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Activation of estrogen receptor-alpha by E2 or EGF induces temporally distinct patterns of large-scale chromatin modification and mRNA transcription.

Berno V, Amazit L, Hinojos C, Zhong J, Mancini MG, Sharp ZD, Mancini MA - PLoS ONE (2008)

Bottom Line: Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results.Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation.Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America. valeria.bernoembl.it

ABSTRACT
Estrogen receptor-alpha (ER) transcription function is regulated in a ligand-dependent (e.g., estradiol, E2) or ligand-independent (e.g., growth factors) manner. Our laboratory seeks to understand these two modes of action. Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results. Data show differential recruitment of GFP-ER to the array, with the AF1 domain playing a vital role in EGF-mediated responsiveness. Temporal analyses of large-scale chromatin dynamics, and accumulation of array-localized reporter mRNA over 24 hours showed that the EGF response consists of a single pulse of reporter mRNA accumulation concomitant with transient increase in array decondensation. Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation. Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

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ER transcriptional activity at the promoter array.PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4-hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP-ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean ±SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03.
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pone-0002286-g004: ER transcriptional activity at the promoter array.PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4-hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP-ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean ±SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03.

Mentions: To examine the relationship between array size and transcriptional responses to EGF, E2 or 4HT, we next used RNA FISH to measure dsRED2skl mRNA accumulation at the array (Figure 4A). Restorative deconvolved image stacks from GFP-ER and hybridization signals (array-associated transcripts) were collected and quantified as a measure of co-localizing reporter mRNA. First we examined the entire range of the expression within the transiently transfected cell population to determine if there was a cell-to-cell correlation between GFP-ER expression levels and the quantified FISH signal. In the PRL-HeLa cells that were non-transfected (fluorescence mean intensity was <∼500, Figure 4B, yellow circles), there was a low level of FISH signal detectable at the array (Figure 4A, inset value). Cell gating based upon ER expression level indicated a suppression of reporter transcript accumulation in cells overexpressing the receptor (GFP-ER mean intensity in the nucleus ∼>8000, Figure 4B, blue circles; intensity value >1.5fold compared to MCF-7 cells). In the remaining cells with low levels of GFP-ER (∼4000±2000, Figure 4B, red circles), FISH signals were not linearly dependent on fluorescence (receptor) levels, suggesting possible involvement of both expression level and an asynchronous cell population [22]. These observations led us to analyze cells with low levels of fluorescence (mean intensity<∼6000) in the following experiments.


Activation of estrogen receptor-alpha by E2 or EGF induces temporally distinct patterns of large-scale chromatin modification and mRNA transcription.

Berno V, Amazit L, Hinojos C, Zhong J, Mancini MG, Sharp ZD, Mancini MA - PLoS ONE (2008)

ER transcriptional activity at the promoter array.PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4-hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP-ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean ±SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2386239&req=5

pone-0002286-g004: ER transcriptional activity at the promoter array.PRL-HeLa cells transiently expressing GFP-ER were treated with E2, EGF, 4-hydroxytamoxifen (4HT) or ethanolic vehicle for the indicated time. Subsequent to ligand treatment the cells were fixed and subjected to an RNA FISH protocol using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. A. Representative images of a single cell for each treatment. The presence of transcripts at the promoter array is identified by accumulated signal above the level for the nucleoplasm. The inset values (red type) represent the amount of transcript at 2 hours, relative to vehicle controls. B. Representative image of PRL-HeLa cells transfected with GFP-ER (green) exemplify the heterogeneity of ER expression levels. The cells show the RNA FISH signal associated with the array in the cell population (red signal) (I). The nuclear GFP-ER mean of fluorescence and RNA FISH array signal were plotted for vehicle- and E2-treated cells. Each symbol represents the measurements from a singe cell (II). C. To quantify FISH signal over 24 hours the total intensity of signal at the array (minus background signal) was determined by cumulative summation of 20 planes. Data represent the mean ±SEM of three different experiments graphed as fold induction over mean time-matched vehicle-treated control cells. D. This panel represents the earlier time points for E2 and EGF treatment. Fold activation between EGF and E2 at 30 minutes was significantly different, with a p value of 0.03.
Mentions: To examine the relationship between array size and transcriptional responses to EGF, E2 or 4HT, we next used RNA FISH to measure dsRED2skl mRNA accumulation at the array (Figure 4A). Restorative deconvolved image stacks from GFP-ER and hybridization signals (array-associated transcripts) were collected and quantified as a measure of co-localizing reporter mRNA. First we examined the entire range of the expression within the transiently transfected cell population to determine if there was a cell-to-cell correlation between GFP-ER expression levels and the quantified FISH signal. In the PRL-HeLa cells that were non-transfected (fluorescence mean intensity was <∼500, Figure 4B, yellow circles), there was a low level of FISH signal detectable at the array (Figure 4A, inset value). Cell gating based upon ER expression level indicated a suppression of reporter transcript accumulation in cells overexpressing the receptor (GFP-ER mean intensity in the nucleus ∼>8000, Figure 4B, blue circles; intensity value >1.5fold compared to MCF-7 cells). In the remaining cells with low levels of GFP-ER (∼4000±2000, Figure 4B, red circles), FISH signals were not linearly dependent on fluorescence (receptor) levels, suggesting possible involvement of both expression level and an asynchronous cell population [22]. These observations led us to analyze cells with low levels of fluorescence (mean intensity<∼6000) in the following experiments.

Bottom Line: Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results.Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation.Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America. valeria.bernoembl.it

ABSTRACT
Estrogen receptor-alpha (ER) transcription function is regulated in a ligand-dependent (e.g., estradiol, E2) or ligand-independent (e.g., growth factors) manner. Our laboratory seeks to understand these two modes of action. Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results. Data show differential recruitment of GFP-ER to the array, with the AF1 domain playing a vital role in EGF-mediated responsiveness. Temporal analyses of large-scale chromatin dynamics, and accumulation of array-localized reporter mRNA over 24 hours showed that the EGF response consists of a single pulse of reporter mRNA accumulation concomitant with transient increase in array decondensation. Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation. Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

Show MeSH
Related in: MedlinePlus