Epigenetic Activation of the Human Growth Hormone Gene Cluster during Placental Cytotrophoblast Differentiation

Induction kinetics of the placental hGH-hCS genes in BeWo cells. (A) Histone deacetylase inhibition with TSA failed to induce BeWo cell gene expression. The cells were treated with the indicated concentrations of TSA for 12 h. Total RNA was analyzed by Northern blotting using ethanol as a vehicle control. Hybridization probes used are indicated to the right of each panel. (B) FSK-induced BeWo cell syncytialization resulted in only a moderate increase in hGH-hCS expression. BeWo cells were induced to syncytialize by culturing them in the presence of 80 μM FSK for 4 days. Total RNA from the treated cells was used for Northern blot analyses. The syncytialization was confirmed by hCGβ induction. Dimethyl sulfoxide (DMSO) was the vehicle control. (C) Placental hGH-hCS expression was induced by long-term culture of BeWo cells. The cells were plated at a high density and incubated without chemical additives for 32 days. Total RNA (20 μg) was collected every 4 days for analysis by Northern blotting. (D) The BeWo hGH-hCS transcript profile after 32 day of culture was qualitatively similar to that in term placentas. RT-PCR analysis was performed as described for Fig. 1C but with total RNA (5 μg) from BeWo cells at days 0 and 32 of culture and from human term placental villi. hCS-A, hCS-B, and hGH-V mRNAs were detected in all three samples; no hGH-N or hCS-L signals were observed. (E) Matrigel coating of the culture plates enhanced hGH-hCS activation in BeWo cells. The BeWo cells were cultured on Matrigel-coated dishes for 32 days, and total RNA was collected every 8 days, as described for panel C above. The Northern blot analysis showed higher and faster induction of hGH-hCS genes than that shown in panel C when cells were grown on Matrigel.

hGH-hCS genes were induced in ex vivo cultures of primary human placental CTBs. (A) Expression of the hGH cluster genes in primary placental CTB cell culture. The CTBs were cultured in the presence of second-trimester maternal serum for 6 days to induce spontaneous differentiation into syncytial cells capable of expressing the hGH-hCS genes. Total RNA was prepared from daily aliquots and analyzed by Northern blotting. Probes used are indicated to the right of the autoradiographs. (B) hCS-A, hCS-B, and hGH-V are expressed by the induced CTBs. RT-PCR analysis was conducted with RNA from CTBs at days 0 and 2 of culture. The PCR products were digested with TaqI and analyzed as described for Fig. 1C. The hCS-A and hCS-B mRNAs were detected at day 0, and their expression and that of hGH-V were induced by day 2. (C) Quantitative comparison of hGH-hCS expression levels among the placental model systems used in this study. Northern blot analysis was performed on total RNA from human term placenta, cultured primary CTBs at day 6 (CTB 6 d), and day 32 BeWo cells (BeWo 32 d). Relative hGH-hCS mRNA levels, estimated using the rpL32 signals as a loading control, are shown at the bottom.

DNase I-HS mapping in the BeWo, CTB, and STB chromatins. Nuclei from the indicated cells were digested with DNase I, genomic DNA was purified from each sample, and the DNAs were digested with EcoRI and subjected to Southern blot hybridization analysis. The positions corresponding to HSIII, HSIV, and HSV are indicated by arrows at the left. An open arrowhead points to a nonspecific band that was detected before the DNase I treatment (time zero). At the bottom is a diagram of the EcoRI fragment showing the probe position and the sizes of each fragment after DNase I digestion. The results reveal the presence of HSIV and the placenta-specific HS in day 0 and 32 BeWo cells as well as in primary CTBs and STBs. HSIV is absent from the parallel analysis of the human fibroblast line CCDsk-25, which does not express any of the hGH cluster genes. A diagram of the relative fragment sizes is at the bottom.

