In recent years, solid evidence has accumulated that insulin-like growth factor-1 (IGF-1) and 2 (IGF-2) regulate many biological processes in normal and malignant cells. Recently, more light has been shed on the epigenetic mechanisms regulating expression of genes involved in IGF signaling (IFS) and it has become evident that these mechanisms are crucial for initiation of embryogenesis, maintaining the quiescence of pluripotent stem cells deposited in adult tissues (for example, very-small embryonic-like stem cells), the aging process, and the malignant transformation of cells. The expression of several genes involved in IFS is regulated at the epigenetic level by imprinting/methylation within differentially methylated regions (DMRs), which regulate their expression from paternal or maternal chromosomes. The most important role in the regulation of IFS gene expression is played by the Igf-2-H19 locus, which encodes the autocrine/paracrine mitogen IGF-2 and the H19 gene, which gives rise to a non-coding RNA precursor of several microRNAs that negatively affect cell proliferation. Among these, miR-675 has recently been demonstrated to downregulate expression of the IGF-1 receptor. The proper imprinting of DMRs at the Igf-2-H19 locus, with methylation of the paternal chromosome and a lack of methylation on the maternal chromosome, regulates expression of these genes so that Igf-2 is transcribed only from the paternal chromosome and H19 (including miR-675) only from the maternal chromosome. In this review, we will discuss the relevance of (i) proper somatic imprinting, (ii) erasure of imprinting and (iii) loss of imprinting within the DMRs at the Igf-2-H19 locus to the expression of genes involved in IFS, and the consequences of these alternative patterns of imprinting for stem cell biology.
Among the 3.0–3.5 × 104 genes in the mammalian genome, there are ∼80 genes that are paternally imprinted and expressed from the maternally or paternally derived chromosome only. This pattern of expression regulates the appropriate dosage and level of expression of these genes in mammalian cells.1 The expression of imprinted genes is regulated by the imposition of epigenetic marks by DNA methylation within differentially methylated regions (DMRs), which are regulatory CpG-rich cis-elements at the gene locus.2 Most imprinted genes are methylated in mouse on maternally derived chromosomes, and only four, Igf-2-H19, RasGrf1, Dlk1/Dio3 and Zdbf2, are methylated on paternally derived chromosomes.3
According to the parent–offspring conflict theory, while paternally expressed imprinted genes enhance embryo growth and size of the offspring, the maternally expressed genes inhibit cell proliferation and somewhat negatively affect its size.1 Based on this, during pregnancy, the father, through proper expression of paternally imprinted genes, contributes to body size and muscle mass of the developing fetus and ‘wants’ the mother to devote as much of her resources as possible towards the growth of his child. By contrast, the mother wants to conserve as much of her resources as possible toward future births (without compromising the health of the fetus she is currently carrying) by epigenetic modulation of genes bearing maternal imprinting marks.1
Evidence has accumulated that among the imprinted genes, the most important is insulin-like growth factor 2 (Igf-2)-H19. This tandem gene is imprinted both in mice and humans and regulates IGF-2 and insulin-like growth factor-1 (IGF-1) signaling, which affects many vital aspects of cell biology.4 In particular, while the Igf-2 locus encodes IGF-2, which is an autocrine/paracrine mitogen, transcription of H19 gives rise to a non-coding RNA (non-coding RNA) that is a precursor of several microRNAs that negatively affect cell proliferation. For example, it has recently been demonstrated that miR-675 is involved in downregulation of expression of the IGF-1 receptor (IGF-1R).5 The foregoing indicates the dual role of this ‘yin-yang locus’, which involves opposite functional effects of Igf-2 and H19 genes on cell proliferation, and suggests its important role in initiation and regulation of embryonic development.1 Furthermore, recent evidence indicates that erasure of imprinting (hypomethylation) of the DMRs at the Igf-2-H19 locus on both chromosomes, which leads to downregulation of Igf-2 and upregulation of H19 expression, has an important role in regulating the quiescence of pluripotent stem cells residing in adult tissues and thus may be involved in the regulation of life span.6, 7, 8, 9 On the other hand, loss of imprinting (hypermethylation) of DMRs at this locus on both chromosomes results in Igf-2 overexpression and H19 downregulation and is a phenomenon observed in several malignancies.10
In this review, we will discuss the biological consequences of changes in imprinting at the Igf-2-H19 locus. We envision that the changes in expression of genes encoded at this locus are a kind of ‘genetic Rosetta Stone’ that allows one to better understand development, aging and cancerogenesis.
