Identification of HTRA4 as a Transcriptional Target of p63 in Trophoblast

Abstract

The placenta plays a crucial role in pregnancy success. ΔNp63α (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and is involved in CTB maintenance and differentiation. We examined the mechanisms of action of p63 by identifying its downstream targets. Gene expression changes were evaluated following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. High-temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, was identified as a gene reciprocally regulated by p63, and its expression was characterized in primary human placental tissues by RNA-sequencing and in situ hybridization. Potential p63 DNA-binding motifs were identified in the HTRA4 promoter, and p63 occupancy at some of these sites was confirmed using chromatin immunoprecipitation, followed by quantitative PCR in both JEG3 and trophoblast stem cells. These data begin to identify members of the transcriptional network downstream of p63, thus laying the groundwork for probing mechanisms by which this important transcription factor regulates trophoblast stemness and differentiation.

Figure 1

Identification of genes downstream of ΔNp63α (p63). A: Heat map of gene expression changes following ΔNp63α overexpression in JEG3 cells. B: Heat map of gene expression changes following p63 knockdown in JEG3 cells. The color scale represents the measured gene expression intensity, with red representing high and green representing low gene expression. Selected genes are highlighted to the right of each heat map; HTRA4 (boldfaced), a gene reciprocally regulated by p63 overexpression and downregulation, is further evaluated in this study. CMV, cytomegaloviral promoter; sh-p63, p63-specific short hairpin RNA; sh-Scramble, nonspecific short hairpin RNA.

Figure 2

High-temperature requirement A4 (HTRA4) expression in primary human placental cells and tissues. A: Tumor protein p63 (TP63) and HTRA4 show an inverse gene expression pattern by RNA-sequencing (RNA-seq) analysis in primary cytotrophoblasts [CTBs; identified by high epidermal growth factor receptor (EGFR) and integrin alpha-6 (ITGA6) expression] compared with extravillous trophoblasts [EVTs; identified by high human leukocyte antigen G (HLAG) and matrix metalloproteinase 9 (MMP9) expression], isolated from first-trimester placenta. B: Similarly, re-analysis of RNA-seq data from Okae et al shows TP63 to be highly expressed in CTB, whereas HTRA4 is more highly expressed in differentiated trophoblast [EVT and syncytiotrophoblast (STB)] of the first-trimester placenta. Unpaired t-test and one-way analysis of variance was applied for cell expression comparison tests, and P values are shown. C:In situ hybridization of primary human placental tissues across gestation at indicated weeks (w) of gestation, using HTRA4-specific probes. HTRA4 expression is enriched in both STB of floating villi (V) and cell column EVT (CC) in first trimester, but remained only in EVT (basal plate) at later gestational ages (20 weeks and beyond). All panels show tissue at a total magnification of ×100, except for the top right panel, which is a magnified version of the red boxed area in the top left panel (magnified ×400); within the top right panel, arrows show HTRA4 expression in villous STB and arrowheads highlight absence of HTRA4 in villous CTB. ∗P < 0.05, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Figure 3

The ΔNp63α (p63) transcription factor occupies the ΔNp63α and high-temperature requirement A4 (HTRA4) promoters. A: Map of p63 transcription factor binding sites (1 to 8) within the ΔNp63 promoter; primer sets spanning these sites are shown under the map, labeled A through D (D was used as a negative control). B: p63 Binding motif consensus within ΔNp63α promoter. Top panel: Human p63 binding motif consensus. Bottom panel:ΔNp63α promoter p63 consensus. P value for the p63 DNA motifs at ΔNp63 promoter is 1.48 × 10^–3. C and D: Chromatin immunoprecipitation (ChIP) analysis showing fold enrichment of p63 binding (over IgG control) at each region in the TP63 promoter in JEG3 (C) and trophoblast stem cells (TSCs; D). E: Map of p63 transcription factor binding sites (1 to 9) within the HTRA4 promoter. Primer sets spanning these sites are shown under the map, labeled (A to G (C was used as a negative control). F: The p63 binding motif consensus within the HTRA4 promoter. Top panel: Human p63 binding motif consensus. Bottom panel: HTRA4 promoter p63 binding motif consensus. P value for p63 DNA motifs at HTRA4 promoter is 1.48 × 10^–4. G and H: ChIP analysis showing fold enrichment of p63 binding (over IgG control) at each region in the HTRA4 promoter in JEG3 (G) and TSC (H). ∗P < 0.05.