Stimulation of GCMa transcriptional activity by cAMP/PKA signaling is attributed to CBP- mediated acetylation of GCMa

Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan.
Molecular and Cellular Biology (Impact Factor: 4.78). 11/2005; 25(19):8401-14. DOI: 10.1128/MCB.25.19.8401-8414.2005
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Human GCMa is a zinc-containing transcription factor primarily expressed in placenta. GCMa regulates expression of syncytin gene, which encodes for a placenta-specific membrane protein that mediates trophoblastic fusion and the formation of syncytiotrophoblast
layer required for efficient fetal-maternal exchange of nutrients and oxygen. The adenylate cyclase activator, forskolin,
stimulates syncytin gene expression and cell fusion in cultured placental cells. Here we present evidence that cyclic AMP (cAMP) signaling pathway
activates the syncytin gene expression by regulating GCMa activity. We found that forskolin and protein kinase A (PKA) enhances GCMa-mediated transcriptional
activation. Furthermore, PKA treatment stimulates the association of GCMa with CBP and increases GCMa acetylation. CBP primarily
acetylates GCMa at lysine367, lysine406, and lysine409 in the transactivation domain (TAD). We found that acetylation of these residues is required to protect GCMa from ubiquitination
and increases the TAD stability with a concomitant increase in transcriptional activity, supporting the importance of acetylation
in PKA-dependent GCMa activation. Our results reveal a novel regulation of GCMa activity by cAMP-dependent protein acetylation
and provide a molecular mechanism by which cAMP signaling regulates trophoblastic fusion.

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Available from: Hungwen Chen, Jul 31, 2014
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    • "In this scenario, protein kinase A (PKA) activated by cAMP facilitates the association of GCM1 and dualspecificity phosphatase 23, which dephosphorylates Ser322 and promotes interaction between the CERB-binding protein (CBP) and GCM1 (Lin et al., 2011). As a result, CBP acetylates and stabilizes GCM1 and functions as a co-activator of GCM1-mediated transcriptional activation (Chang et al., 2005). Protein acetylation is a reversible modification in which histone deacetylases (HDACs) are crucial for removing acetyl groups from modified lysine residues. "
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    ABSTRACT: The placental transcription factor GCM1 and its target gene syncytin-1 are involved in cAMP-stimulated trophoblastic fusion for syncytiotrophoblast formation. GCM1 DNA-binding activity is inhibited by sumoylation, whereas GCM1 stability is decreased by deacetylation. cAMP enhances GCM1 desumoylation through the Epac1/Rap1/CaMKI signaling cascade and CaMKI is known to downregulate class IIa HDAC activity. In this paper, we study whether the Epac1/Rap1/CaMKI signaling cascade regulates GCM1 activity and placental cell fusion through class IIa HDACs. Interaction and co-localization of GCM1 and HDAC5 were characterized by co-immunoprecipitation analysis and immunofluorescence microscopy. Regulation of GCM1 transcription activity and syncytin-1 expression by HDAC5 was studied by transient expression. Phospho-specific antibodies against HDAC5, RNA interference, and immunofluorescence microscopy were used to examine the derepression of GCM1 activity, syncytin-1 expression, and cell-cell fusion by Epac1/Rap1/CaMKI signaling cascade in placental BeWo cells expressing constitutively active Epac1 and CaMKI. We demonstrate that both GCM1 and HDAC5 are expressed in the syncytiotrophoblast layer of full-term placenta and the nuclei of BeWo cells. The interaction between HDAC5 and GCM1 facilitates GCM1 deacetylation and suppresses its transcriptional activity. In contrast, Epac1 stimulates HDAC5 phosphorylation on Ser259 and Ser498 in a Rap1- and CaMKI-dependent manner leading to nuclear export of HDAC5 and thereby derepression of GCM1 transcriptional activity. Importantly, HDAC5 suppresses syncytin-1 expression and cell-cell fusion in BeWo cells, which is counteracted by Epac1 and CaMKI. Our results reveal a new layer of regulation of GCM1 activity and placental cell fusion through the Epac1/Rap1/CaMKI signaling cascade by restraining HDAC5 from interacting with and mediating GCM1 deacetylation.
