SETDB1 in Early Embryos

The histone methyltransferase SETDB1 contributes to the silencing of local chromatin and the target specificity appears to be determined through various proteins that SETDB1 interacts with. This fundamental function endows SETDB1 with specialized roles in embryonic cells. Keeping the genomic and transcriptomic integrity via proviral silencing and maintaining the pluripotency by repressing the differentiat ion-associated genes have been demonstrated as the roles of SETDB1 in embryonic stem cells. In early developing embryos, SETDB1 exhibits characteristic nuclear mobilizations that might account for its pleiotropic roles in these rapidly changing cells as well. Early lethality of SETDB1-null embryos, along with other immunolocalization findings, suggests that SETDB1 is necessary for reprogramming and preparing the genomes of zygotes and pluripotent cells for the post-implantation developmental program. Introduction SETDB1 (SET domain, bifurcated 1), also known as ESET/ KMT1E, is a histone H3 lysine 9 (H3K9)-specific methyltransferase that is involved in the transcriptional silencing of euchromatic genes. Since it was identified via its interaction with Erg in a yeast two-hybrid screen (Yang et al., 2002), SETDB1 has been shown to bind to various proteins, including KAP-1 (Schultz et al., 2002), SP3 (Stielow et al., 2008), mAM (Wang et al., 2003), DNMT3A (Li et al., 2006), OCT4 (Yeap et al., 2009; Yuan et al., 2009), and the MBD1/CAF-1 chaperone complex (Loyola et al., 2009; Sarraf and Stancheva, 2004), which suggests that SETDB1 is involved in multiple biological processes. In support of this suggestion, recent studies demonstrate that SETDB1 is necessary for proviral silencing and the selfrenewal and lineage specification of embryonic stem cells (ESCs). In addition, SETDB1 is attracting increasing interest owing to its genic amplification and therefore increased expression in patients with melanoma (Ceol et al., 2011; Macgregor et al., 2011) and in lung cancer cells (Rodriguez-Paredes et al., 2013). SETDB1 has also been implicated in neural diseases and development (Chase et al., 2013; Ryu et al., 2006; Tan et al., 2012). Given these findings, the role of SETDB1 is likely to be distinct from that of other H3K9-specific methyltransferases, such as SUV39H1, GLP, and G9A. Briefly, SUV39H1 (also known as KMT1A) is crucial for the establishment of pericentric and telomeric heterochromatin (Peters et al., 2003; Peters et al., 2001; Schotta et al., 2002) and for rDNA silencing (Peng and Karpen, 2007). G9A (also named Eu-HMTase2/ KMT1C), forms a heterodimer with GLP (Ueda et al., 2006), which methylates not only H3K9 but also a number of non-histone substrates, including p53, ACINUS, and LEPTIN (Huang et al., 2010; Lee et al., 2010; Rathert et al., 2008), and also functions in germ cell development (Tachibana et al., 2007). Hence, these methyltransferases seem to methylate different genomic regions and be involved in distinct cellular processes, although there are likely to be collaborations between them in some circumstances (Brower-Toland et al., 2009; Fritsch et al., 2010; Yoon et al., 2008). SETDB1 is a genome-wide chromatin modifier that achieves target specificity via its protein partners. Hence, the existing data on SETDB1 function are, in reality, focused on the regulation of gene expression by transcription factors. However, several lines of evidence suggest that SETDB1 is involved also in other cellular processes than the transcriptional regulation. For example, SETDB1 is an essential structural component of promyelocytic leukemia-nuclear body (PML-NB) (Cho et al., 2011), which is a large proteinaceous structure involved in many diverse cellular processes (reviewed in (Bernardi and Pandolfi, 2007; de The et al., 2012)). Two studies have reported that SETDB1 is localized partly in the cytoplasm and partly in the nucleus (Cho et al., 2013; Loyola et al., 2006). Taken together, the evidence suggests that SETDB1 has additional roles that remain uncharacterized. Here, I will discuss the behavior and the possible function of SETDB1 in cells in early developmental stages, including preimplantation-stage embryos and their derivative pluripotent stem cells, focusing on the immunocytochemical observations, which provide insights into the roles of SETDB1. The molecular mechanisms of gene repression by SETDB1 are outside the scope of this article. Structural features in SETDB1 A protein’s structure is predictive of its behavior. SETDB1 has a structure composed of evolutionarily conserved SET, pre-SET, and post-SET domain responsible for histone methylation (Rea et al., 2000) (Fig. 1A). The preand postSET domains are necessary for SET domain catalytic activity (Schultz et al., 2002). As denoted in the name of the protein, the SET domain of SETDB1 is interrupted by the insertion of several hundred amino acids. Such a split in the SET domain is unique to SETDB1 and is conserved across organisms including humans, worms, and flies. It is currently unknown whether this intervening sequence is required for the catalytic activity of SETDB1, has a particular role in SETDB1 function, or is simply a way of bringing the two parts of split SET domain closer together. SETDB1 also possesses a putative methyl-CpGCurr. Issues Mol. Biol. (2015) 17: 1-10. horizonpress.com/cimb !1 SETDB1 in Early Embryos and Embryonic Stem Cells SETDB1 in Early Embryos Kang binding domain (MBD) and two consecutive Tudor domains. The MBD domain of SETDB1 is of particular interest because it represents the intra-molecular coupling of a methyl-CpG ‘reader’ to the H3K9 methylation ‘writer’, suggesting an interdependent mechanism for the establishment and/or propagation of these two epigenetic marks. However, it remains unknown whether the MBD of SETDB1 can selectively bind methylated DNA. Notably, the MBD domain of SETDB1 contains two DNA-interacting arginine residues that are conserved in the MBD domains of MBD1 (Ohki et al., 2001) and MeCP2 (Ho et al., 2008), suggesting that the MBD domain is functional. Tudor domain proteins are functionally assigned to bind methylated arginine or lysine residues on both histone and non-histone substrates and are implicated in diverse biological processes including RNA metabolism and germ cell development (reviewed in (Chen et al., 2011; Siomi et al., 2010)). The Tudor domain of SETDB1 in mammals is present in a tandem double configuration (Fig. 1A) and, like other double Tudor domain proteins, is thought to recognize certain type(s) of histone lysine methylation. For example, JMJD2A/JHDM3A is a histone demethylase specific for H3K9me3 and H3K36me3 that binds to H3K4me3 (Huang et al., 2006), and mammalian p53 binding protein (53BP1), which is implicated in DNA damage repair, binds to H4K20me1 and H4K20me2 (Botuyan et al., 2006), also reviewed in (Pek et al., 2012; Taverna et al., 2007). The double Tudor domains of SETDB1 might be required for binding to chromatin with double strength, or may bind different histone modifications (Botuyan et al., 2006), each with distinct preference to a certain ligand. It remains unknown which lysine residue(s) and which state(s) of lysine methylation the Tudor domains of SETDB1 recognize, or whether a totally different role is played by these domains. Nuclear SETDB1 and cytoplasmic SETDB1 Microscopic observations suggest that transcriptional regulation via promoter methylation may not be the only function of SETDB1. Immunostaining of SETDB1 in Curr. Issues Mol. Biol. (2015) 17: 1-10. horizonpress.com/cimb !2 A