Expression and Transcriptional Regulation of Individual Pregnancy-specific Glycoprotein Genes in Differentiating Trophoblast Cells

Human pregnancy-specific glycoproteins (PSGs), encoded by eleven highly conserved genes, are the major placental polypeptides. Low PSG levels in maternal circulation have been associated with complicated pregnancies. However, expression of each PSG gene and their regulation during cytotrophoblast cell differentiation remain poorly explored. Herein, we analyze the expression of five PSG genes and demonstrate that they are almost undetectable in undifferentiated trophoblast, but are all transcribed in differentiated cells. Among them, PSG1, PSG3 and PSG5 genes achieve high mRNA levels while PSG7 and PSG9 are poorly expressed. In addition, total PSG proteins and transcripts markedly increase during trophoblast differentiation, preceding morphological syncytialization and βhCG expression. The 5′ regulatory region contributes to the transcriptional control of PSG gene induction in trophoblast cells undergoing differentiation. This responsive region in PSG3 maps within a 130 bp promoter sequence, which overlaps the transcription start site and requires a functional Retinoic Acid Responsive Element (RARE) and a GA-binding protein (GABP) consensus site for basal and differentiation-dependent promoter activity, respectively. Present findings provide novel data for understanding the control of PSG gene expression and demonstrate that their proteins and transcripts represent early markers of trophoblast differentiation.Fil: Camolotto, Soledad Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Racca, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Rena, Viviana Celeste del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Nores, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Patrito, Luis Clemente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Genti de Raimondi, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Panzetta-Dutari, Graciela Maria del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

PSG Gene Expression Is Up-Regulated by Lysine Acetylation Involving Histone and Nonhistone Proteins

Background: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. Methodology/Principal Findings: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs) up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 59regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, 2147/2140), was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA). This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT) function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. Conclusions/Significance: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.Fil: Camolotto, Soledad Andrea. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina;Fil: Racca, Ana Cristina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina;Fil: Ridano, Magali Evelin. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina;Fil: Genti-Raimondi, Susana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina;Fil: Panzetta-Dutari, Graciela Maria del Valle. Universidad Nacional de Córdoba. Facultad de Cs.Químicas. Departamento de Bioquímica Clínica; Argentina

A novel regulator of human villous trophoblast fusion: the Krüppel-like factor 6

Cell–cell fusion is an essential event during life. Throughout human pregnancy, the syncytiotrophoblast (STB) layer of the placenta is formed by continuous fusion of the underlying villous cytotrophoblasts, thus maintaining placental functionality. Defects in this process are associated with pathologies like pre-eclampsia and intrauterine growth restriction. Krüppel-like factor 6 (KLF6) is a transcription factor highly expressed in human and murine placenta. However, KLF6 functions in trophoblast cells remain largely unexplored. The aim of this work was to address the role of KLF6 during STB formation. KLF6 knockdown through small interfering RNA experiments hindered cell–cell fusion revealed by immunofluorescence microscopy in human primary villous cytotrophoblast as well as in the human placental-derived BeWo cell line. Furthermore, KLF6 silencing led to a decrease in the expression of the fusogenic protein Syncytin-1 and the cell cycle regulator p21Cip1/Waf1measured by quantitative RT-PCR and western blot assays. On the contrary, transcript levels of genes that encode for proteins involved in STB formation such as Syncytin-1, Syncytin-2, Connexin-43 and Zonula Occludens-1 increased when KLF6 was overexpressed in differentiating villous cytotrophoblasts and in non-fusing placental-derived JEG-3 cells. Interestingly, the expression of two trophoblast biochemical differentiation markers, βhCG and PSG3, were not reduced after KLF6 silencing in differentiating trophoblast cells. Present results support the notion that KLF6 is a relevant participant in cytotrophoblast fusion.Fil: Racca, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ridano, Magali Evelin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Camolotto, Soledad Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Genti de Raimondi, Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Panzetta-Dutari, Graciela Maria del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin