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  • br Materials and methods br Results

    2018-11-06


    Materials and methods
    Results
    Discussion The zinc finger protein family is the most abundant protein family in mammals (Lander et al., 2001). The members of this family are subdivided further according to the presence of additional conserved structural regions/motifs (Bellefroid et al., 1991; Collins et al., 2001; Rosati et al., 1991). The KRAB domain-containing zinc finger proteins belong to one such sub-family, account for almost one-third of all zinc finger proteins and constitute the single largest class of transcription factors in the mammalian genome (Looman et al., 2002). Even after three decades of their initial discovery, the function of many KRAB-zinc finger proteins is still largely unknown. Our previous comparative transcriptome analysis between undifferentiated pluripotent stem Talabostat mesylate manufacturer and their differentiated counterparts led to the finding that Zfp819, a member of the KRAB-zinc finger proteins, is specifically expressed in pluripotent stem cells (Meyer et al., 2010). Since the function of Zfp819 is completely unknown, we set out in the present study to systematically analyze the Zfp819 expression pattern in pluripotent stem cells and adult mouse tissues and to determine its function. Our results show that Zfp819 is expressed preferentially in pluripotent stem cells and involved in transcriptional repression of ERVs and maintenance of genomic integrity. We demonstrated that the C-terminal part of Zfp819 harboring several zinc finger motifs is the region responsible for its localization to the nucleus. However, we were unable to identify any well-known NLS in this region, highlighting the presence of novel or non-canonical NLS. Recently, Lee et al. (2006) identified three non-canonical NLS motifs named as PY-NLS, which are recognized by Kapβ2, a nucleocytoplasmic trafficking protein. We identified and characterized four PY-NLS motifs in Zfp819, all of which showed efficient nuclear localization of the tagged EGFP. The open chromatin structure designated by DNA hypomethylation and the presence of active histone modifications is a hallmark of promoter regions of pluripotency-associated genes such as Oct3/4, Sox2, and Nanog (Meshorer and Misteli, 2006; Mikkelsen et al., 2007; Doi et al., 2009). Our DNA methylation and histone modification analyses of the Zfp819 promoter region revealed the presence of open chromatin suggesting that this novel gene might play an essential role in pluripotent cells. It is known that the members of the core pluripotency transcription factor network such as Oct3/4, Sox2, and Nanog mutually cooperate to activate the expression of genes involved in maintenance of pluripotency (Loh et al., 2006; Chen et al., 2008; Kim et al., 2008; Marson et al., 2008). Our confirmation of Oct3/4, Sox2, and Nanog binding to the DCRM-2 of Zfp819 further strengthens that the expression of this gene is in turn regulated by the pluripotency network. Intriguingly, we observed an inverse correlation in expression of some of pluripotency marker genes when the expression of Zfp819 was modulated, suggesting that Zfp819 might also function in the transcriptional regulation of these pluripotency factors. It is well known that many KRAB zinc finger proteins function as transcriptional repressors through their interaction with KAP1 (Friedman et al., 1996). The transcriptional regulator KAP1 is known to maintain the pluripotency and also required for the terminal differentiation of ESCs (Cammas et al., 2002, 2004; Hu et al., 2009). Moreover, KAP1 was shown to be important for maintenance of genomic integrity of ESCs by constitutive repression of ERVs (Rowe et al., 2010). In line with these observations, we found a very high activation of ERVs upon Zfp819 knockdown and could show the interaction of Zfp819 with KAP1, suggesting that Zfp819 might recruit KAP1 to the subset of ERVs and to maintain their repression in ESCs. Recently, we identified Chd4 as one of the interaction partners of Zfp819 in yeast two-hybrid screening experiments and subsequently confirmed this interaction in both in vivo and in vitro approaches (Zheng et al., 2012). Chd4 is a component of the nucleosome-remodeling and histone deacetylase (NuRD) complex, which also consists of Chd3, Hdac1/2, Mta1 and Mta2 (Lai and Wade, 2011). The genetic depletion of Chd4 is known to impair the recruitment of DNA damage response proteins and result in a high sensitivity to DNA damaging agents (Larsen et al., 2010). Both KAP1 and Chd4 have been implicated in DNA damage repair, where they might provide a local chromatin configuration which is necessary for efficient signaling activities and repair (Li et al., 2007; White et al., 2006; Urquhart et al., 2011). It has been shown that in response to genotoxic stress, ATM phosphorylates KAP1, which facilitates its co-localization with several DNA damage response proteins and induces chromatin decondensation (White et al., 2006; Hu et al., 2012). The phosphorylated KAP1 is known to interact with Oct4 and to regulate the expression of pluripotency-related genes in ES cells (Seki et al., 2010). Moreover, it was reported that Oct4 is localized to induced DNA damage regions and this localization occurs simultaneously with the phosphorylation of H2A.X (γH2A.X) (Bartova et al., 2011). These observations suggest that Oct4 not only function in transcriptional regulation of pluripotency genes but also function in genomic integrity maintenance mechanisms of pluripotent cells. In line with this hypothesis, Fong et al. (2011) have identified XPC-RAD23B-CETN2 (XPC) nucleotide excision repair machinery as a stem cell co-activator complex (SCC) for Oct4 and Sox2 mediated transcriptional regulation and suggested the involvement of this multi-protein complex in safeguarding the genomic integrity (Fong et al., 2011). We observed a very high level of spontaneous DNA damage on the basis of high levels of γH2A.X in unstressed Zfp819-KD cells, indicating that these cells are inefficient in DNA damage repair or more prone to DNA damage. In this context, the observed upregulation of Oct4 in Zfp819-KD cells might lead to efficient recruitment of Oct4 to the sites of DNA damage and strive to maintain the genomic integrity. In contrast, the upregulation of various differentiation marker genes in Zfp819-KD cells might indicate that the genomic integrity compromised cells might undergo differentiation mediated apoptosis (Lin et al., 2005). Then, we induced DNA damage in these cells and found the prolonged presence of DNA damage foci, supporting the assumption that Zfp819-KD cells are inefficient in repairing the damage. Similar to these results, Chd4-deficient cells were shown to have reduced DNA damage repair ability (Smeenk et al., 2010), suggesting that both Zfp819 and Chd4 might have overlapping function. Further experiments aimed at identification of Zfp819 targets and in-depth characterization of identified interaction proteins might provide the functional role of Zfp819 in regulation of pluripotency and differentiation networks.