Cell Cycle. 2015;14(10):1596-610. doi: 10.1080/15384101.2015.1026485.
Destabilization of pluripotency in the absence of Mad2l2.
Pirouz M, Rahjouei A, Shamsi F, Eckermann KN, Salinas-Riester G, Pommerenke C, Kessel M.
The induction and maintenance of pluripotency requires the expression of several core factors at appropriate levels (Oct4, Sox2, Klf4, Prdm14). A subset of these proteins (Oct4, Sox2, Prdm14) also plays crucial roles for the establishment of primordial germ cells (PGCs). Here we demonstrate that the Mad2l2 (MAD2B, Rev7) gene product is not only required by PGCs, but also by pluripotent embryonic stem cells (ESCs), depending on the growth conditions. Mad2l2(-/-) ESCs were unstable in LIF/serum medium, and differentiated into primitive endoderm. However, they could be stably propagated using small molecule inhibitors of MAPK signaling. Several components of the MAPK cascade were up- or downregulated even in undifferentiated Mad2l2(-/-) ESCs. Global levels of repressive histone H3 variants were increased in mutant ESCs, and the epigenetic signatures on pluripotency-, primitive endoderm-, and MAPK-related loci differed. Thus, H3K9me2 repressed the Nanog promoter, while the promoter of Gata4 lost H3K27me3 and became de-repressed in LIF/serum condition. Promoters associated with genes involved in MAPK signaling also showed misregulation of these histone marks. Such epigenetic modifications could be indirect consequences of mutating Mad2l2. However, our previous observations suggested the histone methyltransferases as direct (G9a) or indirect (Ezh2) targets of Mad2l2. In effect, the intricate balance necessary for pluripotency becomes perturbed in the absence of Mad2l2.
KEYWORDS: MAP kinase; Mad2B; Rev7; differentiation; embryonic stem cells; pluripotency; primitive endoderm
Mad2l2 is a chromatin binding protein involved in both cell cycle control and DNA repair. Mad2l2 was previously described as an accessory, non-catalytic subunit of the translesion DNA polymerase zeta, and its knockdown led to hypersensitivity towards DNA damage. Mad2l2 appears to function by binding to a diverse spectrum of proteins via its conserved HORMA domain. Accordingly, functions for Mad2l2 were previously claimed in such diverse processes as DNA repair, cell cycle control, and the regulation of gene expression. Previously, we describe a mouse mutant lacking the Mad2l2 gene. Embryos lose PGCs briefly after their specification, and do not proceed in epigenetic reprogramming. We suggested new functions of Mad2l2 as a regulator of epigenetic reprogramming, which is particularly relevant for primordial germ cells, and therefore required for fertility of males and females . Owing to similarities between PGCs and mouse Embryonic Stem Cells (mESCs) , in this work we investigated role of Mad2l2 in the maintenance of pluripotency in mESCs .
Mad2l2-/- ESCs are unstable and deviate into primitive endoderm cells
Mad2l2-/- ESCs grown in conventional LIF/serum condition, are instable and deviate spontaneously toward PrE fate, a process that is independent of apoptosis, but is caused by impaired regulation of MAPK transcripts as well as pluripotency associated genes. Small molecule inhibitors of FGF signaling made the maintenance of undifferentiated Mad2l2-/- ESCs cultures possible. Knockout ESCs represented a hyperactive MAPK pathway indicated by elevated pErk1/2 and pJNK1/2 protein levels. In the ground state culture (LIF/2i conditions), the undifferentiated ESCs were shielded from differentiation triggers by the blocking of the MAPK pathway. Our gene expression profiles demonstrated that several components of the MAPK pathway were indeed affected by the absence of Mad2l2 even in undifferentiated ESCs adapted to LIF/2i. We observed that positive effectors of MAPK signaling were up-, and negative effectors were downregulated. In the absence of Mad2l2, ESCs become hypersensitive to FGF4 stimulation. Expression of pluripotency associated genes, especially of Nanog, was also downregulated In LIF/serum Mad2l2-/- ESCs that destabilize them for further differentiation . Downregulation of Nanog in mouse and human ESCs leads either to primitive endoderm induction [5, 6] or induction of extraembryonic endoderm-associated genes GATA4, and GATA6, together with trophectoderm-associated genes CDX2, and GATA2 . However, in Mad2l2 deficient ESCs, suppression of the Nanog locus via increased deposition of H3K9me2 is accompanied by misregulation of MAPK genes and epigenetic de-repression of the Gata4 locus that deviate Mad2l2-/- ESCs only to primitive endoderm, but not to trophectoderm.