Histone modifications at the hGH cluster and its LCR in BeWo cells during hGH-hCS activation. (A) Positions of amplimers used in the ChIP assays. The structure of the hGH cluster, linked genes, and the hGH LCR are depicted. P-elements and the putative hCS enhancers are indicated (shaded and open ovals, respectively). Shaded and striped rectangles indicate extensive conserved segments among the PGR units and the hGH-N gene. The amplimer names and their positions are shown below the map. The PGR unit is also shown in an expanded format. (B) The histone H3K4 dimethylation pattern in BeWo cells was established prior to gene activation and did not change after long-term culture. Soluble chromatin was collected from BeWo cells at days 0 and 32 of culture by digesting nuclei with micrococcal nuclease. The ChIP analysis was conducted with anti-dimethylated-histone H3K4 antibody, and the resulting input and bound fractions were subjected to PCR amplification. The bound/input signal ratio at each amplicon (A) was normalized to that at the GAPDH promoter, which was considered 100. The relative histone modification levels in day 0 (shaded bars) and day 32 (black rectangles) BeWo cells are shown. Standard error values are indicated. The α-globin promoter was used as a negative control. (C) Histone H3K4 trimethylation was detected at low levels across the entire locus in BeWo cells at both day 0 and day 32. Histone H3K4 trimethylation patterns, examined by ChIP analysis with anti-trimethylated-H3K4 antibody, were analyzed and plotted as described for panel B. (D) Histone H3 acetylation was significantly induced across the locus during the 32 days of BeWo cell culture. Histone H3 acetylation patterns, using anti-acetylated-histone H3 antibody, were studied as described for panel B. (E) Histone H4 acetylation was significantly induced across the locus during BeWo cell culture. The ChIP assay was performed, analyzed, and plotted as described for panel B but with anti-acetyl histone H4 antibody.

Histone modification at the hGH cluster and its LCR in primary CTBs before and after 4 days of differentiation. (A) Histone H3K4 dimethylation was established after day 4 of culture in primary CTBs. The amplimer positions are the same as those in Fig. 5A. Primary CTBs were isolated from human term placentas and cultured for 4 days in the presence of second-trimester maternal serum. The ChIP assay was performed with anti-dimethylated-histone H3K4 antibody as described for Fig. 5B on chromatin from freshly prepared CTBs (day 0) and after culture (day 4). For comparison, corresponding values for modifications of chromatin isolated from primary placental STBs, previously reported by us (32), are shown. (B) The histone H3K4 trimethylation pattern was largely preset in the freshly prepared, day 0 CTBs. The histone H3K4 trimethylation pattern was examined by ChIP with anti-trimethylated-H3K4 antibody, analyzed, and labeled. (C) Histone H3 acetylation was induced at the hGH locus during differentiation from CTBs to STBs. The histone H3 acetylation patterns were determined by ChIP with anti-acetylated-histone H3 antibody. (D) Histone H4 acetylation was induced at the hGH locus during differentiation of the CTBs. The histone H4 acetylation levels were investigated by ChIP with anti-acetylated-histone H4 antibody. (E) All genes in the hGH cluster were similarly modified in the day 4 CTBs. Histone modification levels at each cluster gene were examined by ChIP. The PCR analysis of the isolated DNA was performed with an amplimer set common to the five genes; the amplified products were digested with four restriction enzymes to distinguish each signal.

Summary of epigenetic modifications during the transition of human placental CTBs to STBs. (Top) The histone modification patterns in freshly prepared primary CTBs are diagrammed below the map. Prior to terminal STB differentiation, all the placental LCR HSs were already formed and the placental genes were expressed at trace levels, whereas hGH-N was totally inactive. Moderate levels of histone H3K4 dimethylation were observed at discrete regions, including HSV, the sites of the promoters of the placental genes, and the sites of the putative enhancers. Histone H3K4 trimethylation was also established at this point within the PGR regions. In contrast, only minimum levels of H3 acetylation were observed across the locus, and H4 acetylation was limited to HSV and at the placental-gene promoters. (Middle) After 4 days of culture, most of the CTBs fused to form a syncytium and the expression of the placental genes, especially hCS-A and hCS-B, was dramatically enhanced. Histone H3K4 di- and trimethylation patterns were well established, and the histone H3/H4 acetylation levels increased modestly at the LCR and at the cluster region in a block encompassing the four placental genes. (Bottom) In the full-term placental STBs, histone H3/H4 acetylation increased to maximum levels and the epigenetic patterns necessary for full activation of the placental genes were completed. The four placental genes are expressed at the highest levels, whereas hGH-N, lacking the epigenetic modifications, remains inactive in STBs.