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

Laursen, Kristian B.; Gudas, Lorraine J.

doi:10.1074/jbc.ra118.001951

Cited by 18 publications

(20 citation statements)

References 60 publications

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“…To define the role of RAR␥ in EtOH-mediated transcription in more depth, we used an ESC line in which both alleles of a target sequence in exon 8 of RAR␥ were deleted by CRISPR knockout (RAR␥E8 Ϫ/Ϫ ) ( Fig. S2A) (26). We cultured WT and RAR␥E8 Ϫ/Ϫ ESCs with 40 mM EtOH for 48 h and found that transcript levels of Hoxa1 (11.6 Ϯ 2.2-fold, p ϭ 0.008), Cyp26a1 (9.1 Ϯ 1.1-fold, p ϭ 0.002), RAR␤2 (6.7 Ϯ 1.8-fold, p ϭ 0.034), Crabp2 (5.3 Ϯ 1.1-fold, p ϭ 0.018), Cdx1 (20.2 Ϯ 4.4-fold, p ϭ 0.012), Hnf1␤, (4.8 Ϯ 1.3-fold, p ϭ 0.044), and the long noncoding RNA Hotairm1 (8.9 Ϯ 1.3-fold, p ϭ 0.003) (30), increased in WT ESCs compared with vehicle-treated cells.…”

Section: Rar␥ Is Required For Ethanol Regulation Of Genes Involved Inmentioning

confidence: 99%

Ethanol promotes differentiation of embryonic stem cells through retinoic acid receptor-γ

Serio

Laursen

Urvalek

et al. 2019

Journal of Biological Chemistry

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Edited by Joel M. Gottesfeld Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ ROL) can be oxidized to all-trans-retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH's effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-␥ (RAR␥) knockout ESC lines generated by CRISPR/ Cas9 and homologous recombination, we demonstrate that EtOH signals via RAR␥ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. We also report that EtOH-mediated increases in homeobox A1 (Hoxa1) and cytochrome P450 family 26 subfamily A member 1 (Cyp26a1) transcripts, direct RA target genes, require the expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1 family member A2 (Aldh1a2), suggesting that EtOH-mediated induction of Hoxa1 and Cyp26a1 requires ROL from the serum. As shown with CRISPR/Cas9 knockout lines, the retinol dehydrogenase gene Rdh10 and a functional RARE in the ROL transporter stimulated by retinoic acid 6 (Stra6) gene are required for EtOH induction of Hoxa1 and Cyp26a1. We conclude that EtOH stimulates stem cell differentiation by increasing the influx and metabolism of ROL for downstream RAR␥-dependent transcription. In stem cells, EtOH may shift cell fate decisions to alter developmental outcomes by increasing endogenous ROL/RA signaling via increased Stra6 expression and ROL oxidation. Complex regulatory circuitry is required for maintaining the properties of stem cells so that symmetric self-renewal is not diverted prematurely to drive differentiation toward terminal cell fates (1). High levels of blood ethanol (EtOH) lead to aberrant regulation of normal differentiation in developing embryos and fetuses, making EtOH a teratogen (2-4). Unraveling the mechanisms by which EtOH interferes with endogenous cell signaling pathways that control differentiation is critical for understanding EtOH-mediated toxic effects which lead to disease states that arise during development, i.e. fetal alcohol spectrum disorders (FASD) 2 (5) and diseases such as cancers, which are caused in part by changes in cell plasticity (6). Of particular importance in stem cell differentiation is the interaction between EtOH and the vitamin A (retinol, ROL) metabolite all-trans-retinoic acid (RA), which lies at the nexus of physiological differentiation of stem cells (7, 8). Dysregulated RA signaling (from either supraphysiological or subphysiological levels) leads to several teratogenic phenotypes in common with EtOH (9-11). Several studies have shown interactions between EtOH and RA signaling (4, 12-16), possibly because of the similarities in metabolism between ROL and EtOH. Both EtOH and ROL metabolism rely on parallel two-step o...

