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Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells.

Bourdeau V, Deschênes J, Laperrière D, Aid M, White JH, Mader S - Nucleic Acids Res. (2007)

Bottom Line: Although siRNA-mediated inhibition of ERalpha expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes.Binding sites for several other transcription factors, including proteins known to tether ERalpha, were enriched in up- and/or down-regulated primary targets.Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Immunology and Cancer and Biochemistry Department, Université de Montréal, C.P. 6128 Succursale Centre Ville, Montréal, QC H3C 3J7, Canada.

ABSTRACT
Estrogen receptors (ERs), which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and about 1300 putative secondary target genes of estradiol in MCF-7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide. Although siRNA-mediated inhibition of ERalpha expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes. Binding sites for several other transcription factors, including proteins known to tether ERalpha, were enriched in up- and/or down-regulated primary targets. Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.

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Distribution of EREs in a 50 kb window around the TSS of estrogen target genes. (A) Number of EREs per gene identified at a 75% cutoff rate using the matrix described in Figure 6A in a 2.5 kb sliding window within 50 kb of genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) up-regulated genes. (B) Number of EREs per gene identified at a 75% cutoff rate in a 2.5 kb sliding window within 50 kb genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) down-regulated genes. Note that all described alternative TSS were considered in this analysis.
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Figure 7: Distribution of EREs in a 50 kb window around the TSS of estrogen target genes. (A) Number of EREs per gene identified at a 75% cutoff rate using the matrix described in Figure 6A in a 2.5 kb sliding window within 50 kb of genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) up-regulated genes. (B) Number of EREs per gene identified at a 75% cutoff rate in a 2.5 kb sliding window within 50 kb genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) down-regulated genes. Note that all described alternative TSS were considered in this analysis.

Mentions: Distribution of EREs in sliding 2.5 kb windows with 500 bp increments between −25 and +25 kb of the TSS indicates that enrichment of EREs (identified at a 75% cutoff) in genes up-regulated in the presence of CHX, but not in those up-regulated in its absence, can be observed within −20 to +20 kb of the TSS (Figure 7A). Finally, enrichment in EREs was higher when considering only ERα-associated chromatin regions (50) found within a 20 kb window around the TSS of different groups of genes, reflecting the capacity of ChIP to accurately pinpoint the regulatory regions in target gene flanking sequences (Figure 6C). However, no enrichment was observed in the only–CHX group of up-regulated genes even when considering only ERα-bound fragments (Figure 6C). Together, these results validate the use of CHX to identify primary E2 target genes.Figure 7.


Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells.

Bourdeau V, Deschênes J, Laperrière D, Aid M, White JH, Mader S - Nucleic Acids Res. (2007)

Distribution of EREs in a 50 kb window around the TSS of estrogen target genes. (A) Number of EREs per gene identified at a 75% cutoff rate using the matrix described in Figure 6A in a 2.5 kb sliding window within 50 kb of genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) up-regulated genes. (B) Number of EREs per gene identified at a 75% cutoff rate in a 2.5 kb sliding window within 50 kb genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) down-regulated genes. Note that all described alternative TSS were considered in this analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2248750&req=5

Figure 7: Distribution of EREs in a 50 kb window around the TSS of estrogen target genes. (A) Number of EREs per gene identified at a 75% cutoff rate using the matrix described in Figure 6A in a 2.5 kb sliding window within 50 kb of genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) up-regulated genes. (B) Number of EREs per gene identified at a 75% cutoff rate in a 2.5 kb sliding window within 50 kb genomic regions centered around the transcriptional start sites (TSS) of primary (+CHX) or total (−CHX) down-regulated genes. Note that all described alternative TSS were considered in this analysis.
Mentions: Distribution of EREs in sliding 2.5 kb windows with 500 bp increments between −25 and +25 kb of the TSS indicates that enrichment of EREs (identified at a 75% cutoff) in genes up-regulated in the presence of CHX, but not in those up-regulated in its absence, can be observed within −20 to +20 kb of the TSS (Figure 7A). Finally, enrichment in EREs was higher when considering only ERα-associated chromatin regions (50) found within a 20 kb window around the TSS of different groups of genes, reflecting the capacity of ChIP to accurately pinpoint the regulatory regions in target gene flanking sequences (Figure 6C). However, no enrichment was observed in the only–CHX group of up-regulated genes even when considering only ERα-bound fragments (Figure 6C). Together, these results validate the use of CHX to identify primary E2 target genes.Figure 7.

Bottom Line: Although siRNA-mediated inhibition of ERalpha expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes.Binding sites for several other transcription factors, including proteins known to tether ERalpha, were enriched in up- and/or down-regulated primary targets.Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research in Immunology and Cancer and Biochemistry Department, Université de Montréal, C.P. 6128 Succursale Centre Ville, Montréal, QC H3C 3J7, Canada.

ABSTRACT
Estrogen receptors (ERs), which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and about 1300 putative secondary target genes of estradiol in MCF-7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide. Although siRNA-mediated inhibition of ERalpha expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes. Binding sites for several other transcription factors, including proteins known to tether ERalpha, were enriched in up- and/or down-regulated primary targets. Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.

Show MeSH
Related in: MedlinePlus