly, imprinting processed seems to be dynamic; some of the imprinted genes remain imprinted constantly, while others exhibit tissue-specific and temporal or time specific pattern of expression. As noted, the imprinted genes could affect the development of mammalian organisms. Imprinting poses serious concerns regarding the use of stem cells in regenerative medicine (Passier and Mummery, 2003). Stem cells are classified into two main classes; ES and iPS cells. Genome imprinting affects all aspects of stem cells from establishment to differentiation. Accordingly, here we have collected recently available information on the genome imprinting in stem cells and highlighted the need for assessing genome imprinting during iPS cells study and applications. 2. Regulation of gene expression through genome imprinting Genomic imprinting is a cis-acting event, affecting that expression of an allele located on only one of the two chromosomes based on a specific pattern (Barlow and Bartolomei, 2014). Although genomic imprinting works through repression of gene expression, it is not necessary a silencing process. Accordingly, any regulatory elements including promoters (Vu and Hoffman, 1994), enhancers (Webber et al., 1998), splicing junctions (Croteau et al., 2003) or polyadenylation sequences could be targeted by imprinting machinery (Wood et al., 2008). Although several different mechanisms for the impacts of imprinting on the gene expression have been proposed in different studies, the most common scheme is presented in Fig. 2. Repression of gene expression via direct methylation of the promoter region is a widespread mechanism of imprinting (Fig. 2A). In this 2
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Fig. 2. At least four models for the imprinting mechanism have been proposed. A) direct methylation/demethylation of the regulatory regions for many imprinted genes. B) Inhibition of gene expression with a competition between methylation and binding activity of a protein (CTCF, CCCTC-binding factor) to the ICR (Imprinting Control Region) sequence. Binding of CTCF prevents methylation and allow expression of the gene 2 and the downstream enhancer would be occupied. This blocks the enhancer activity on gene 1. In contrast, methylation on the ICR region prevent expression from gene 2 and the enhancer is free to activate the gene 1. C) Direct repression by PEG3, a newly identified transcriptional repressor. PEG3 occupies the binding site of the CTCF in the ICR, it would prevent gene expression either with a mechanism similar to part b or a new mechanism shown here. D) The role of non-coding RNAs in the imprinting mechanism; expression from a gene in opposite direction produces an antisense transcript that in turns silences other genes in the cluster. While, methylation of the promoter region of the ncRNA gene would prevent suppression, allowing expression of the imprinted genes from the counterpart locus.
3. The status of imprinted genes in somatic cells
hES cells, variable expression for the imprinted loci has been observed (Park et al., 2014), which might be due to disrupted DNA methylation pattern at these loci (Kim et al., 2007). Indeed, the imprinted gene expression patterns vary between mice cloned by nuclear transfer and the ES cell as a result of instability in the epigenetic marks at the imprinted loci (Humpherys et al., 2001). Besides, cloned mice derived from ES cells of the same subclone showed significant differences in the imprinted gene expression (Humpherys et al., 2001). However, some studies suggest that epigenetic instability is not a common phenomenon and is apparently a cell-line dependent event (Rugg-Gunn et al., 2007). Moreover, in some stem cells, the mono-allelic expression has been detected even in the absence of DNA methylation (Umlauf et al., 2004). These conflicting reports might indicate the existence of some other mechanisms rather than a generally accepted DNA methylation model for maintaining imprinting loci. Some explanatory models proposed for the widespread loss of imprinting (LOI) in hES cells (Rugg-Gunn et al., 2007). First, global reshuffling of the transcription programs could result in differential gene expression of many genes including imprinted genes. Second, differences in expression show differential methylation patterns in the maternal and paternal genome. Third, differential genes expression could be due to the growth advantage that the affected gene provide to hES cells. A similar mechanism could explain the variation observed in the imprinted gene expression in the iPS cells, where growth advantages count most in the cell culture. Notably, the extent of epigenetic stability of hES cells could be experimentally examined by measuring the expression level of the imprinted genes. While some imprinted genes such as Snrpn, Ipw, and Kcnq1ot1 shown to be highly stable in the hES cells, others including H19, Igf2 and Meg3 show significant variations in the degree of imprinting from cells to cells. Such measures could indicate the general status of epigenetic modifications in the ES cells (Rugg-Gunn et al., 2007).