Nanog and Gata4/6 play pivotal roles for the segregation of pluripotent epiblast and primitive endoderm in the early blastocyst. Recently Gata4 was reported to block somatic cell reprogramming by direct repression of Nanog, endorsing their mutual inhibition . Therefore, fine-tuning of Nanog and Gata4 expression is critical for the maintenance of pluripotency in ESCs as well as for its induction, which appeared to be deficient in Mad2l2-/- ESCs.
We observed appearance of Oct4+/Nanog– cells in the periphery of Mad2l2 deficient ESC colonies. These transient cells are unstable and primed for differentiation. Therefore, they rapidly slip through differentiation and first become Oct4+/Nanog–/Gata4+ (similar to primitive endoderm cells at the early blastocysts) and then Oct4–/Nanog–/Gata4+/Sox17+ cells (similar to fully differentiated primitive endoderm cells at the late blastocysts) (Figure 1). During commitment to primitive endoderm lineage, Mad2l2-/- cells acquire a typical differentiated cell cycle and gene expression profiles, epigenetic signature that is distinct from those in undifferentiated wild type ESCs. Thus, Mad2l2 is essential for self-renewal in mouse ESCs and safeguards their pluripotency.
Figure 1. Mad2l2 deficient ESCs are unstable and deviate into primitive endoderm
Top: Spontaneous appearance of differentiated ESCs at the periphery of Mad2l2 knockout colonies. Bottom: These differentiated cells in knockout cultures are positive for Gata4 and Sox17, the two primitive endoderm markers.
Mis-regulation of Nanog promoter as well as MAPK-associated transcripts is the cause of transient appearance of Oct4+/Nanog–/Gata4+ cells, which further differentiate into Oct4–/Nanog–/Gata4+/Sox17+ primitive endoderm cells. These cells lack characteristics of pluripotent cells including typical cell cycle profile, low abundance of repressive histone marks, chimera formation potential, etc. .
Mad2l2: a connection between DNA damage, epigenetic reprogramming, and pluripotency
In conclusion, Mad2l2 is essential for epigenetic reprogramming of mouse early PGCs as well as for pluripotency of mouse ESCs. These functions of Mad2l2 appear unrelated, only in the first glance, to its well-documented role in DNA repair. However, these functions are likely to have evolved together, and can be considered as the two sides of a same coin: the “genome integrity/fidelity”. Mad2l2 is an accessory subunit of DNA polymerase zeta, which points to its role in genome integrity upon DNA damage introduction. It is also essential for genome fidelity of PGCs and pluripotent cells. Both cell types are under an intensive selective pressure to ensure no transmission of genetically or epigenetically defective cells to the next generation or to the embryo proper, respectively. As the result, DNA damage-susceptible Mad2l2-/- PGCs fail to undergo epigenetic reprogramming, and are eliminated before having chance to contribute to the germline development. This happens by prompt apoptosis of all Mad2l2-/- PGCs. In contrast, this phenotype has a less penetrance in pluripotent cells, since only half of the early Mad2l2-/- embryos are lost in vivo. In ESCs, non-authentic Mad2l2-/- cells are eliminated by spontaneous differentiation and loss of pluripotency, in order to avoid a possibility for the generation of DNA damage-susceptible individuals. Thus, Mad2l2 is a molecule in which mechanisms underlying PGC development, pluripotency, and DNA damage are connected to ensure development of authentic PGCs and pluripotent stem cells.
- Pirouz, M., S. Pilarski, and M. Kessel, A critical function of Mad2l2 in primordial germ cell development of mice. PLoS Genet, 2013. 9(8): p. e1003712.
- Pirouz, M., A. Klimke, and M. Kessel, The reciprocal relationship between primordial germ cells and pluripotent stem cells. J Mol Med (Berl), 2012. 90(7): p. 753-61.
- Pirouz, M., et al., Destabilization of pluripotency in the absence of Mad2l2. Cell Cycle, 2015. 14(10): p. 1596-610.
- Kalmar, T., et al., Regulated fluctuations in nanog expression mediate cell fate decisions in embryonic stem cells. PLoS Biol, 2009. 7(7): p. e1000149.
- Hough, S.R., et al., Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog. Stem Cells, 2006. 24(6): p. 1467-75.
- Mitsui, K., et al., The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 2003. 113(5): p. 631-42.
- Hyslop, L., et al., Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells, 2005. 23(8): p. 1035-43.
- Serrano, F., et al., Gata4 blocks somatic cell reprogramming by directly repressing nanog. Stem Cells, 2013. 31(1): p. 71-82.
Mehdi Pirouz; PhD
Stem Cell Program, Boston Children’s Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA