SIRT1-mediated deacetylation of CRABPII regulates cellular retinoic acid signaling and modulates embryonic stem cell differentiation.
ABSTRACT: Retinoid homeostasis is critical for normal embryonic development. Both the deficiency and excess of these compounds are associated with congenital malformations. Here we demonstrate that SIRT1, the most conserved mammalian NAD?-dependent protein deacetylase, contributes to homeostatic retinoic acid (RA) signaling and modulates mouse embryonic stem cell (mESC) differentiation in part through deacetylation of cellular retinoic acid binding protein II (CRABPII). We show that RA-mediated acetylation of CRABPII at K102 is essential for its nuclear accumulation and subsequent activation of RA signaling. SIRT1 interacts with and deacetylates CRABPII, regulating its subcellular localization. Consequently, SIRT1 deficiency induces hyperacetylation and nuclear accumulation of CRABPII, enhancing RA signaling and accelerating mESC differentiation in response to RA. Consistently, SIRT1 deficiency is associated with elevated RA signaling and development defects in mice. Our findings reveal a molecular mechanism that regulates RA signaling and highlight the importance of SIRT1 in regulation of ESC pluripotency and embryogenesis.
Project description:Retinoid homeostasis is critical for normal embryonic development, and both the deficiency and excess of these compounds are associated with congenital malformations. Here we found that SIRT1, the most conserved mammalian NAD+-dependent deacetylase, contributes to the maintenance of homeostatic retinoic acid (RA) signaling and modulates mouse embryonic stem cell (mESC) differentiation. Our data show that SIRT1 deficiency enhances RA signaling, thereby accelerating mES cell differentiation in response to RA. Our findings highlight the importance of SIRT1 in transcriptional regulation of ESC pluripotency and embryogenesis. Three pairs of sh-Control and sh-SIRT1 E14 mESC cells (with dulpicate for each sample) were treated with vehicle ethanol or with 20 nM of RA for 2 days. Total RNA was isolated using a Qiagen RNA easy mini kit with on-column DNAseI treatment. RNA quality was validated with the Agilent 2100 Bioanalyzer in the microarray facility. Three-pairs of ethanol treated samples, and 4 RA treated sh-Control, and 6 RA treated sh-SIRT1 samples were analyzed by Agilent Whole Mouse Genome 4x44 formate oligo arrays (014868) (Agilent Technologies) following the Agilent 1-color microarray-based gene expression analysis protocol.
Project description:Cellular retinoic acid-binding protein II (CRABP-II) undergoes nuclear translocation upon binding of retinoic acid (RA). In the nucleus, CRABP-II directly binds to the nuclear receptor RAR to form a complex through which RA is "channeled" from the binding protein to the receptor. CRABP-II thus facilitates the ligation of RAR and markedly enhances its transcriptional activity. The primary sequence of CRABP-II contains three putative SUMOylation sites, centered at K45, K87, and K102. We show here that RA induces interactions of CRABP-II with the E2 SUMO ligase Ubc9 and triggers SUMOylation of the protein both in vitro and in cultured cells. Mutagenesis analyses demonstrate that K102 is the sole CRABP-II residue to be SUMOylated in response to RA. Mutation of this residue abolishes the ability of CRABP-II to undergo nuclear translocation in response RA and thus impairs CRABP-II-mediated activation of RAR. Additional observations demonstrate that apo-CRABP-II is associated with endoplasmic reticulum (ER), and that RA triggers the dissociation of CRABP-II from this location. Furthermore, we show that RA-induced dissociation of CRABP-II from the ER requires SUMOylation of K102. Hence, SUMOylation of K102 in response to RA binding is critical for dissociation of CRABP-II from ER and, consequently, for mobilization of the protein to nucleus and for its cooperation with RAR.
Project description:BACKGROUND AND OBJECTIVES:SIRT1, a histone diacetylase, modify transactivation function of various transcription factor including p53 and NF-?B. p53 and NF-?B is involved in in vitro differentiation of mouse embryonic stem cells (mESC) into mouse embryoid body (mEB). These suggest that SIRT1 might affect in vitro differentiation of mESC into mEB by regulation of p53 and NF-?B. METHODS AND RESULTS:In this study we analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC. To examine SIRT1-specific gene in mESC, this study conducted microarray-based differential gene expression analysis between wild and SIRT1 knockout mESC. Comparing their gene expression patterns, this study determined a list of genes regulated by SIRT1. cDNA microarray data-set analysis revealed that genes associated with transcription and signal transduction are significantly modified in SIRT1 knockout mESC. cDNA microarray data-set analysis between mESC and EB in wild and SIRT1 showed that SIRT1 inhibits p53 signaling pathway but not affect NF-?B signaling pathway. CONCLUSIONS:This study suggests that SIRT1 modify mESC differentiation by regulation of p53 transcriptional activity.
