AUBPs are critical regulators of developmental gene expression which respond to inter- and intracellular signals. Some specific examples of this regulation include the repression of Notch1 in response to PI3K/Erk MAP kinase signals during T-cell development (93), repression of TNFalpha in response to p38/Erk MAP kinase signals during inflammation (94), and the stabilization of p21 in response to ATR/ATM kinase activation after DNA damage (95). Here we demonstrate AUBPs mechanistically connect FGF/Erk MAP kinase signaling to the regulation of pluripotency, self-renewal and differentiation in mESCs. More specifically, control over Brf1 expression rapidly regulates the expression of key pluripotency-associated genes, reduces the capacity to self-renew, and enhances mesendoderm differentiation upon LIF withdrawal.
Why has Brf1 been selected for implementing FGF-dependent cellular responses in mESCs? One possibility is that, owing to its rapid transcriptional response to FGF signaling and its short protein and mRNA half-life (1.5 hours and 1 hour, respectively), Brf1 is capable of tracking dynamic changes in Erk MAP kinase activity. Brf1 directly affects mRNA abundance, and provides stem cell populations with a mechanism to quickly respond to changes in FGF signaling, without necessarily altering underlying transcriptional states. However, the benefit of these dynamical properties to the biology of pluripotent or differentiating mESCs still remains unclear. Brf1 also provides a regulation that is similar to miRNAs, which also provide mRNA-level repression while maintaining flexibility in target selection. Interestingly, Zfp36 AUBPs and miRNAs are known to cooperate in regulating some mRNA targets (96), indicating a potential point
of convergence between these two regulatory mechanisms. Future work will address why this system is particularly well adapted to serve as a regulator in mESCs, and as a mediator of FGF/Erk MAP kinase signaling.
Whether Brf1 plays a similar role in the embryo is unclear. We note that the regulatory effects of Brf1 in cell culture mimic the developmental response to FGF/Erk MAP kinase signaling at different stages of embryonic development. For example, FGF4 signaling promotes extra-embryonic endoderm differentiation in the inner cell mass partly by destabilizing the expression of pluripotency genes (97). Brf1 may participate in this process by repressing Nanog and other pluripotency regulators. At a slightly later developmental stage, and one that is more relevant to mESCs, both FGF4 and FGF8 are required for mesoderm induction (98, 99). The expression of Brachyury, a regulator of mesoderm morphogenesis, is enhanced by FGF4 and FGF8 signaling through unknown mechanisms (100). Our results show that Brf1 expression during differentiation in mESCs similarly enhances Brachyury and other primitive streak markers. This regulatory connection could explain why Brf1 knockout mice also exhibit the same gross defects in chorio-allantoic fusion, neural tube closure and placental organization at mid-gestation (E11) as FGFR2 knockouts, an indication of shared regulation and function (74, 101).
Although the regulation of FGF/Erk MAP kinase signaling likely differs between the embryonic and cell culture context, these observations implicate Brf1 as an important FGF/Erk MAP kinase-inducible regulator of development in both systems.
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