Proper imprinting of the Igf-2-H19 locus and initiation of embryogenesis
The tandem Igf-2-H19 locus is located on chromosome 11p15 in humans and chromosome 7 in mice and, as mentioned above, is paternally imprinted both in humans and in mice. This preservation across species suggests the importance of its involvement in mammalian development.
Figure 1a is a schematically simplified structure for this locus, showing that the regulatory DMR is methylated on the paternal chromosome and erased on the maternal chromosome. Accordingly, the filled lollypops at the DMR regulatory region of the paternal chromosome depict methylation, and open lollypops on the maternal chromosome indicate lack of methylation. If the DMR is methylated, it cannot bind the regulatory DNA-binding zinc finger insulator protein, CTCF, which establishes a functional boundary between the Igf-2 and H19 transcription regions.11, 12 The binding of CTCF has immediate consequences for expression of these loci. As expression of both Igf-2 and H19 is regulated by a 3′-distal enhancer (shown as a red box), the presence of CTCF bound to the DMR at the maternal locus prevents transcription of Igf-2, and in this situation, only H19 is transcribed to RNA. In contrast, the presence of a methylated DMR on the paternal chromosome prevents binding of CTCF, and in this situation, the 3′-distal enhancer promotes transcription of mRNA from the Igf-2 locus. This ensures a proper balance in expression of both genes: Igf-2 from the paternal and H19 from the maternal chromosome (Figure 1a). Overall, four CTCF-binding sites have been identified so far in the murine Igf-2-H19 DMR and seven in its human counterpart.13, 14 Interestingly, the human Igf-2-H19 DMR is not able to function when introduced as a transgene into the murine genome, which suggests some species differences that tune the regulation of this DMR.15
To explain the biological consequences of the gene expression encoded by this locus, the IGF-2 protein product of the Igf-2 gene stimulates cells in both an autocrine and paracrine way after binding to IGF-1R and with lower affinity to the insulin receptor.16 Cells also express the high-affinity-binding IGF-2 receptor (IGF-2R); however, this is a non-signaling receptor that simply binds IGF-2 and prevents its signaling through IGF-1R and insulin receptor.17 On other hand H19, as mentioned above, transcribes a long, 2.3-kb, ncRNA that is evolutionarily conserved at the nucleotide level in humans and rodents and not translated to protein. Instead, it is processed into small microRNAs18 of which miR-675 as mentioned above negatively regulates the expression of IGF-1R.5 In addition, in situ hybridization of the H19 ncRNA revealed that it is detectable in cytoplasmic ribonucleoprotein particles, which suggests that the H19-derived microRNAs are involved in ribosomal function and translation. However, the loss of H19 is not lethal in mice, and such animals display an organ overgrowth phenotype similar to babies with Beckwith–Wiedemann syndrome.19 On the other hand, overexpression of H19 is a dominant lethal mutation and mouse embryos overexpressing H19 die after embryonic day 14. This may reflect its overall suppressive role in early stages of development involving suppression of IGF-1R expression,17 as well as negative regulation of other yet-to-be identified targets.
The close coupling of Igf-2 and H19 expression is explained by the fact that these two genes share the same 3′-gene enhancer (shown in Figure 1a as red boxes), and it has been reported that deletion of this 3′-enhancer results in downregulation of both Igf-2 and H19 expression.20 However, there are also some indications that the 3′-enhancer has a more robust effect on expression of H19 than Igf-2, which could be explained by the fact that (i) H19 has a stronger promoter than Igf-2 and/or (ii) the H19 gene is physically closer to the 3′-enhancer than Igf-2 (Figure 1).20 Interestingly, it has recently been postulated that the H19 locus is also a source of antisense RNA (H91 RNA), which regulates expression of Igf-2 by interacting with a novel promoter for this gene.21 This latter effect adds more complexity to the regulation of the Igf-2-H19 locus, but as it has been described so far only in myoblasts, its biological significance is still awaiting further validation in stem cells.