Project description:Retinoic acid (RA) is a potent morphogen required for embryonic development. RA is formed in a multistep process from vitamin A (retinol); RA acts in a paracrine fashion to shape the developing eye and is essential for normal optic vesicle and anterior segment formation. Perturbation in RA-signaling can result in severe ocular developmental diseases-including microphthalmia, anophthalmia, and coloboma. RA-signaling is also essential for embryonic development and life, as indicated by the significant consequences of mutations in genes involved in RA-signaling. The requirement of RA-signaling for normal development is further supported by the manifestation of severe pathologies in animal models of RA deficiency-such as ventral lens rotation, failure of optic cup formation, and embryonic and postnatal lethality. In this review, we summarize RA-signaling, recent advances in our understanding of this pathway in eye development, and the requirement of RA-signaling for embryonic development (e.g., organogenesis and limb bud development) and life.
Project description:SIRT1 is increasingly recognized as a critical regulator of stress responses, replicative senescence, inflammation, metabolism, and aging. SIRT1 expression is regulated transcriptionally and post-transcriptionally, and its enzymatic activity is controlled by NAD+ levels and interacting proteins. We found that SIRT1 protein levels were much higher in mouse embryonic stem cells (mESCs) than in differentiated tissues. miRNAs post-transcriptionally downregulated SIRT1 during mESC differentiation and maintained low levels of SIRT1 expression in differentiated tissues. Specifically, miR-181a and b, miR-9, miR-204, miR-199b, and miR-135a suppressed SIRT1 protein expression. Inhibition of mir-9, the SIRT1-targeting miRNA induced earliest during mESC differentiation, prevented SIRT1 downregulation. Conversely, SIRT1 protein levels were upregulated post-transcriptionally during the reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. The regulation of SIRT1 protein levels by miRNAs might provide new opportunities for therapeutic tissue-specific modulation of SIRT1 expression and for reprogramming of somatic cells into iPS cells.
Project description:All- trans-retinoic acid (RA), a vitamin A metabolite, is an important signaling molecule required for the proper development of the heart. The epicardium is the main source of RA in the embryonic heart, yet the cardiogenic functions of epicardial-produced RA are not fully understood. Here, we investigated the roles of RA signaling in the embryonic epicardium using in vivo and in vitro models of excess or deficiency of RA. Our results suggested that RA signaling facilitates the cytoskeletal rearrangement required for the epicardial-to-mesenchymal transition of epicardial cells. In vivo treatment with an inhibitor of RA synthesis delayed the migration of epicardial-derived precursor cells (EPDCs) into the myocardium; the opposite was seen in the case of dehydrogenase/reductase superfamily (DHRS)3-deficient embryos, a mouse model of RA excess. Analysis of the behavior of epicardial cells exposed to RA receptor agonists or inhibitors of RA synthesis in vitro revealed that appropriate levels of RA are important in orchestrating the platelet-derived growth factor-induced loss of epithelial character, cytoskeletal remodeling, and migration, necessary for the infiltration of the myocardium by EPDCs. To understand the molecular mechanisms by which RA regulates epicardial cytoskeletal rearrangement, we used a whole transcriptome profiling approach, which in combination with pull-down and inhibition assays, demonstrated that the Ras homolog gene family, member A (RhoA) pathway is required for the morphologic changes induced by RA in epicardial cells. Collectively, these data demonstrate that RA regulates the cytoskeletal rearrangement of epicardial cells via a signaling cascade that involves the RhoA pathway.-Wang, S., Yu, J., Jones, J. W., Pierzchalski, K., Kane, M. A., Trainor, P. A., Xavier-Neto, J., Moise, A. R. Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells.
Project description:Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires retinoic acid (RA) and its coactivator Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signaling and controls embryonic symmetry. We demonstrate that Wdr5, Hdac1, and Hdac2 are required for RA signaling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite formation characteristic of RA-deficiency. We also identify the Rere-binding histone methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA polymerase II recruitment. Our work identifies a protein complex linking key epigenetic regulators acting in the molecular control of embryonic bilateral symmetry.Retinoic acid (RA) regulates the maintenance of somitogenesis symmetry. Here, the authors use a proteomic approach to identify a protein complex of Wdr5, Hdac1, Hdac2 that act together with RA and coactivator Rere/Atrophin2 and a histone methyltransferase Ehmt2 to regulate embryonic symmetry.
Project description:There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.