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First published online 12 September 2006
doi: 10.1242/jcs.03161

Journal of Cell Science 119, 4101-4116 (2006)
Published by The Company of Biologists 2006
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Estrogen-receptor-{alpha} exchange and chromatin dynamics are ligand- and domain-dependent

Z. Dave Sharp1,*, Maureen G. Mancini2,*, Cruz A. Hinojos2,*, Fangyan Dai2, Valeria Berno2, Adam T. Szafran2, Kelly P. Smith3, Tanmay T. Lele4, Donald E. Ingber4 and Michael A. Mancini2,{ddagger}

1 Molecular Medicine, University of Texas Instititue of Biotechnology, San Antonio, TX, USA
2 Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
3 Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA
4 Vascular Biology Program, Departments of Pathology and Surgery, Children's Hospital and Harvard Medical School, Boston, MA, USA


Figure 1
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  		Fig. 1. PRL-based array construction and testing. (A) Sequence of the prolactin promoter (+1 to -66) with one high-affinity Pit-1-binding site (1P, italics and underlined) and enhancer (-1807 to -1498) that contains four Pit-1-binding sites (1D-4D, italics and underlined), and five EREs (PRL1-5, Bold text). (B) Sequence of the synergy element containing the Pit-1 1D site and two EREs (PRL1 and 5). (C) Schema showing the essential elements of the reporter constructions. Transcription start site, proximal promoter and enhancer sequence are shown in A. Xn indicates multimerized synergy elements denoting variable numbers of repeats (8, 13, 26, 52, 104).

 

Figure 2
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  		Fig. 2. Ligands regulate large-scale chromatin structure. (A-D) GFP-ER was transiently expressed in PRL-HeLa cells, and then treated with (A) ethanol, (B) E2, (C) 4HT or (D) ICI for 2 hours prior to fixation. Decondensed arrays are seen in vehicle- and E2-treated cells, although E2 treatment results in further decondensation, and condensed arrays are seen in 4HT- and ICI-treated cells. (E) Cells transiently expressing GFP-ER were treated with either ethanol (ETOH, vehicle) or 10 nM of ligand (E2, 4HT, ICI) for 2 hours. After fixing and counter-staining with DAPI, cells were imaged and array size was quantified using HTM as described in Materials and Methods. Note that array size correlates positively with transcription signal obtained by FISH (Fig. 3). For cells analyzed in A-D, n >=200; for HTM, n=500.

 

Figure 3
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  		Fig. 3. Real-time visualization of alterations in chromatin structure of the GFP-ER-targeted PRL array in response to E2 or 4HT. Time-lapse images were obtained by deconvolution microscopy using live cells transiently expressing GFP-ER. Ligand addition is indicated after t=0, and frames are labeled with time points after t=0. The images were deconvolved and those shown are projected image stacks. (A) Typical data from PRL-HeLa cells treated with E2. (B) Data from PRL-HeLa cells treated with 4HT. n=10 for cells analyzed. The size bar indicates length in microns.

 

Figure 4
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  		Fig. 4. Single-cell analyses of the transcriptional response of the PRL array to ligands. (A,B) Cells were (A) mock-transfected or (B) transiently transfected with GFP-ER expression plasmid. Cells in B were treated with vehicle or the indicated ligands (10 nM) for 2.5 hours, processed for FISH and imaged as described in Materials and Methods. GFP-ER signal is shown in green, mRNA FISH signal is shown in red. Overlay includes DAPI-stained nuclei (blue). (C) The FISH signals at the array were quantified as described in Materials and Methods and shown as bar graphs. Treatments (30 minutes, white bars and 2 hours, black bars) are indicated below the graphs; `Non' and `Transfected' indicate mock- and transfected cells, respectively. FISH signals at the array were quantified in 20 cells for each treatment as relative intensity of the cells treated with vehicle and are shown as bar graphs. While there is constitutive level of FISH signal (A), it increases significantly in cells with arrays demarcated with GFP-ER. *P=0.007, E2-treated GFP-ER-expressing cells at 30 minutes compared with vehicle controls. **P<0.001, E2-treated GRP-ER-expressing cells at 2 hours compared with vehicle controls. ***P=0.0016, non-transfected cells compared with GFP-ER-expressing cells. #P=0.0378, 30 minutes compared with cells treated 2 hours with E2. §P<0.001, antagonist-treated GFP-ER-expressing cells versus vehicle controls.

