G9a-mediated histone methylation inhibits TP53BP2 transcription in trophoblasts
Histone modifications play important roles in the regulation of gene transcription34(). We used ENCODE Histone Modification Tracks embedded in the UCSC Genome Browser and identified 7 histone modifications (H3K4me1, H3K4me2, H3K4me3, H3K9me2, H3K9me3, H3K27me3 and H3K36me3) in the TP53BP2 promoter region. Among these histone modifications, H3K4me2 and H3K4me3 presented the greatest number of enrichment peaks (Figure 7A ). By performing a ChIP assay, we only detected a significant reduction in H3K9me2 enrichment at the TP53BP2 promoter in HTR8/SVneo cells under hypoxia, but not in other cells (Figure 7B ). Immunofluorescence staining revealed a significant decrease in H3K9me2 levels in placental trophoblasts from PE pregnancies (Figure 7C ). This result also suggested the importance of H3K9me2 in TP53BP2 transcription in the trophoblasts of PE pregnancies. Furthermore, by measuring the levels of several widely recognized histone methyltransferases (HMTs), including G9a, LSD1, SUV39H1 and SUV39H2, we detected significant decreases in G9a levels in placentas from PE pregnancies and in HTR8/SVneo cells under hypoxia (Figure 7D, E ). In vitro assays demonstrated that G9a knockdown in HTR8/SVneo cells (Figure S7 ) significantly upregulated TP53BP2 transcription under hypoxia (Figure 7F ). Similarly, treatment with BIX-01294 (a G9a-specific inhibitor) also increased the level of TP53BP2 in HTR8/SVneo cells under hypoxia (Figure 7G ). These data demonstrated that G9a can inhibit TP53BP2 expression in the placentas of PE pregnancies.