In somatic cells, the allele-specific expression has been described for many of the imprinted genes in a tissue-depended pattern (Kohama et al., 2011). In the brain, for example, an interesting pattern of imprinting has been detected. Maternal gene expression occurs in the developing brain, while mostly paternal gene expression found in the adult brain (Gregg et al., 2010). The crucial role of epigenetic modifications, specifically genome imprinting, is the subject of many studies during gametogenesis, pre- and post-fertilization and early development of embryos. Thanks to these studies acceptable level of success achieved in the assisted reproduction technology (ART) (Fraser and Lin, 2016). It is known that global epigenetic remodeling in the primordial germ cell (PGC) is required to establish an epigenetic ground state. Therefore, incomplete epigenetic remodeling might be one of the reasons for inefficiencies in overall germ cells production, inefficient meiotic progression and the incomplete imprinting events observed for in vitro mediated differentiation (Durruthy et al., 2014). Hypo-methylated in the ICRs of imprinted genes such as SNRPN, H19/IGF2, and IGF2R has been observed at early stages of development in the embryos derived by somatic cell nuclear transfer (SCNT) and IVF techniques. Therefore, epigenetic erasure might occur at the imprinted loci during in vitro reprogramming and culture of oocytes or embryos (Smith et al., 2015). However, epigenetic anomalies appear to be less evident at the imprinted ICRs later during development; suggesting either proper re-methylation or survival of less affected embryos to the later stages of development (Smith et al., 2015). In somatic cells, the imprinted gene H19, its new antisense transcript 91H, and its miRNA are involved in the regulation the function of IGF receptor and in cell cycle progression (Vennin et al., 2013). Furthermore, it has been shown that Peg10 and Peg 4 imprinted genes are involved in PGC reprogramming and their epigenetic status linked to Tet1 function (Yamaguchi et al., 2013). Therefore, it has been suggested that imprinting has a vital role in somatic cells both at regulation and maintenance levels. Besides, an imprinted gene network (IGN) has been detected in adult somatic stem cells when compared to their differentiated progeny (Berg et al., 2011), suggesting the role of imprinting in general cell growth and tissue homeostasis.
5. The status of imprinted genes in the iPS cells Currently, the impacts of the reprogramming procedures on the status of epigenetic marks, including imprinted genes, have not been fully understood in the iPS cells. Additionally, conflicting reports make it difficult to determine if cell type of origin would affect the dynamics of epigenetic and imprinting reprogramming. However, it has been suggested that the self-renewal of the stem cells derived from different types of adult tissues depends on the status of imprinted genes (Zacharek et al., 2011). On the other hand, imprinted genes play an important role during development and involved in some types of diseases. It is, therefore, crucial to uncover the consequences of cellular reprogramming on these loci in the iPS cells (Tomizawa and Sasaki, 2012). Similar imprinting pattern exists between iPS cells and their somatic cell of origin (Rouhani et al., 2014). Accordingly, some studies suggest that loss of imprinting during reprogramming is mediated by TETs at the H19 locus, similar to ES cells, and depends on the epigenetic marks of the precursor cells (Bermejo-Álvarez et al., 2015). Surprisingly, imprinting errors are observed in iPS clones, indicating the presence of epigenetic anomalies that are related to the reprogramming process.
4. The status of imprinted genes in ES cells The status of imprinting has not been fully established in the ES cells because of these cells affected by global epigenetic remodeling during embryonic developmental stages (Rugg-Gunn et al., 2007). Apparently, maintaining self-renewal properties of adult stem cells require epigenetic modification in the imprinted genes (Kelsey, 2011). Additionally, knockdown of some imprinting genes related long non-coding RNAs affects the stem cell function and self-renewal (Luo et al., 2015), where stem cell differentiation is shown to be under direct control of the noncoding RNAs (Luo et al., 2015). Furthermore, a direct role of maternally-imprinted locus Dlk1-Gtl2 is well documented in the self-renewal of hematopoietic stem cell (HSC) (Qian et al., 2016). On the other hand, several defects have been observed in the genomic imprinting loci in the mouse and human embryonic stem (hES) cells (John and Helen, 2008). Unexpectedly, during differentiation of 4
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Fig. 3. The impacts of imprinting on different aspects of stem cell biology as indicated in the literature. Embryonic stem cells (A), somatic cells (B) and iPS cells (C) were compared. Self-renewal, cell growth, differentiation, homeostasis, and development are affected by imprinting status in ESCs, iPS and their somatic derivatives. The most updated reference for each step is given in the brackets.
(He et al., 2014). Of note, in mouse, the expression of Dlk1-Dio3 cluster is shown to be altered in iPS cells (Stadtfeld et al., 2010). Surprisingly, upon reprogramming, culture conditions could affect the epigenetic patterns, imprinting and the properties of the resulting iPS cells. For example, ascorbic acid inhibits the removal of Dlk1-Dio3 imprinting pattern and enhances the generation of iPS cells from B cells (Stadtfeld et al., 2012). Hence, genomic imprinting is known to variably lose during reprogramming of mouse iPS cells (Takikawa et al., 2013). In human induced Pluripotent Stem (hiPS) cells, dysregulation of NNAT, a key imprinted gene, has been observed (Teichroeb et al.,
These epigenetic abnormalities could be responsible for the variable phenotypes and low success rates of iPS differentiation and cloning (Bressan et al., 2014). Furthermore, in mouse primordial germ cells and their cell culture models, resistance to global genome DNA demethylation have been identified, which are linked to imprinting regions (Miyoshi et al., 2016). Despite many similarities between iPS cells and ES cells, there exist significant differences in overall methylation pattern between these two cell types. Clearly, iPS cells are shown to partly retain the methylation pattern of the original somatic cells. In addition, these cells gain unexpected de novo methylation upon or during cellular reprogramming
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cases have been observed during neural lineage differentiation from human somatic iPSCs (Lee et al., 2019). The imprinting genes expression status could also be used as a benchmark for identifications of those cells that would have full developmental potential (Dirks et al., 2019). Some strategies have been suggested for detection of correct imprinting states in hiPSCs (Perrera and Martello, 2019). First and the most applicable approach would be measuring and comparing the expression of a set of imprinted genes in the parental and progeny cell lines to assess the stability of the imprinting processes. This could be easily achieved via quantitative RT-PCR or RNA-sequencing. The difficult parts might be compiling a list of imprinting genes to be used as benchmark for each types of cell lines and tissues. Second types of assay could be detection of mono or biallelic expression for the imprinted genes via SNPs identification (Santoni et al., 2017). This approach relies on RNA-sequencing technology and therefore has some limitations when one produces numerous iPSCs in a single experiment. Additionally, stem cells, derivatives should be also assessed for correct imprinting gene expression prior to therapeutic uses. Therefore, monitoring the status of imprinting should be included in the experimental design before generating iPSCs, during differentiation into other types of cells and in the terminally differentiated progenies before their inclusion in the clinical studies.