    Molecular Human Reproduction 07/2013; 19(11). DOI:10.1093/molehr/gat050 · 3.75 Impact Factor
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    • "Only for JAR a minor additive effect could be seen with the combined use of AZA and TSA for Syncytin-1 and hCG. Chuang et al. could show that treatment with TSA up-regulated the placenta specific transcription factor GCMa [65]. Upstream of the 5′LTR of ERVW-1 two GCMa binding sites were identified which can increase Syncytin-1 expression and cell fusion in JEG3 and BeWo [66]. "
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    ABSTRACT: Terminal differentiation of villous cytotrophoblasts (CT) ends in formation of the multinucleated syncytiotrophoblast representing the fetal-maternal interface. Aberrations during this cell-fusion process are associated with Intrauterine Growth Restriction (IUGR), Preeclampsia (PE) and High Elevated Liver and Low Platelets (HELLP) Syndrome. Syncytin-1, the envelope gene of the human Endogenous Retrovirus ERVW-1, is one of the most important genes involved in cell-fusion and showed decreased gene expression during these pathological pregnancies. The aim of this study was to determine the methylation pattern of the entire promoter of ERVW-1 and to correlate these findings with the expression profile of Syncytin-1 in the placental syndromes. 14 isolated villous cytotrophoblasts from control (n = 3), IUGR (n = 3), PE (n = 3), PE/IUGR (n = 3) and HELLP/IUGR (n = 2) placentae were used to determine the mean methylation level (ML) for the ERVW-1 promoter region. ML rose significantly from 29% in control CTs to 49% in IUGR, 53% in PE, 47% in PE/IUGR and 64% in HELLP/IUGR indicating an epigenetic down-regulation of Syncytin-1 by promoter hypermethylation. DNA demethylation of the trophoblast like cell lines BeWo, JEG-3 and JAR with 5-AZA-2'desoxycytidine (AZA) showed an increased Syncytin-1 expression and fusion ability in all cell lines. Promoter activity of the 5'LTR could be inhibited by hypermethylation 42-fold using a luciferase based reporter-gene assay. Finally overexpression of the methyltransferases DNMT3a and LSH could be responsible for a decreased Syncytin-1 expression by promoter hypermethylation of ERVW-1. Our study linked decreased Syncytin-1 expression to an epigenetic hypermethylation of the entire promoter of ERVW-1. Based on our findings we are predicting a broad aberrant epigenetic DNA-methylation pattern in pathological placentae affecting placentogenesis, but also the development of the fetus and the mother during pregnancy.
    PLoS ONE 02/2013; 8(2):e56145. DOI:10.1371/journal.pone.0056145 · 3.23 Impact Factor
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    • "The FLAG-tagged Gcm tool constitutes a useful sensor to characterize post-transcriptional regulatory events. First, since several bands appear on Western Blots revealing the Gcm–FLAG product (Fig. 1C) and the GCMa vertebrate protein is regulated by phosphorylation, which seems to affect its activity and stability (Schreiber et al., 1997; Chang et al., 2005; Knerr et al., 2005; Yang et al., 2005; Chiang et al., 2009; Yasui et al. 2012), Fig. 6. Characterization of peritracheal cells. "
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    ABSTRACT: In Drosophila, the transcription factor Gcm/Glide plays a key role in cell fate determination and cellular differentiation. In light of its crucial biological impact, major efforts have been put for analyzing its properties as master regulator, from both structural and functional points of view. The lack of efficient antibodies specific to the Gcm/Glide protein, however, precluded thorough analysesof its regulation and activity in vivo. In order to relieve such restraints, we designed an epitope-tagging approach to "FLAG"-recognize and analyzethefunctional protein both in vitro(exogenous Gcm/Glide) and in vivo (endogenous protein). We here i) reveal a tight interconnection between the small RNA and the Gcm/Glide pathways. AGO1 and miR-1 are Gcm/Glide targets whereas miR-279 negatively controls Gcm/Glide expression ii) identify a novel cell population, peritracheal cells, expressing and requiring Gcm/Glide. Peritracheal cells are non-neuronalneurosecretory cellsthat are essential in ecdysis. In addition to emphasizing the importance of following the distribution and the activity of endogenous proteins in vivo, thisstudy provides new insights and a novel frame to understand the Gcm/Glide biology.
    Developmental Biology 12/2012; 375(1). DOI:10.1016/j.ydbio.2012.12.014 · 3.55 Impact Factor
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