Evidence has accumulated that the dual yin-yang role of this master locus is relevant to several biological functions, including normal fetal development, as the properly balanced expression of Igf-2 and H19 is required for initiation of embryogenesis.22 Accordingly, imprinting at the Igf-2-H19 locus is one of the major factors preventing parthenogenetic development in mammals, and the biological importance of this locus is demonstrated by the creation of viable bimaternal mice derived from two female sets of chromosomes.22 These mice are created by fusion of two haploid nuclei, one from a non-growing and the other from a fully growing oocyte, into a diploid bimaternal zygote. As female chromosomes have unmethylated DMRs at the Igf-2-H19 locus (Figure 1a), which leads to overexpression of inhibitory H19 ncRNA in this ‘zygote-like’ totipotent cell, the crucial step in creating bimaternal mice is an appropriate genetic modulation of the Igf-2-H19 locus from one of the maternally derived chromosomes, which promotes expression of IGF-2 and thus properly balanced dosage of IGF-2/H19.22
In sum, proper imprinting of the Igf-2-H19 locus is required for balanced expression of both genes in normal embryonic development and is maintained later on in all somatic cells of the growing embryo and postnatal infant. Thereafter, in all the adult tissues except rare population of developmental early cells that will be discussed below, the somatic cells express proper somatic imprinting, as depicted in Figure 1a.
Erasure of imprinting at Igf-2-H19 loci regulates the quiescence of pluripotent stem cells residing in adult tissues
In contrast to somatic cells, imprinting within DMRs at Igf-2-H19 loci is erased during early embryogenesis in primordial germ cells (PGCs).23 Figure 1b depicts the consequences of this erasure of methylation within the DMRs at the Igf-2-H19 locus, which leads to downregulation of growth-promoting IGF-2 from both paternal and maternal chromosomes, and overexpression from these chromosomes of proliferation-limiting H19 ncRNA. This is an important regulatory mechanism that keeps PGCs quiescent and prevents them from teratoma formation. A similar phenomenon also occurs in very-small embryonic-like stem cells (VSELs), which share several markers with migrating PGCs24, 25 and are deposited during development in developing organs, including adult bone marrow (BM).26, 27, 28 Figure 2a shows a representative hypomethylation state of the DMR for the Igf-2-H19 locus in murine BM-derived VSELs and normal (∼50%) methylation observed in hematopoietic stem cells (HSCs). These changes in methylation of DMRs at this locus result, as shown in Figure 2b, in downregulation of IGF-2 mRNA and upregulation of H19 ncRNA in VSELs. The biological significance of this epigenetic modification in PGCs and VSELs will be discussed below.
Erasure of Igf-2-H19 imprinting in PGCs
As the precursor cells for gametes (oocytes or sperm), PGCs are the most important cell population, because they transfer parental DNA and mitochondria to the next generation. PGCs become specified as the first population of stem cells during embryogenesis in the proximal part of the epiblast, which forms all three layers of the trilaminar germ disc of the embryo proper in a process called gastrulation.29 After being specified, PGCs migrate to the extra-embryonic tissues, enter through the primitive streak the embryo proper, and migrate to the genital ridges.30 As has been demonstrated, during this migration process PGCs erase the methylation at several maternally and paternally imprinted loci, including paternal imprinting at the Igf-2-H19 locus. This mechanism of erasure of imprinted marks has several important consequences. First, after the erasure of imprinting, PGCs (i) are quiescent and unable to proliferate in vitro, (ii) do not form teratomas, (iii) do not complement blastocyst development and (iv) are not capable of performing as DNA donors in therapeutic cloning using their harvested nuclei. However, all these limitations in the pluripotency of PGCs are reversed when their imprinting is reestablished, as seen, for example, during ex vivo generation of embryonic germ cells from PGCs.31, 32 These embryonic germ cells that recover proper somatic imprinting behave as embryonic stem cells (ESCs) in all of the assays listed above.29
During normal development, the proper somatic pattern of imprinting in germline cells is established when PGCs, after colonization of the genital ridges, differentiate into the precursors of gametes.33 However, this occurs both in female germline (paternal imprinting) and male germline (maternal imprinting) cells, first after a meiotic division in which the precursors of gametes containing diploid chromosomes give rise to progeny that possess the haploid number of chromosomes and are not able to proliferate. However, when haploid male and female gametes fuse during fertilization, the chromosomes in the diploid zygote have proper complementary imprinting, including at the Igf-2-H19 loci.
