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  • Introduction Open chromatin is a global constitutive feature

    2018-10-24

    Introduction Open chromatin is a global, constitutive feature of naive embryonic stem ON-01910 (ESCs), which are characterized by low levels of repressive histones and DNA methylation (Gaspar-Maia et al., 2011; Meshorer and Misteli, 2006). Diverse factors are required to maintain ESC chromatin in a decondensed state. They are important to stabilize pluripotency and to maintain the potential for self-renewal and differentiation. DPPA3 (also called PGC7 or Stella) is one of the factors known to modulate epigenetic processes in both embryonic and germ cell development (Payer et al., 2003). It is a highly specific, maternal effect gene required for early embryonic cells and primordial germ cells (PGCs), and demarcates naive versus primed pluripotency (Hayashi et al., 2008; Ohinata et al., 2008). Heterogeneous expression of DPPA3 was detected in ESC populations growing in Lif/serum as well as LIF/2i medium (Hayashi et al., 2008; Singer et al., 2014). DPPA3 heterogeneity was concluded to result from a combination of low burst frequencies and large burst sizes (Singer et al., 2014). DPPA3 is known to be involved in the maintenance of the low level of DNA methylation typical for early embryonic cells as well as ESCs (Hayashi et al., 2008). At the same time, it protects imprinted loci against demethylation (Nakamura et al., 2007). DPPA3 is required for the generation of fully reprogrammed pluripotent stem cells, in particular for the erasure of the epigenetic memory encoded in the DNA methylation pattern (Xu et al., 2015b). Both ESCs and PGCs, the two cell types characterized by the expression of DPPA3, require the MAD2L2 protein for the proper establishment of their epigenetic status (Pirouz et al., 2013, 2015). In the absence of Mad2L2 PGCs do not reprogram toward a more flexible chromatin configuration after their induction, and ESCs are not able to propagate their typical open chromatin, which is necessary for stable pluripotency. MAD2L2 (also called REV7 or MAD2B) was originally identified by its function in DNA repair, playing an accessory role in translesion DNA repair and an inhibitory role in the resection of 5′ ends after DNA double-strand breaks and at unprotected telomeres (Boersma et al., 2015; Xu et al., 2015a). As a downstream component of the DNA damage response pathway, MAD2L2 promotes non-homologous end joining versus homologous recombination. The role of MAD2L2 in DNA repair, ESC development, and PGC development is tightly connected to the epigenetic status of the respective cells. To investigate the relationship between MAD2L2 and the formation of open chromatin we compared wild-type and Mad2l2-deficient ESCs, employing super-resolution microscopy, transcriptome sequencing, methylome sequencing, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis. We identified Dppa3 as a downstream target of MAD2L2, and derived a model explaining the epigenetic effects of MAD2L2.
    Results
    Discussion The absence of MAD2L2 in mutant ESCs corresponds with the low MAD2L2 levels observed in somatic cells (Figure 4E). Cells with a low level of MAD2L2 have in common that the inhibitory binding of MAD2L2 to EHMT1 and EHMT2 is reduced (Pirouz et al., 2013). Thus, histone H3 can be efficiently methylated on residue K9, and H3K9me2 can then form a docking place for further epigenetic regulators. DNA methyltransferase DNMT1 is recruited together with UHRF1 and EHMT2 at the replication fork, where it will also bind to proliferating cell nuclear antigen, and cooperatively to hemimethylated DNA (Esteve et al., 2006; Liu et al., 2013; Rothbart et al., 2012). Together, these and other interacting factors represent an assembly of both chromatin writers and readers, which maintain and establish a cell-specific chromatin configuration (Liu et al., 2013; Shinkai and Tachibana, 2011). Collectively, the low levels of MAD2L2 and DPPA3 correspond to high levels of repressive histone modifications, substantial DNA methylation, and thus a closed chromatin configuration. In contrast, MAD2l2 levels are high in wild-type ESCs and PGCLCs, downstream of a cascade resembling the DNA damage response pathway (Figures 1A–1I and S2A). In such cells MAD2L2 can directly bind to both EHMT1 and EHMT2, suppress EHMT2 expression, and thus inhibit histone H3K9 and DNA methylation (Pirouz et al., 2013). By antagonizing the formation of H3K9me2, MAD2L2 will derepress the Dppa3 gene. DPPA3 can then displace DNMT1 from UHRF1 at the replication fork, so that the growing strand cannot be methylated (Figure 4F; Funaki et al., 2014). In any case, the combined presence of MAD2L2 and DPPA3 in ESCs correlates with their generally low levels of both DNA and histone H3K9 methylation, i.e., a generally open chromatin configuration. In conclusion, the global promotion of open chromatin, and the inhibition of heterochromatin in ESCs and germ cells (PGCLCs and PGCs) depend critically on MAD2L2. The local opening of chromatin observed around DNA breaks may involve MAD2L2 and a similar mechanism (Noon et al., 2010). It is conceivable that the major importance of MAD2L2 lies in its epigenetic functions, be it in a stem or germ cell context, or in response to DNA damage.