In summary we show that calreticulin

In summary, we show that calreticulin and TGF-β signaling intersect downstream of TGF-β receptors I and II. The presence of calreticulin permits TGF-β-mediated inactivation of GSK3β and thus promotes SNAIL2/SLUG-regulated repression of E-cadherin. This condition is sine qua non for the expression and activation of EMT components and, further downstream, cardiac factors during cardiomyocyte differentiation. We demonstrate here, that regulated Ca2+ signaling from calreticulin to calcineurin is critical for unabated TGF-β signaling necessary for the exit from pluripotency and cadherin switch during EMT, and commencement of cardiomyogenesis from mouse ESCs.
Experimental Procedures
Author Contributions
Acknowledgments We are indebted to Dr. Joanne E. Murphy-Ullrich and Dr. Sylvia Papp for critical reading of the manuscript. M.O. is a member of the Ontario Stem Cell initiative and a member of the Heart & Stroke/Richard Lewar Centre of Excellence. This work was supported by grants from CIHRMOP-102549 and MOP-106461 to M.O.
Introduction Early mouse zygotes differentiate in the trophectoderm and inner cell mass (ICM). Mouse embryonic stem 5-fluorocytosine (ESCs) are derived from the ICM in mouse embryos (Evans and Kaufman, 1981; Martin, 1981). ESCs are usually maintained in serum supplemented with leukemia inhibitory factor (LIF), which keeps them in a pluripotent state capable of self-renewal (Smith et al., 1988; Williams et al., 1988) through the activation of JAK-STAT3 signaling (Matsuda et al., 1999; Niwa et al., 1998). Serum/LIF generates a heterogeneous population of ESCs by causing auto-inductive stimulation of the MAPK/ERK pathway by fibroblast growth factor (FGF) 4 (Kunath et al., 2007; Stavridis et al., 2007; Ying et al., 2008). Ying et al. (2008) proposed that LIF and bone morphogenetic protein signals act downstream of the ERK pathway to block ESC commitment. To maintain a ground state in ESCs, they used three selective small-molecule inhibitors, SU5402, PD0325901, and CHIR99021, to inhibit FGF receptor (FGFR) tyrosine kinases, the ERK pathway, and Wnt signaling, respectively (Ying et al., 2008). Later, the use of two inhibitors, PD0325901 and CHIR99021 (called 2i), with LIF to block the MAPK/ERK and glycogen synthase kinase 3β (GSK3β) pathways was postulated to be sufficient to maintain the ESC ground state (Silva et al., 2008). ESCs cultivated in a serum-free 2i medium with LIF (2i ESCs) exhibit greater pluripotent gene expression than ESCs cultivated in serum with LIF (serum ESCs). Further, 2i ESCs homogeneously express NANOG, which potentiates pluripotent gene transcription by creating a permissive chromatin structure (Marks et al., 2012; Marks and Stunnenberg, 2014; Miyanari and Torres-Padilla, 2012; Silva et al., 2009). In addition, 2i leads to genome-wide DNA hypomethylation due to reduced expression of the DNA methyltransferase 3 (DNMT3) family (Bagci and Fisher, 2013; Leitch et al., 2013). The mechanism by which 2i creates a permissive chromatin structure and downregulates DNMT3 expression remains undefined. DNA methylation by the DNMT family is a heritable epigenetic modification involved in gene silencing, imprinting, and retrotransposon suppression (Baylin, 2005; Jin et al., 2011). In mammals, there are four major members of the DNMT family: DNMT1, DNMT3A, DNMT3B, and DNMT3L. DNMT3A and DNMT3B share similar domain structures: an N-terminal variable region, followed by a PWWP domain, a cysteine-rich zinc-binding domain, and a C-terminal catalytic domain. DNMT3L has no catalytic activity by itself because it lacks the C-terminal catalytic domain (Subramaniam et al., 2014). The DNMT family is dynamically regulated during mouse development (Smith et al., 2012). Global DNA hypomethylation by Dnmt1−/− and Dnmt3a−/−,3b−/− in ESCs blocks differentiation and induces histone hyperacetylation (Lei et al., 1996; Okano et al., 1999). In ESCs, G9a, a nuclear histone lysine methyltransferase (HMT) that methylates histones H1, H3K9, and H3K27 (Tachibana et al., 2001; Wu et al., 2011), recruits DNMT3A/B independently of its HMT activity (Epsztejn-Litman et al., 2008). G9a contains a SET domain with ankyrin repeats and mediates the transfer of one to three methyl groups from S-adenosylmethionine to the amino group of a target lysine (Esteve et al., 2005), resulting in gene silencing (Dillon et al., 2005; Tachibana et al., 2001). G9a also methylates non-histone proteins, such as p53, WIZ, CDYL1, ACINUS, REPTIN, MYOD, and DNMT1, to regulate chromatin structure and transcriptional machinery (Jung et al., 2015; Ling et al., 2012; Rathert et al., 2008). The mechanism underlying G9a-mediated DNMT3A/B regulation in ESCs is not well understood.