2011). Furthermore, in female hiPS cells, many genes located on the Xchromosome show unusual over-expression. These observations suggest that despite a remarkable overall similarity in the transcriptome of the hES cells, even isogenic hiPS cells contain altered gene expression of the imprinted and X-chromosome linked genes (Teichroeb et al., 2011). 6. Knowledge of imprinted genes status is important for successful regenerative medicine Although the epigenetic pattern of freshly developed hiPS cells appears to be different from those of hES cells (Nishino et al., 2011), these differences decline after multiple passages of hiPS cells (Hiura et al., 2013; Pick et al., 2009). In contrast, existing abnormal imprinting would not be removed even after prolonged passaging (Hiura et al., 2013). These observations in addition to the variable expression of imprinted genes among different types of iPS cells suggest that each cell line should be individually assessed before use for regenerative medicine (Pick et al., 2009). Abnormal expression of the imprinted genes would change the dosage of gene products in the stem cells. Consequently, the differentiation and proliferation capacity of the stem cells would be affected. The exact impact of such modification on the fate of the stem cells is unknown. However, a tendency toward specific tissues has been observed, following overexpression of some imprinted genes in ES cells (Prelle et al., 2000). Additionally, as the biallelic expression of several imprinted genes including MEST (Pedersen et al., 1999), MEG3 (Zhang et al., 2003), H19 and IGF2 (Cui et al., 2002), correlate with different types of cancers. These unexpected results raise concerns regarding the use of such cells in cell therapy, where aberrant expression might exist for one or more of these genes (Choi et al., 2017). Surprisingly though, alteration of imprinting status at the Dlk1-Dio3 cluster in ST5 and ST8 iPS cell lines did not result in any considerable changes in the differentiation capacity of these lines to the early hepatic lineage in vitro. However, after further culture to day 19, a statistically significant functional impairment was observed in glycogen storage capacity (Christodoulou et al., 2011). Another example is the expression of the ZFP57, a master regulator in genomic imprinting in the iPS cells derived from MEF cells (Mackay et al., 2008). Similarly, ZFP57 is highly expressed in the undifferentiated ES cells as well (Zuo et al., 2012), albite variation of gene expression from this site in iPS cells might be different.
Abbreviations ART DMRs ES CTCF HSC hES hiPS IGN ICR iPS IGF2 LOI PGC IGF2R SCNT
Assisted reproduction technology Differentially Methylated Regions Embryonic stem Enhancer Blocking Factor Hematopoietic stem cell Human Embryonic Stem Human induced Pluripotent Stem Imprinted gene network Imprinting Control Region Induced pluripotent stem Insulin-like growth factor 2 Loss of imprinting Primordial germ cell Receptor for IGF2 Somatic cell nuclear transfer
Appendix A. Supplementary data 7. Future perspective Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gep.2019.119063.
The extent of imprinting loss or gain remains to be fully explored during of reprogramming of somatic cells to iPS cells. What we understand so far is that although imprinting errors may be rare in the iPS cells, they appear to be resistant to reversal epigenetic modifications. Therefore, persistent alteration of imprinting genes expression in the iPS cells is likely to undermine their use in regenerative medicine (Fig. 3). Many concerns raised regarding the use of iPS cells for medical applications (Fraser and Lin, 2016; Durruthy et al., 2014). Based on current knowledge, we suggest rigorous evaluation of the imprinting genes status in the iPS cells prior to their use as primary stem cells for production of other types of cells. As such, any differentiation protocols should include approaches for evaluation of epigenetic changes, including the status of genomic imprinting (Mishra et al., 2018). However, many studies so far suggest that some imprinted loci appear to be more affected by the loss of imprinting. This implies that targeted assays for specific loci should be established, as the first step in the characterization of newly generated iPS cell lines, and also for those that have been passaged repeatedly. This is required to obtain iPS cells resembling ES cells in epigenetic marks. Recent literature highlighted the need for evaluation of imprinting status in the stem cells to minimize the impacts of aberrant imprinting on the pluripotency. Specific
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