Many studies have focused on the broader role of the Dlk1-Meg3 locus, including the function of the different protein-coding genes in the locus, using different mouse models (da Rocha et al., 2008). For example, the transmembrane glycoprotein Dlk1 has been associated with cellular differentiation (Carlsson et al., 1997; Laborda, 2000; Moon et al., 2002). In addition, studies on IG-DMR and Meg3 KO mice have also shed light on the role of the maternally
A
B
Deletion Model Targeting Strategy Phenotype Maternal KO Phenotype Paternal KO Publication
IG deletion IG-DMR loss of imprinting
lethal after E16
no loss of imprinting viable
Lin et al., 2003
Meg3-DMR/exon1-5 deletion Meg3-DMR + Exon 1-5
lethal 4 weeks after birth
no loss of imprinting 50% die perinatally 25% die few days after birth 25% show growth retardation
Takahashi et al., 2009
Meg3-partDMR/exon1-5 partial deletion Meg3-DMR + Exon 1-5 loss of imprinting pre-/perinatal death
no loss of imprinting growth retardation
Zhou et al., 2010 no loss of imprinting
26
expressed lncRNA Meg3 and the associated ncRNAs (Figure 1.7 B) (Lin et al., 2003; Takahashi et al., 2009; Zhou et al., 2010).
Lin et al. generated a mouse strain with a 4.15 kb deletion covering the IG-DMR (Lin et al., 2003). At E16, heterozygous animals were detected in the expected ratios. Later in embryonic development, the number of heterozygous live embryos was reduced in animals with maternal inheritance of the deleted IG-DMR. In these animals, bidirectional loss of imprinting on both alleles was observed in the locus, leading to loss of expression of the maternally expressed genes and gain of expression of the paternally expressed genes. These deregulations potentially lead to the observed lethality after E16. In contrast, offspring that inherited the targeted deletion from the father did not exhibit any phenotype, and no loss of imprinting was observed. In line, the model of Zhou et al. showed a highly similar phenotype (Zhou et al., 2010). Here, a 5 kb deletion covering part of the Meg3 promoter, part of the Meg3-DMR and exon 1-5 was generated. Paternal deletion did not lead to loss of imprinting or deregulated expression patterns from the maternal or paternal allele. Maternal deletion, however, was associated with loss of imprinting and reactivation of paternal expression patterns, leading to loss of maternal genes and bi-allelic expression of paternal genes. Consequently, mice died perinatally and presented skeletal muscle defects. In contrast, a mouse model generated by Takahashi et al. exhibited a different phenotype (Takahashi et al., 2009). Deletion of a 10 kb region including the Meg3-DMR, the Meg3 promoter and exon 1-5 was performed. Mice with maternal deletion died 4 weeks after birth, but only loss of Meg3 expression was observed. ncRNAs from the maternal locus were still moderately expressed. In contrast, mice inheriting the deletion from the father mostly died perinatally. In these mice, expression of the paternal protein-coding genes was reduced, while ncRNAs from the maternal allele, but not Meg3, were upregulated. In addition, no loss of imprinting was observed, independently of deletion inheritance. Zhou et al. speculated that, in the model of Takahashi et al., the Neomycin promoter from the resistance cassette, which is oriented in the same direction as Meg3, leads to moderate expression of the ncRNA cluster in maternal deletion mice (Takahashi et al., 2009; Zhou et al., 2010). In line, this promoter would drive ncRNA expression from the paternal allele, leading to the observed upregulation of maternal ncRNAs in the paternal deletion mice (Takahashi et al., 2009; Zhou et al., 2010). In summary, Meg3 itself seems to be important to control imprinting of the Dlk1-Meg3 locus. In addition, the different phenotypes observed in these mouse models highlight the importance of study design and result interpretation. Studies in different cancer entities revealed that Meg3 might act as a tumor suppressor (Benetatos et al., 2011; Zhou et al., 2012). Zhang et al. could show that Meg3 expression was reduced in nonfunctioning pituitary tumors (Zhang et al., 2003). Re-expression of the lncRNA
27 in cancer cells led to inhibition of cellular proliferation. Loss of Meg3 expression has also been reported in meningioma, renal cell carcinoma, leukemia, neuroblastoma, pheochromocytoma and Wilms’ tumors (Astuti et al., 2005; Benetatos et al., 2008; Benetatos et al., 2010; Kawakami et al., 2006; Khoury et al., 2010; Zhang et al., 2010a). Interestingly, loss of Meg3 expression in cancer is not caused by mutations or genomic deletions but mainly by methylation of IG- or Meg3-DMR, leading to silencing of the associated maternal locus and reactivation of paternal expression patterns (Benetatos et al., 2011; Zhou et al., 2012). In summary, Meg3 acts as a tumor suppressor gene and is epigenetically silenced in different tumor entities.
In multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, aberrant methylation patterns of DMRs in the Meg3 locus were reported (Benetatos et al., 2008; Benetatos et al., 2010; Khoury et al., 2010). No association was observed between methylation patterns and AML subtypes. However, an increase in Meg3 promoter methylation was associated with decreased overall survival of AML patients. Furthermore, an increase in Dlk1 expression, which is linked to silencing of the maternal expression patterns, was observed in the majority of AML patients (Khoury et al., 2010). In addition, acquired upd of the Dlk1-Meg3 locus has been shown to promote clonal hematopoiesis (Chase et al., 2015). Thus, Meg3 and the Meg3 locus harbor important tumor suppressor functions in the hematopoietic lineage.
Different functional studies have been performed to identify the mode of action of Meg3, mostly in human tumor cell lines (Benetatos et al., 2011). In general, Meg3 seems to suppress Mdm2 expression, leading to an increase in p53 levels (Zhou et al., 2007). Recently, Lyu et al. could show that Meg3 reduces AML cell proliferation via a p53-dependent but also a p53- independent mechanism, presumably by downregulation of DNMT3a (Lyu et al., 2017). They also reported epigenetic silencing of the maternal locus, leading to loss of Meg3 expression. Interestingly, they could show that TET2 activates the Meg3 locus via WT1. Thus, TET2 mutations, which are common in AML, could lead to silencing of Meg3. In addition, Meg3 seems to suppress Rb phosphorylation directly and via activation of p16INK4A, which negatively
regulates Cdk4/6-cyclin D complexes (Zhang et al., 2010c). Thus, in line with the observed tumor suppressor function, Meg3 acts as an inhibitor of cell cycle. Meg3 is also involved in regulating angiogenesis via vascular endothelial growth factor (VEGF) signaling (Zhou et al., 2012). An increase in brain blood vessel development in Meg3 KO mice supports the hypothesis that Meg3 seems to inhibit the VEGF axis, thereby limiting angiogenesis (Gordon et al., 2010). In summary, Meg3 acts as a tumor suppressor by regulating p53 and Rb, thereby inhibiting proliferation, and by inhibition of VEGF, leading to a decrease in angiogenesis.
28
Meg3 expression is stimulated by cAMP response element binding protein (CREB) family activation and binding to cAMP response elements (CRE) located in the Meg3 proximal promoter (Zhao et al., 2006). Overall, care has to be taken when analyzing Meg3 function. Silencing of the maternally expressed genes does not only affect the lncRNA Meg3 but also the other ncRNAs encoded in this locus. In addition, methylation of IG- and Meg3-DMR leads to reactivation of the paternal expression patterns and therefore a 2-fold increase in protein levels of DLK1, RTL2 and DIO3. Thus, it is very difficult to distinguish between primary and secondary effects of Meg3 loss, especially as most studies are correlative and mainly analyze consequences of Meg3 silencing. However, no real mechanistic studies have been performed on how Meg3 is supposed to exert its multiple functions. As lncRNA functions are highly versatile, a better understanding of the mode of action of Meg3 is needed.