Erasure of Igf-2-H19 imprinting in VSELs
Modification of genomic imprinting also has a crucial role in maintaining the pool of pluripotent stem cells residing in adult tissues. Specifically, our group demonstrated that adult murine tissues harbor a population of pluripotent Oct4+SSEA-1+Sca-1+Lin–CD45– cells,26, 27 and a corresponding population of Oct-4+SSEA-4+CD133+Lin–CD45– cells has also been identified in humans.34, 35, 36 We hypothesize that VSELs, are deposited in adult tissues during early embryogenesis and serve as a back-up population of precursor stem cells for more differentiated tissue-committed stem cells.37 Careful molecular analysis of VSELs has revealed that their quiescence in adult BM and premature depletion from the tissues is controlled by epigenetic changes to imprinted genes, including the Igf-2-H19 locus, which is erased in murine VSELs24, 25 similarly as seen in PGCs (Figure 1b and Figure 2).
In addition to erasure at the Igf-2-H19 locus, murine VSELs also modify expression of other imprinted genes, but not all these epigenetic changes are identical to those seen in PGCs.24, 25 For example, we observed that murine BM-sorted VSELs, like PGCs, erase the paternally methylated imprints (for example, DMRs at the Igf-2-H19 and RasGrf1 loci), while in contrast to PGCs, hypermethylate the maternally methylated imprints (for example, DMRs at Igf-2R). Thus, the changes in expression of these genes in mouse additionally impairs IGF signaling (IFS), because hypermethylation of the DMR at the Igf-2R locus leads to overexpression of IGF-2R, which, as mentioned above, is a non-signaling receptor that binds IGF-2 and prevents its interaction with the signaling receptors IGF-1R and insulin receptor. On the other hand, erasure of the DMR at the RasGrf1 locus in mice leads to downregulation of RasGRF1, which is a small guanosine triphosphate (GTP) exchange factor for Ras involved in proper signal transduction from activated IGF-1R and Ins-R. It is important to point out that in contrast to the Igf-2-H19 locus, which is imprinted both in mice and humans,38 Igf-2R and RasGrf1 loci are imprinted in murine but not in human cells, even when IGF-2R is highly expressed by human VSELs. Based on this difference, murine VSELs regulate more genes involved in IFS by imprinting than their human counterparts (Figure 3).
The epigenetic modification of these imprinted loci (including Igf-2-H19) explains why VSELs, like PGCs, despite expressing several markers of pluripotency such as (i) an open chromatin structure at the promoters for Oct-4 and Nanog, (ii) bivalent domains at developmentally important homeobox-domain-containing genes, (iii) reactivation of the X chromosome in female VSELs and (iv) in vitro differentiation into cells from all three germ layers, do not complement blastocyst development after injection into the pre-implantation blastocyst and do not grow teratomas in immunodeficient mice.24, 25, 26, 27, 28, 39
The fact that erasure of imprinting at the Igf-2-H19 locus may have a crucial role in keeping VSELs quiescent in adult tissues has important practical implications. Specifically, we envision that reestablishment of proper expression of the IGF-2/H19 ratio in VSELs will be crucial for effective expansion of these cells ex vivo for potential application in regenerative medicine. Supporting the feasibility of this goal, the IGF-2/H19 ratio is also perturbed in parthenogenetic stem cells, and it has recently been demonstrated in two independent reports that downregulation of H19 ncRNA in these cells significantly improves their ex vivo expansion.40
Loss of imprinting at Igf-2-H19 loci and malignant transformation
A growing body of evidence suggests that cancer originates in the stem/progenitor cell compartment as a result of mutations accumulating over a lifetime.41 These mutations are maintained in stem cell compartments, and self-renewing stem cells may be subjected to additional mutations and epigenetic changes so that the genome is destabilized and uncontrolled neoplastic proliferation is initiated. Interestingly, during the 19th and early 20th centuries, several investigators proposed that cancer develops in populations of embryonic-like cells that are left in a dormant state in developing organs during embryogenesis.41 This ‘embryonic rest hypothesis of cancer origin’ suggested that adult tissues contain embryonic remnants that normally lie dormant, but that can be activated to become cancerous. Based on the presence of PGCs and VSELs in adult tissues, it is tempting to hypothesize that these cells could be the missing link that reconciles this past theory of cancer origin with current theories envisioning cancer as a stem cell disorder. However, this hypothesis needs more experimental corroboration.