 

Figure 5
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  		Fig. 5. Colocalization of SRC-3 coactivator and GFP-ER at the PRL array. PRL-HeLa cells transiently expressing GFP-ER were treated with vehicle or the ligands indicated (10 nM, 2 hours). Afterwards the cells were immunostained for SRC-3. The fluorescent signal origin is labeled above the panels and merged images include DAPI-stained nuclei. n>=200 for cells analyzed. The size bar indicates length in microns.

 

Figure 6
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  		Fig. 6. Colocalization of BRG1 chromatin modifier and GFP-ER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for BRG1. Cells were treated and images were obtained and presented as in Fig. 4. n>=200 for cells analyzed. The size bar indicates length in microns.

 

Figure 7
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  		Fig. 7. Colocalization of RNAPII large subunit and GFP-ER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for RNAPII. Cells were treated and images were obtained and presented as in Fig. 4. n>=200 for cells analyzed. The size bar indicates length in microns.

 

Figure 8
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  		Fig. 8. Colocalization of acetylated histone H3 (acH3) and GFP-ER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for acH3. Cells were treated and images were obtained and presented as in Fig. 4. For cells analyzed, n>=200.

 

Figure 9
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  		Fig. 9. Colocalization of histone dimethylated at lysine residue (dimethK4) and GFP-ER at the PRL array. Representative PRL-HeLa cell images transiently expressing GFP-ER and immunostained for dimethK4. Cells were treated and images were obtained and presented as in Fig. 4. n>=200 for cells analyzed.

 

Figure 10
Figure 10
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  		Fig. 10. ER and Pit-1 mobility over PRL array. (A) Representative time-lapse images of photobleaching and recovery of GFP-ER-targeted arrays (FRAP). FRAP was used to examine GFP-ER at the PRL array and in the surrounding nucleoplasm (images not shown). PRL-HeLa cells transiently expressing GFP-ER were treated with vehicle or ligands at 10 nM for 2 hours. Pre-bleach and bleach refer to cells before and after photobleaching. Time in seconds after bleaching is indicated. (B) Representative time-lapse images of nuclear photobleaching and loss of GFP-ER from targeted arrays (inverseFRAP). In these experiments, PRL-HeLa cells transiently expressing GFP-ER were treated with E2 (Estrogen) or 4HT (Tamoxifen). Pre and Post refer to images acquired prior to and immediately post photobleaching; time after post-bleach is indicated in seconds. (C) PRL-HeLa cells transiently expressing GFP-Pit-1 were analyzed by FRAP as described in A. (D) FRAP recovery curves are shown for nucleoplasm (left) and PRL array (right) in cells transiently expressing GFP-ER and treated with vehicle (No ligand), E2, 4HT (Tam) and ICI. The initial fluorescence immediately before the bleach was normalized to 1 and the curve starts from the time point immediately after the bleach. Note that non-ligand-dependent interactions greatly slowed the recovery of receptor over the PRL array compared with nucleoplasm, but E2 did not additionally decrease recovery times of GFP-ER at the array. 4HT lead to faster recovery both on and off the array. ICI, as expected, immobilized GFP-ER in the nucleus. (E) iFRAP loss curves were obtained in a similar manner, except that prebleach values were retained. PRL-HeLa cells transiently expressing GFP-ER were treated with vehicle, E2 or 4HT (10 nM) prior to photobleaching. The fluorescence at the PRL array was averaged over a 60-second time frame. The initial fluorescence immediately after photobleaching was normalized to 1 and relative fluorescence before bleaching was set to zero. koff was slower with E2 than no ligand or 4HT. n=30 for FRAP analysis under each condition.

 

Figure 11
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  		Fig. 11. ER domains required for PRL array targeting. (A) Schematic of ER functional domains with residue coordinates of the deletion mutants used in these experiments. (B) Representative images of GFP-ER282, GFP-ER534 and GFP-ER554 colocalized with endogenous RNAPII or acetylated histone H3 by immunofluorescence. Each truncation was transiently expressed in PRL-HeLa cells and treated with vehicle or E2 (10 nM, 2 hrs) as indicated. GFP-X refers to the GFP signal captured from each receptor. DAPI indicates counterstained nuclei. (C) Comparison of GFP-ER and GFP-ER truncation mobility within the nucleoplasm or the array. PRL-HeLa cells were treated with vehicle or E2. Bulk denotes recovery times in the nucleoplasm and array identifies recovery times at the PRL array. Half-time of recovery was determined and is shown as a bar graph (see Materials and Methods). n=30 for FRAP analysis under each condition,

 

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© The Company of Biologists Ltd 2006