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Section: Rar␥ Is Required For Ethanol Regulation Of Genes Involved Inmentioning

confidence: 99%

Ethanol promotes differentiation of embryonic stem cells through retinoic acid receptor-γ

Serio

Laursen

Urvalek

et al. 2019

Journal of Biological Chemistry

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“…We have previously shown that EtOH‐mediated differentiation of ESCs requires RA:RAR γ ‐dependent transcription (Serio et al., ). To determine whether the effects of AcH on ESC differentiation are likewise dependent on RA signaling, we used RAR γ −/− ESCs previously generated in our laboratory (Laursen and Gudas, ). We treated WT, Aldh2 −/− , and RAR γ −/− ESCs with either 40 mM EtOH or 1 mM AcH to determine whether AcH‐dependent increases in differentiation‐associated transcripts proceed via RAR γ and are thus dependent on RA signaling.…”

Section: Resultsmentioning

confidence: 99%

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation

Serio

Gross

et al. 2019

Alcoholism Clin & Exp Res

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Background: Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation.Methods: We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation.Results: By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARb2) upon EtOH treatment of WT and Acss2 À/À ESCs, but not Aldh2 À/À ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-c (RARc) is required for both EtOH-and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARc signaling in AcH-induced ESC differentiation.Conclusions: ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARc.

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“…Genetic deletion of a single RAR is not embryonically lethal, most likely due to a compensatory effect by other RARs; however, double knockouts of Rara/Rarb, Rara/Rarg, or Rarb/Rarg die in utero . Though, it has been proposed that RA may also carry out biological activities independent of RARs via alternate receptors (Kruse et al, 2008;Shaw, Elholm, & Noy, 2003;Zhou, Suino-Powell, et al, 2011), the findings have not been independently validated and recent studies of triple RARα, β, and γ-ablated cells argue that in stem cells, RA functions via its cognate RAR receptors (Borland et al, 2008(Borland et al, , 2011Laursen & Gudas, 2018;Rieck, Meissner, Ries, Muller-Brusselbach, & Muller, 2008). RXRβ appears to be required for spermatogenesis while RXRα is required for the normal development of the eye and the growth of the compact layer of the myocardium (Kastner et al, 1994Merki et al, 2005;Ruiz-Lozano et al, 1998;Sucov et al, 1994).…”

Section: Retinoic Acid As Ligand Of Nuclear Hormone Receptorsmentioning

confidence: 99%

“…The fact that a transcriptionally silent RXRα (lacking the activation domains AF1 and AF2) supports myocardial growth argues that, in this instance, RXRα functions as the heterodimeric partner of another nuclear receptor and not as a homodimer (Mascrez, Ghyselinck, Chambon, & Mark, 2009). Though, it has been proposed that RA may also carry out biological activities independent of RARs via alternate receptors (Kruse et al, 2008;Shaw, Elholm, & Noy, 2003;Zhou, Suino-Powell, et al, 2011), the findings have not been independently validated and recent studies of triple RARα, β, and γ-ablated cells argue that in stem cells, RA functions via its cognate RAR receptors (Borland et al, 2008(Borland et al, , 2011Laursen & Gudas, 2018;Rieck, Meissner, Ries, Muller-Brusselbach, & Muller, 2008).…”

Section: Retinoic Acid As Ligand Of Nuclear Hormone Receptorsmentioning

confidence: 99%

Recent insights on the role and regulation of retinoic acid signaling during epicardial development

Wang

Moise

2019

Genesis

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The vitamin A metabolite, retinoic acid, carries out essential and conserved roles in vertebrate heart development. Retinoic acid signals via retinoic acid receptors (RAR)/retinoid X receptors (RXRs) heterodimers to induce the expression of genes that control cell fate specification, proliferation, and differentiation. Alterations in retinoic acid levels are often associated with congenital heart defects. Therefore, embryonic levels of retinoic acid need to be carefully regulated through the activity of enzymes, binding proteins and transporters involved in vitamin A metabolism. Here, we review evidence of the complex mechanisms that control the fetal uptake and synthesis of retinoic acid from vitamin A precursors. Next, we highlight recent evidence of the role of retinoic acid in orchestrating myocardial compact zone growth and coronary vascular development.

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

Cited by 18 publications

References 60 publications

Ethanol promotes differentiation of embryonic stem cells through retinoic acid receptor-γ

Ethanol promotes differentiation of embryonic stem cells through retinoic acid receptor-γ

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation

Recent insights on the role and regulation of retinoic acid signaling during epicardial development

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