Nevertheless, hypermethylation of the Igf-2-H19 locus on both chromosomes, which is called loss of imprinting (in contrast to erasure of imprinting), results in Igf-2 overexpression (Figure 1c) and is observed as an epigenetic change in several malignancies (for example, rhabdomyosarcoma and nephroblastoma) where overexpressed IGF-2 acts as an autocrine growth factor for tumor cells. The best example of this mechanism is Beckwith–Wiedemann syndrome, which is associated with the development of several pediatric sarcomas.10 A similar loss of imprinting, however, has been also reported in pediatric sarcomas developing independently as part of Beckwith–Wiedemann syndrome.42, 43
VSELs—imprinting of the Igf-2-H19 locus, IFS and the implications for aging and cancerogenesis
The above-mentioned changes in expression of imprinted genes in VSELs and, in particular, the common epigenetic change, erasure of imprinting at the Igf-2-H19 locus, observed in both murine and human VSELs, leads to significant attenuation of IFS (Figure 3) in these cells.24, 25 As a result, due to epigenetic changes in imprinted genes, VSELs are protected from autocrine and paracrine IFS, which would otherwise lead to their premature depletion from adult tissues, as well as potentially trigger uncontrolled proliferation leading to teratoma formation. This attenuation of IFS in VSELs may have important implications, both for aging and cancerogenesis.
VSELs and a novel view of aging
IFS negatively correlates with life span in different species, including mice and humans.44, 45, 46, 47, 48 We have already reported that VSELs can be specified into HSCs and have proposed that in one marrow (BM) they correspond to the most primitive precursors for HSCs.49, 50 If this is also true for VSELs residing in other organs (for example, liver, skeletal muscles and epidermis), they could also be a potential back-up population for other types of tissue-committed stem cells, though more evidence is needed. Nevertheless, as the erasure of Igf-2-H19 imprinting in VSELs negatively affects IFS signaling, it potentially maintains their quiescent state and protects them from premature depletion from the tissues. Based on this, we proposed a novel hypothesis that relates aging, longevity and IFS to the abundance and function of pluripotent VSELs deposited in adult tissues. A decrease in the number of these cells should negatively affect pools of tissue-committed stem cells in various organs and have an impact on tissue rejuvenation and life span.47, 51, 52 In support of this expectation, we observed a significantly higher number of VSELs and HSCs in the BM of long-living murine strains (for example, Laron dwarfs and Ames dwarfs) whose longevity is explained by low levels of circulating IGF-1 and decreased IFS.7 It is known that IFS involves TORC1 (TOR (target of rapamycin) complex 1)—ribosomal protein S6K (S6 kinase), and that this TORC1–S6K axis controls several basic cellular processes, including transcription, translation, protein and lipid synthesis, cell growth/size and cell metabolism that affect aging.53 It explains why inhibition of TORC1/S6K pathway by rapamycin has been shown to efficiently extend life span in several experimental animal models in vivo.53 Similar effect on TORC1/S6K signaling is achieved in VSELs by erasure of imprinting at Igf-2-H19 locus.
