Combinatorial knockout of RARα, RARβ, and RARγ completely abrogates transcriptional responses to retinoic acid in murine embryonic stem cells

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.

Keywords: CRISPR/Cas; differentiation; embryonic stem cell; pluripotency marker; retinoic acid receptor; retinol; transcriptional regulation; vitamin A.

Figure 1.

Generation of RAR single, double, and triple knockout cell lines. A, strategy and experimental outline. We generated an RAR TKO ES cell line by sequentially abrogating RARγ (33643), RARβ (34523), and RARα (40479) using transiently introduced CRISPR vectors. B, genotyping of RAR KO, DKO, and TKO. In brief, we used PCR to amplify the CRISPR target and assayed for the absence of specific restriction sites (MscI, PcuII, and SacI) present in the WT sequence. C, allelic mapping of introduced CRISPR edits. Two alleles for each gene are depicted and deleted bases are specified by lowercase letters. Note that each genome edit introduces a frameshift mutation in the targeted allele. The CRISPR guide-RNA target sites are indicated above (note that the RARβ guide-RNA targets the complementary strand). Restriction sites employed for genome-edit detections are underlined. Allelic mapping of the additional RAR TKO clone (40453) is described in Fig. S1. D, transcript levels of Cdx1, RARβ2, Cyp26a1, and Hoxa1 RA-responsive genes in CCE WT cells, RARγ knockout (33643), RARγ;RARβ double knockout (34523), and RARγ;RARβ;RARα triple knockout (40479) cells treated with vehicle or RA for 24 or 48 h. *, p ≤ 0.05; **, p ≤ 0.01.

Figure 2.

Growth and transcriptome analysis in WT and RAR TKO ES cells. A, cell numbers in RA-treated conditions relative to untreated conditions. Note that WT cells and RARγ knockout cells (33643) growth arrest in response to RA, whereas no growth arrest is observed for the RAR triple knockout cell lines (40479 and 40453). B, unsupervised clustering analysis groups WT cells into untreated and RA-treated populations, whereas the TKO cells group into clusters of the specific biological repeats. C, genome-wide plot of the effects of RA in WT (left) and RAR TKO (triple knockout ES cells, right). Note that the TKO untreated versus RA-treated cells depict a mirrored distribution around a log -fold change of 1 (10⁰). Statistically significant changes are marked in red (p < 0.01). The few changes observed in transcript levels between vehicle and RA-treated TKO cells were not statistically significant, and were generally associated with low read coverage. Consequently, these nonsignificant differences most likely reflect variations in sequencing coverage. D, transcriptome reads for the top 20 most RA-responsive genes in WT ES cells (up- or down-regulated, left and right, respectively) plotted relative to untreated cells (set as 1.0). Note the absence of RA-responsiveness in the RAR triple knockout cells.

Figure 3.

Transcript levels of RA-responsive genes in WT and RAR TKO ES cells. A, transcript levels of RA-responsive differentiation markers (Cyp26a1, Hoxa1, Cdx1, Stra8, CoupTF1, and Meis1). B, transcript levels of RA-responsive stem cell markers (Nanog, Oct4, Zfp42, Sox2, Sall4, and Klf4) in untreated and RA-treated RAR triple knockout cells. RA-treatment was with 1.0 μm RA for 24 h. The transcriptome data set is derived from triplicate independent experiments, each starting with freshly thawed cells (*, p value ≤ 0.05 relative to untreated cells, or as indicated; ***, p value ≤ 0.001).

Figure 4.

Transcript levels of RARs, RXRs, and imprinted genes in WT and RAR TKO ES cells. A, transcript levels of RAR-dependent imprinted genes (Stmn2, Mest, Tex13, Mobp, Gtl2, and H19). B, transcript levels of RAR and RXR nuclear receptors (RARα, RARβ, RARγ, RXRα, RXRβ, and RXRγ) in untreated and RA-treated RAR triple knockout cells. RA treatment was with 1.0 μm RA for 24 h. The transcriptome data set is derived from triplicate independent experiments, each starting with freshly thawed cells (*, p value ≤ 0.05 relative to untreated cells, or as indicated; **, p value ≤ 0.01; ***, p value ≤ 0.001).