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. 2022 Mar 8;11:e71094. doi: 10.7554/eLife.71094

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© 2022, Ashley et al

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Figure 5. — (a) Placental fragments were exposed for 8 hr to 25(OH)D₃, which led to limited alterations in DNA methylation compared to incubation with control buffer. Shown are two examples of clusters of hypermethylated CpGs (highlighted in yellow), where the blue bars represent the array’s beta value for individual CpGs. (b) The promoters of the upregulated genes identified in the RNA-seq data displayed lower methylation than those of downregulated genes in both control and 25(OH)D₃-treated conditions. To extend these observations, we performed ChIP-seq on syncytialized cytotrophoblast cells incubated with 20 μM 25(OH)D₃ or control cell culture medium for 24 hr (n = 2 placentas). (c) Representative confocal microscopy image of cytotrophoblast cells cultured for 90 hr and stained with DAPI (blue; nuclei) and desmoplakin (green), present on the cell surface. Multiple nuclei within a single-cell demonstrate syncytialization has occurred. (d) The promoters of upregulated genes (identified in the RNA-seq data) displayed higher levels of both H3K4me3 and H3K27ac than those seen at downregulated genes. (e) Examples of specific upregulated (KLHL11) and downregulated (ACOD1) genes, showing no changes in the enrichment of H3K4me3 or H3K27ac at the promoter (highlighted in yellow) when comparing control and vitamin D conditions.

Figure 5—figure supplement 1. — (a) Placental fragments were exposed for 8 hr to 25(OH)D₃, which led to limited alterations in DNA methylation compared to incubation with control buffer. Shown are two examples of clusters of hypermethylated CpGs (highlighted in yellow), where the blue bars represent the array’s beta value for individual CpGs. (b) The promoters of the upregulated genes identified in the RNA-seq data displayed lower methylation than those of downregulated genes in both control and 25(OH)D₃-treated conditions. To extend these observations, we performed ChIP-seq on syncytialized cytotrophoblast cells incubated with 20 μM 25(OH)D₃ or control cell culture medium for 24 hr (n = 2 placentas). (c) Representative confocal microscopy image of cytotrophoblast cells cultured for 90 hr and stained with DAPI (blue; nuclei) and desmoplakin (green), present on the cell surface. Multiple nuclei within a single-cell demonstrate syncytialization has occurred. (d) The promoters of upregulated genes (identified in the RNA-seq data) displayed higher levels of both H3K4me3 and H3K27ac than those seen at downregulated genes. (e) Examples of specific upregulated (KLHL11) and downregulated (ACOD1) genes, showing no changes in the enrichment of H3K4me3 or H3K27ac at the promoter (highlighted in yellow) when comparing control and vitamin D conditions.