Estrogen-receptor- exchange and chromatin dynamics are ligand- and domain-dependent

JCS03161 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) Transient co-transfection assays demonstrating transcriptional responsiveness of the PRL promoter/enhancer reporter vector to GFP-Pit-1 expression. The 1X, 13X, 26X and 52X refers to the number of synergy element repeats that are 5’ of the PRL promoter/enhancer (dm66/enh) as shown in panel C. Reporter only refers to mock transfection controls. (B) Luciferase assays of a stably integrated prolactin promoter/enhancer luciferase reporter plasmid (no additional synergy elements) in HeLa cells. Cells were transfected with plasmids directing expression of Pit-1 or ER either alone or together as indicated below each bar graph. Each transfection also contained a β-Gal expression plasmid to control for transfection efficiency. Treatment of the cells with vehicle (−) or E2 (+) is indicated. β-Gal below the bar graphs indicates control transfections in which the β-Gal expression plasmid was transfected alone. The experiment confirmed Pit-1/ER synergistic activation of the PRL promoter/enhancer reporter in a stably integrated chromosomal setting in HeLa cells. (C) PRLHeLa cells transiently expressing GFP-ER (Top two panels) and MCF7 cells (Bottom panel) were immunostained with anti-ER and labeled for immunofluorescence with Alexa Fluor-546-conjugated secondary antibody. ER immunofluorescence was detected using the same exposure time (0.176 seconds; 100% ND filter) for both cell lines. The mean nucleoplasm immunofluorescence intensity units for GFP-ER in PRL-HeLa are 2375.9 ± 176.2 s.e.m. and 1599.8 ± 158.2 in MCF7 cells (n= 6 for each cell line).

• Supplemental Figure 2 -

  Fig. S2. Visualizing stable chromosomal integrants of PRL-HeLa (p52XPRL-dsRED2-SKL). This stable integrant containing an expanded synergy element (52×) was generated as described in Material and Methods. (A) Images obtained during the initial colony screening. The PRL-HeLa line was transiently transfected with GPP-Pit-1 and GFP-ER (green) to maximize fluorescent signal in the nucleus and optimize dsRED2-SKL expression (red cytoplasmic dots). The three cells shown were all in the same field and represent two transfected cells (green) and one negative cell; collectively, this approached showed GFP-ER−Pit-1-transfected cells (green) can exhibit an intranuclear ‘array’ that expressed dsRED2skl (red). After fixation, the cells were counterstained with DAPI (blue). (B) Fluorescent in situ hybridization (FISH, see methods) was performed to detect the plasmid DNA integrants (top left panel) and reporter mRNA (bottom left panels). In this experiment the cells were cotransfected with GFP-Pit-1 and GFP-ER (middle panels). Right panel shows image overlays. Note that fluorescent foci colocalize with FISH foci signals. In our early experiments, array size and shape varied considerably, and was correlated with expression levels of activators. For example, when expression levels of ER and Pit-1 are high (as it is in this FISH experiment), there is a visible reduction in array size, attributable we speculate to transcription squelching. (C) PRL-HeLa cells transiently expressing GFP-ER were treated with vehicle, 10 nM E2, or 10 nM 4HT for 2.5 hours. After scoring, cells having an array (indicated by nuclear foci of fluorescence) were graphed as percentage of the total cells with fluorescent nuclei. (D) Shown is a representative example of PRL-HeLa cells co-transfected with plasmids expressing CFP-ER (red) and YFP-Pit-1 (green). DAPI (blue) is the counter stain.

• Supplemental Figure 3 -

  Fig. S3. Colocalization of SRC-1 and GFP-ER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for SRC-1. The cells were treated with vehicle or the ligands indicated (10 nM, 2 hours). The cells were imaged using a Delta Vision restorative microscope (63× 1.4 NA apochromat objective). Shown are projections of stacked images (0.2 μm Z-step, with a total of 2 μm). The signal origin is shown above the panel columns and the treatment condition is shown to the left of the panel rows. Merged images include DAPI-stained nuclei.

• Supplemental Figure 4 -

  Fig. S4. Colocalization of cyclin T1 transcription-elongation factor and GFP-ER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for cyclin T1. The cells were treated and the images were obtained and presented as in supplementary material Fig. S3.

• Supplemental Figure 5 -

  Fig. S5. Colocalization of CDK9 transcription-elongation factor and GFPER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for CDK9. The cells were treated and the images were obtained and presented as in supplementary material Fig. S3.