Fig 2: Effect of HDAC inhibition on histone acetylation and syncytiotrophoblast development.BeWo trophoblasts were exposed to the broad spectrum HDAC inhibitor trichostatin A (TSA, 0–20 nM), and then induced to differentiate for 48 h. a Levels of AcH2BK5, AcH3K27, AcH3K14, histone H3, CGB, and β-Actin (loading control) were determined by western blotting. b Transcript levels of ERVW1, ERVFRD-1, OVOL1, CGB, HSD11B2, and TP63 in cells cultured in undifferentiated (black bar) and differentiation (blue bars) conditions with or without various doses of TSA. c Percentage of fused cells following culture in undifferentiated or differentiation conditions with or without 20 nM TSA. Representative images of E-cadherin (red) and CGB (green) are shown to the left of the graph. Nuclei were counterstained using DAPI (blue). Scale bar represents 80 μm. Graphs represent means ± SEM. Data significantly different from the Ctrl (0 nM TSA) cells cultured in differentiation conditions are indicated by an asterisk (*P < 0.05; n = 3 in (b), n = 9 images from three experiments in (c)).

Fig 3: Effect of selective HDAC inhibitors on syncytiotrophoblast development.a RT-PCR depicting expression of HDAC1-10 in primary term cytotrophoblasts (CTB) and BeWo trophoblasts. RNA18SN1 was used to ensure that a similar quantity of cDNA was used in PCR reactions. b Percentage of BeWo trophoblasts that underwent syncytialization following culture in undifferentiated conditions (black bar), or differentiation conditions (blue bars) in the presence of FK228, MS275, LMK235, BRD4345, CAY10683, or RGFP966. Please note that only the HDAC1/HDAC2 inhibitor FK228 significantly inhibited cytotrophoblast differentiation. Representative images of E-cadherin (red) and CGB (green) in undifferentiated and differentiation conditions with or without 5 nM FK228 is shown to the right of the graph. Nuclei were counterstained using DAPI (blue). Scale bar represents 80 µm. c Transcript levels of ERVW1, ERVFRD-1, OVOL1, CGB, HSD11B2, and TP63 in cells cultured in undifferentiated and differentiation conditions with or without various doses of FK228. Graphs represent means ± SEM. Data significantly different from control cells cultured in differentiation conditions are indicated by an asterisk (*P < 0.05; n = 9 images from three experiments in (b), n = at least 4 in (c)).

Fig 4: Reduced histone acetylation during syncytialization of human trophoblast stem cells.a Transcript levels of ERVFRD-1, CGB, HSD11B2, and TP63 in human trophoblast stem cells cultured in stem conditions, and following 5 days culture in conditions that promote syncytiotrophoblast formation. b Detection of CGB in human trophoblast stem cells cultured in stem and differentiation conditions. Nuclei were counterstained using DAPI (blue). c Levels of AcH2BK5, AcH3K9, AcH3K27, AcH3K14, AcH3K18, AcH3, and total H3 were determined by western blotting. Densitometric analysis relative to total histone H3 on day 1 and day 4 of syncytialization is shown to the right of the western blots. The dotted line represents signal intensity in undifferentiated cells. Graphs represent means ± SEM. Data significantly different from undifferentiated cells are indicated by an asterisk (P < 0.05; n = 3). Scale bars represent 100 μm.

Fig 5: Dynamic changes in genomic AcH3 binding during trophoblast differentiation.a Pie chart showing total number of AcH3 peaks, and the number of peaks that increased or decreased AcH3 binding during syncytiotrophoblast development by at least twofold (Padj < 0.01). b Top 10 chromosomal regions exhibiting decreased (black bars) and increased (blue bars) AcH3 binding during differentiation. Genes located proximate (<1 kb) to these chromosomal areas are included above each bar. c Representative genome browser views of chromosomal regions proximate to LHX4, (ΔN)TP63, TEAD4, CGB, OVOL1, and SYDE1, which exhibit differential AcH3 binding in undifferentiated and differentiation conditions. d Immunohistochemistry for TEAD4 and CGB on first trimester human placental sections. Arrowheads denote syncytiotrophoblast; arrows point to cytotrophoblasts. Scale bars represent 50 μm.