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    • BRIGHT was previously identified by

    2018-11-12

    BRIGHT was previously identified by mass spectrometry (MS) as a secondary binding partner of enforced expression of NANOG in mESCs (Wang et al., 2006), but no follow-up analysis was performed. Employing coimmunoprecipitation in mESCs, we confirmed endogenous interactions of BRIGHT with NANOG as well as with OCT4 and SOX2 (Figure 4A). These data, along with our inability to detect any of these interactions when each was overexpressed pairwise in somatic purinergic receptor (data not shown), prompted a parallel examination of BRIGHT function in mESCs. BRIGHT transcript expression increased significantly following in vitro differentiation, an inverse correlation with core factor expression (Figure 4B). Differentiation led to increased BRIGHT protein accumulation within the nuclear matrix (Figure 4C), the region shown to be the most active site of BRIGHT transcriptional activity (Zong et al., 2000). In mESCs, purinergic receptor stable overexpression of BRIGHT at levels below those required to initiate differentiation (Figure S5A) led to its recruitment to Oct4, Sox2, and Nanog promoters (Figure 4D). Thus, reciprocal to what is observed for loss of BRIGHT in somatic cells, we reasoned that an increase in levels of BRIGHT, in association with ESC-specific interacting proteins, might repress core pluripotency factor transcription. Accordingly, the endogenous loci of Oct4, Sox2, and Nanog were downregulated following overexpression of BRIGHT in undifferentiated mESCs and in the mouse embryonic carcinoma cell line p19 (Figure 4E; Figure S4B). Employing luciferase reporters that contained the promoter/enhancer regions shown in Figure 3B, we observed strong repression following transient BRIGHT overexpression, regardless of the mESC differentiation state (Figure 4F; Figure S4C). These data indicate that BRIGHT directly represses transcription of core pluripotency factors and suggest a role for BRIGHT as an activator of differentiation.
    Discussion We have demonstrated that complete loss of Bright expression in MEFs is alone sufficient for both induction of somatic cell reprogramming and for increased efficiency of conventional iPSC reprogramming. We suggest that at least three separate steps contribute to the mechanism by which Bright loss facilitates reprogramming. First, Bright KO-MEFs are refractory to cellular senescence, promoting somatic self-renewal (Figure 1B). Telomere shortening and activation of Rb or p53 are key senescence-inducing factors (Zhao and Daley, 2008). Neither cell cycle nor signature transcripts of these families were significantly altered in KO-MEFs (data not shown). However, that BRIGHT interacts with and is activated by p53 (Lestari et al., 2012), a previously established barrier to reprogramming (Li et al., 2009), suggests that Bright loss bypasses senescence through a mechanism other than transcriptional derepression of pluripotency factors, alleviating the requirement for derepression as the initiating step to reprogramming. Unlike loss or mutation of p53, Bright KO-MEFs do not undergo genomic instability at a level detectable by karyotype (data not shown). Second, loss of Bright leads to direct derepression of key regulators of pluripotency. This conclusion is supported by our observations that (1) BRIGHT upregulation and nuclear matrix localization (Figures 4B and 4C) accompany mESC differentiation and the well-established downregulation of OCT4, SOX2, and NANOG in mESCs (De Miguel et al., 2010), (2) BRIGHT is recruited to promoter/enhancer regions of these factors in MEFs and mESCs (Figures 3A and 4D), and (3) BRIGHT overexpression in mESCs represses both endogenous loci and reporter transcription of Oct4, Sox2, and Nanog (Figures 4E and 4F). Loss of BRIGHT repression, in conjunction with activation of the leukemia-inhibitory factor (LIF) signaling pathway, may be key to BRIGHT-mediated reprogramming. Third, loss of Bright in MEFs might disrupt signaling pathways shown to antagonize pluripotency through core factor repression. One such pathway, Activin/TGF-β, is upstream of BRIGHT in human lung (Lin et al., 2008) and in Xenopus gastrulation, where BRIGHT is required for mesoderm differentiation (Callery et al., 2005). Likely additional, as-yet-uncharacterized signal pathways are altered by loss of Bright following transfer of KO-MEFs to LIF-augmented cultures. These data further suggest that a normal function of BRIGHT is to promote and maintain cell differentiation.