Abstract
All-trans-retinoic acid (RA), a potent inducer of cellular differentiation, functions as a ligand for retinoic acid receptors (RARα, β, and γ). RARs are activated by ligand binding, which induces transcription of direct genomic targets. However, whether embryonic stem cells respond to RA through routes that do not involve RARs is unknown. Here, we used CRISPR technology to introduce biallelic frameshift mutations in RARα, RARβ, and RARγ, thereby abrogating all RAR functions in murine embryonic stem cells. We then evaluated RA-responsiveness of the RAR-null cells using RNA-Seq transcriptome analysis. We found that the RAR-null cells display no changes in transcripts in response to RA, demonstrating that the RARs are essential for the regulation of all transcripts in murine embryonic stem cells in response to RA. Our key finding, that in embryonic stem cells the transcriptional effects of RA all depend on RARs, addresses a long-standing topic of discussion in the field of retinoic acid signaling.
Highlights
All-trans–retinoic acid (RA), a potent inducer of cellular differentiation, functions as a ligand for retinoic acid receptors (RAR␣, , and ␥)
We found that RA disrupts the leukemia inhibitory factor receptor (LIFR) signaling, altering cellular phosphorylation [8], but the exact mechanisms of RA-dependent transcriptional gene repression have yet to be determined
We evaluated transcript levels in WT and RAR triple knockout (TKO) ES cells, and found that transcript levels of RAR␣, RAR, and RAR␥ were increased in WT cells, but unchanged in the TKO cells in response to RA (Fig. 4B)
Summary
All-trans–retinoic acid (RA), a potent inducer of cellular differentiation, functions as a ligand for retinoic acid receptors (RAR␣, , and ␥). In HepG2 cells, the translocation of Rbp (Srbp) bound retinol to Rbp (Crbp1) was demonstrated to activate the Jak/ Stat phosphorylation cascade through the Stra membrane receptor [40] It remains to be determined whether this intersects with the LIFR pathway, but if so, this pathway could potentially account for the altered cellular phosphorylation in response to vitamin A. RA may elicit transcriptional responses 1) through RARs ␣, , and ␥; 2) through nuclear receptors other than the RARs; and 3) through a nongenomic phosphorylation cascade, possibly involving RARs located in the cytoplasm [38] These three routes of RA signaling were identified in various biochemical assays and cell types, yet it remains to be determined how many of these routes are functional in ES cells. In murine ES cells the RARs provide the only route for RA to regulate transcript levels
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