By contrast, compared with normally aging littermates, the number of VSELs and HSCs is significantly reduced in short-living mouse strains (for example, growth hormone-overexpressing transgenic mice) with high levels of circulating IGF-1 and thus elevated IFS.8, 9
VSELs and their potential involvement in cancerogenesis
Elevated IFS is also well known to be involved in development of malignancies.54 Specifically, both obesity and high-caloric uptake, which are associated with highly active IFS, are risk factors for cancer development. Experimental animals with high levels of circulating IGF-1 are not only short-lived but also have a high incidence of cancer.48 On the other hand, long-living animals, such as the Laron dwarf and Ames dwarf mice mentioned above, with low levels of circulating IGF-1, have a much lower incidence of tumor development.48 Importantly, this animal data also correlates very well with the human Laron dwarf mutation, where affected individuals have a very-low level of circulating IGF-1 and at the same time are highly prone to cancer development.55, 56
Based on the observations that predisposition to malignancies in mice correlates with VSEL numbers in their tissues.41 To explain this, we envision that chronic stimulation of VSELs by IFS may potentially activate these cells in an uncontrolled way and promote their malignant transformation. Therefore, it is also likely that some human tumors may originate in VSELs, and IFS may have an important promoting role.57 Again, we envision two possible mechanisms. First, VSELs exposed to constant high circulating levels of IGF-1 could transform into neoplastic cells and second, as will be discussed below, they could transform because of a loss of imprinting at the Igf-2-H19 locus, which would expose them to the autocrine IGF-2 loop and restore normal expression of IGF-1R. Currently, we are testing this hypothesis in appropriate animal models.
Evidence has accumulated that the imprinted Igf-2-H19 tandem gene has a pleiotropic role in several biological processes, including quiescence of VSELs deposited in adult organs. It is important to mention that we have observed that with increasing age, the DMRs at the Igf-2-H19 locus become gradually methylated, and thus VSELs become more sensitive to IFS over time.6, 7, 8 This phenomenon may contribute to their age-related depletion, as well as render them more sensitive to IFS and put them at risk of malignant transformation. Thus, modification of expression at the Igf-2-H19 locus may have an important role in inhibiting aging processes and preventing cancerogenesis. Furthermore, we envision that proper methylation of the DMR at this locus, which is erased in VSELs,24, 25 will be crucial for development of ex vivo strategies for expansion of these cells for the purposes of regenerative medicine.57
In addition to imprinting, expression at the Igf-2-H19 locus is tightly regulated by the CTCF protein, which is involved in the balanced expression of IGF-2 and H19 from the paternal and maternal chromosomes. Of note, an interesting mechanism has been described in which elevated level of IGF-2 in senescent human epithelial cells is the result of a reduction in CTCF expression, which controls the Igf-2-H19 locus. As reported, a decrease in the intracellular CTCF level, leading to lower occupancy of DMRs by CTCF within the Igf-H19 locus, resulted in a 10-fold increase in intracellular Igf-2 expression.58 Therefore, modulation of CTCF expression could also be an option for regulating IFS in VSELs. Furthermore, it is likely that, in addition to CTCF, other proteins are also involved in regulation of this locus that still await identification.
Finally, the status of imprinted genes has been also investigated in some leukemias,54, 59, 60, 61, 62 but taking into consideration the important role IFS has in the development of normal and malignant HSCs,63, 64 more work is needed to study imprinting of the Igf-2-H19 locus in normal and pathological conditions. Another important question is effect of IFS on telomers length. Potential involvement of IGF-1 in this process65, 66 suggests that imprinting status at Igf-2-H19 locus, by modulating via H19 expression of IGF-1R, may have here an important and underappreciated role.
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This work was supported by NIH grant 2R01 DK074720, the Stella and Henry Endowment and Maestro 2011/02/A/NZ4/00035 grant to MZR.
The authors declare no conflict of interest.
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Ratajczak, M., Shin, DM., Schneider, G. et al. Parental imprinting regulates insulin-like growth factor signaling: a Rosetta Stone for understanding the biology of pluripotent stem cells, aging and cancerogenesis. Leukemia 27, 773–779 (2013). https://doi.org/10.1038/leu.2012.322
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