Table 1.
Patient Characteristics.
Table 2.
Quantitative real time-PCR primer sequences.
Table 3.
PCR primer sequences used in ChIP analysis.
Figure 1.
Diagrammatic representation of a 5000 bp fragment encompassing the human GCM1 promoter as well as the transcription and translation initiation sites.
Orange bars indicate Gene2Promoter software identified DREAM binding sites (Pu/CNGTCAPuPuPu/C). Blue bars show all putative core DRE sites (GTCA). The transcription initiation site is denoted with an arrow. The translation initiation site is represented by ATG (3197 bp from the transcription initiation site). The enlarged 43 bp sequence, located about 1250 bp upstream from the transcription initiation site is responsible for DREAM binding to the GCM1 promoter. The promoter region was amplified with 5 sets of primers (1–5) during ChIP analysis (See Figure 3). The locations of the amplicons are depicted with the vertical black lines.
Table 4.
3′ end biotin-labeled probes used in EMSA.
Figure 2.
DREAM expression in human placenta of first, second and third trimester.
Expression pattern of DREAM was examined using immunofluorescence in 8 week (A), 16 week (B) and 38 week (C) placentas. DREAM is stained green and nuclei are stained blue (DAPI). Each image is a representative of 50 fields examined (original magnification, ×400). (D) Quantitative analysis of nuclear expression of DREAM in the trophoblast layer. Number of DREAM-positive nuclei is expressed as a percentage of the total number of nuclei in the trophoblast layer. Nuclear expression decreases with advancing gestation. (E) Total placental DREAM mRNA expression was monitored using qRT-PCR. Data are presented as mean+\−SEM from 3–4 placentas per group (D) and 6–11 placentas per group (E). *** p<0.001.
Figure 3.
ChIP analysis of the GCM1 human promoter.
(A) ChIP assay was validated with positive and negative controls as indicated. (B) Five designed primer sets were validated using a BAC clone containing the human GCM1 promoter as DNA template. (C) As a negative control rabbit IgG-immuno-precipitated DNA was subjected to PCR amplification with the five primer sets. (D) DREAM-immuno-precipitated fraction was subjected to PCR amplification with the five sets of primers. Amplification was observed with primer sets 4 and 5.
Figure 4.
EMSA of 43 bp sequence from the GCM1 promoter.
(A) Binding to a 43 bp 3′ end biotin-labeled probe was tested on nuclear fraction from DREAM-overexpressing cells (control) produced two bandsdenoted with arrows)s. 1∶100 and 1∶1000 competition with unlabeled probe was performed. The probe in combination with the nuclear extract isolated from DREAM siRNA-treated cells resulted in decreased abundance of both bands. Successful super-shift analysis with anti-Histidine tag Ab(A) and anti-DREAM Ab(B) was performed. (B) Mutation analysis of the DRE sites in the 43 bp sequence. Mutated probes were tested using nuclear extracts from DREAM-over-expressing BeWo cells. Mutations 1–3 did not alter the binding DREAM to the probe. The binding of mutated probes 4 and 5 to nuclear extract from DREAM overexpressing BeWo cells resulted in reduced signal.
Figure 5.
Changes in DREAM expression modified GCM1 levels in BeWo cells.
(A) DREAM silencing of BeWo cells increased GCM1 mRNA levels. (B) DREAM over-expression in BeWo cells reduced GCM1 mRNA levels. Data presented as mean+\−SEM from 4 independent experiments with each value determined in triplicate. Each treated time point was compared to an equivalent non-silenced time point control. All non-silenced control time points are set as 1. *** p<0.001, * p<0.05. NS, non-silenced.
Figure 6.
DREAM silencing in first trimester floating placental villous explants.
(A) Increased GCM1 expression in placental explants after siRNA-mediated DREAM silencing. Results are mean+\−SEM from 5 placentas per group with each value determined in triplicate. Each treated time point was compared to an equivalent non-silenced time point control. All non-silenced control time points are set as 1. Histological assessment of DREAM siRNA-treated first trimester floating villous explants revealed reduced expression of DREAM (B, C) and Ki-67 (D, E) in siRNA-treated explants as compared to non-silenced control explants following 2 days of culture. Semi-thin histology of non-silenced control (F) and DREAM siRNA-treated (G) explants with the cytotrophoblast layer delineated. Following siRNA treatment, villous cytotrophoblast layer became dis-continuous (arrows) and showed nuclear condensation in some areas (*). Each field is a representative of 50 samples examined (original magnification; B, C = 1000×; D, E, F, G = 400×). *** p<0.001, * p<0.05. NS, non-silenced.
Figure 7.
Apoptosis and proliferation assays in DREAM siRNA-treated explants.
TUNNEL staining was performed in (A) t = 0 hr control, (B) 100 nM non-silenced control and (C) 100 nM DREAM siRNA-treated explants cultured for 48 hrs. Pictures are representatives of 30 images from 5 independent experiments. Positive nuclear staining was observed only in DREAM siRNA-treated explants. (D–E) BrdU staining in non-silenced control (D) as compared to DREAM siRNA-treated (E) explants. BrdU incorporation was visualized with anti-BrdU immunohistochemistry. BrdU (10 µM) was added to the culture media of the explants for the duration of each of the experiments. (F) Proliferation was measured as the number of BrdU-positive trophoblast nuclei per total number of trophoblast nuclei and normalized to the control. The control was set at a 100% Results are mean+\−SEM from 5 placentas per group with each value determined in triplicate. *** p<0.001. IVS, inter-villous space; VT, villous trophoblast; ST, stroma. Magnification A–C 1000×, D–E 400×.
Figure 8.
Nimodipine and ionomycin treatment of first trimester floating villous explants.
(A) Diagrammatic representation of DREAM and DRE interactions under altered intracellular calcium concentrations. (B) Reduced GCM1 mRNA levels after ionomycin treatment. Data presented as mean ± SEM from 5 independent experiments. (C) BrdU incorporation was used to measure altered cytotrophoblast rate of proliferation in nimodipine and ionomycin-treated first trimester placental explants. Proliferation was measured as the number of BrdU-positive trophoblast nuclei per total number of trophoblast nuclei and normalized to their respective controls. The control was set at a 100% * p<0.05. DRE, downstream regulatory element; Nimo, nimodipine; Iono, ionomycin.
Figure 9.
Increased DREAM expression in severe preeclamptic (sPE) placenta and immuno-precipitation analysis of sumoylated DREAM.
(A) qPCR analysis of DREAM levels. Results are mean+\−SEM from 6 placentas per group. DREAM immunohistochemistry was performed on the RCWIH BioBank TMA #1 array containing 8 sPE samples and 6 age-matched controls. Representative images from control (B) and sPE (C) are shown. Strong nuclear immunostaining was observed in syncytial knots of the sPE placentas (arrows). (D) DREAM IP with rabbit polyclonal antibody 1014 or normal rabbit IgG was subjected to immunoblot analysis with anti-SUMO1 Ab. Arrows represent specific sumoylated DREAM bands. Asterisk labels a non-specific immuno-reactive band. Input (10%) after incubation with the DREAM Ab. One representative pair of control (C) and sPE placental tissue is shown. (E) Graph representing the ratio of IP bands intensity versus input from 6 controls and 6 sPE samples. * p<0.05. hmw, high molecular weight; PE, preeclampsia. Magnification B–C 1000×.