Phosphorylation of the retinoic acid receptor RARγ2 is crucial for the neuronal differentiation of mouse embryonic stem cells

Abstract

BSTRACTRetinoic acid (RA) plays key roles in cell differentiation and growth arrest by activating nuclear RA receptors (RARs) (a, b and c), which are ligand-dependent transcription factors. RARs are also phosphorylated in response to RA. Here, we investigated the in vivo relevance of the phosphorylation of RARs during RA-induced neuronal differentiation of mouse embryonic stem cells (mESCs). Using ESCs where the genes encoding each RAR subtype had been inactivated, and stable rescue lines expressing RARs mutated in phospho-acceptor sites, we show that RA-induced neuronal differentiation involves RARc2 and requires RARc2 phosphorylation. By gene expression profiling, we found that the phosphorylated form of RARc2 regulates a small subset of genes through binding an unusual RA response element consisting of two direct repeats with a seven-base-pair spacer. These new findings suggest an important role for RARc phosphorylation during cell differentiation and pave the way for further investigations during embryonic development.

Nuclear and Extra-Nuclear Effects of Retinoid Acid Receptors: How They Are Interconnected

“…To validate such a hypothesis, RNA-seq and ChIP experiments were performed with mutant mouse embryonic stem cell (mESC) lines expressing RAR phosphomutants in a RAR null background. Such experiments highlighted direct target genes whose expression is controlled by phosphorylation of the N-terminal serine residue of RARγ [2]. These studies also revealed that only RAREs with specific spacings recruit the phosphorylated form of RARγ in response to RA [2].…”

“…Such experiments highlighted direct target genes whose expression is controlled by phosphorylation of the N-terminal serine residue of RARγ [2]. These studies also revealed that only RAREs with specific spacings recruit the phosphorylated form of RARγ in response to RA [2]. Then, the question was how phosphorylation of the N-terminal serine could promote recruitment of RARγ to specific RAREs in response to RA.…”

Retinoic Acid and the Development of the Endoderm

“…For instance, RAR-DNA interactions appear after the addition of RA [70], suggesting that receptors lacking RA are not necessarily bound to DNA or that unbound receptors do not immunoprecipitate well. In addition, RAR phosphorylation has demonstrated the importance in regulating the activity of the receptor [71,72]. Specifically, deleting an RARγ2 in mouse ES cells and replacing it with the same receptor, but with mutated phospho-acceptor sites, demonstrated that phosphorylation was necessary for differentiation of these cells into neurons.…”

A Brn2–Zic1 axis specifies the neuronal fate of retinoic-acid-treated embryonic stem cells

“…Retinoic acid can be used to differentiate mouse embryonic stem cells (ESCs) into a highly homogenous population of glutamatergic neurons (Bibel et al, 2007(Bibel et al, , 2004Götz and Barde, 2005) mimicking the generation of cortical glutamatergic neurons from Pax6-expressing progeny in the developing mouse cortex (Götz and Barde, 2005). The initial trigger for differentiation is the activation of target genes by the retinoic acid receptors (RARs), in particular the RARγ2 isoform (Al Tanoury et al, 2014). ESCs can also be differentiated into neuronal precursor cells by other techniques, such as by treatment with Fgf2 (Okabe et al, 1996;Wichterle et al, 2002).…”

“…Although the initial events in retinoic-acid-induced differentiation have been well characterised (Mahony et al, 2011;Moutier et al, 2012;Al Tanoury et al, 2014), the downstream transcription factors required for specifying neural fate are unknown. Here, we show that Brn2 is essential for retinoic-acidinduced neuronal differentiation of ESCs.…”