Interrelationships between ERα and MAPK signaling: phosphorylation of ERK1 and ERK2 and interaction of ERKs and ERα after hormone treatment of cells.
To characterize the interrelationships between ERα and MAPK signaling, we first examined the phosphorylation of ERK1 and ERK2, downstream effector kinases in the MAPK pathway, and observed robust phosphorylation after estradiol (E2) treatment of cells, which peaked by 15 min (~6-fold increase) and still remained quite elevated at 45 min (Fig. ). We also observed in coimmunoprecipitation experiments that ERK1 and ERK2 interacted with ERα upon hormone treatment and that the interaction of ERK2 and ERα appeared to be stronger (Fig. ). We observed the same interaction in samples with extensive DNase treatment before coimmunoprecipitation (data not shown), implying that ERα and ERK2 are coming down via a protein-protein interaction and are not coimmunoprecipitating because they both might be binding to DNA.
FIG. 1. cDNA microarray gene expression analysis after ERK1 or ERK2 knockdown in MCF-7 cells and effects of kinase depletion on estradiol (E2)-mediated gene regulation. (A) Time course of MAPK activation by E2. MCF-7 cells were treated with 10 nM E2 for the indicated (more ...) Gene expression microarray analysis in breast cancer cells depleted of ERK1 or ERK2 and examination of the impact of ERK knockdown on estrogen-stimulated cell proliferation and cell cycle-associated genes.
Based on our finding that estrogen enhanced the interaction of ERK1 and ERK2 with ERα, we sought to determine what effect these protein kinases might have on the pattern of ERα-mediated gene regulation. Using small interfering RNA (siRNA), we specifically depleted MCF-7 breast cancer cells of each kinase, using siGENOME reagents that contain a pool of four siRNAs targeting the gene of interest (siGENOME pool) or the individual siRNAs present in the pool. As shown in Fig. , we observed very specific knockdown of each kinase. The knockdown of ERK1 or ERK2 did not affect ERα levels in MCF-7 cells, and likewise knockdown of ERα did not alter ERK1 or ERK2 levels (Fig. ). These data indicate that we can obtain a very efficient and selective knockdown of either kinase without cross-regulation or compensation by changes in the levels of ERα, ERK1, or ERK2 proteins.
To examine the effects of ERK1 and ERK2 on E2-regulated gene expression, MCF-7 cells were transfected with a control siRNA or with the pool of four siRNAs (siGENOME) targeting ERK1 or ERK2 for 60 h and were then treated with vehicle or E2 for 4 or 24 h. After RNA isolation and processing, we utilized Affymetrix Hu-133A2 Genechips to evaluate global gene expression profiles (Fig. ). Estrogen treatment resulted in the regulation of over 400 and 1,400 genes in control cells, at 4 and 24 h, respectively, as reported previously (7
), as well as in cells with knockdown of ERK1 or ERK2, but the genes regulated in the three cases showed some notable differences (Fig. ). Of the estrogen-regulated genes, approximately 160 (at 4 h) and 690 (at 24 h) were regulated only in cells with reduced ERK2, and approximately 60 (at 4 h) or 290 (at 24 h) were regulated selectively in cells with reduced ERK1, indicating differential gene regulation by E2 that is determined by the level of each kinase.
Using web-based Panther and ClueGO software, we analyzed the groups of genes whose estrogen regulation were most impacted by ERK2 or ERK1 knockdown, and we found that knockdown of each kinase affected different gene categories. ERK2 knockdown predominantly affected mitosis, DNA repair, and DNA metabolism-related genes (see Fig. S1 at www.life.illinois.edu/bkatzlab/supplementalfigure1.eps
), whereas ERK1 knockdown had less of an effect on these groups of genes. In exploring this aspect further, cell proliferation assays showed that ERK2 knockdown fully blocked the E2-mediated increase in cell number (Fig. ). The depletion of ERK1 had a smaller impact on cell proliferation.
FIG. 2. ERK2 controls E2-regulated cell proliferation and the expression of proliferation associated genes. (A) ERK2 is critical for E2-stimulated cell proliferation. MCF-7 cells were plated at 1,000 cells/well in 96-well plates. Cells were transfected with 20 (more ...)
We also verified E2-stimulated expression of several M-phase genes identified in our microarray analysis in siCtrl cells, including Ki-67, CCNB1, MYBL2, AURKB, and Survivin (BIRC5), which are part of a 21-gene signature used to predict the risk of breast cancer recurrence in patients on tamoxifen therapy (35
). These genes are upregulated by E2, and knockdown of ERK2, but not ERK1 (data not shown), completely blocked their stimulation by E2, further supporting a major role for ERK2 in estrogen enhancement of cell proliferation (Fig. ). When we assessed the expression of S-phase genes required for DNA synthesis, we also observed reduced expression and loss of E2 regulation with ERK2 depletion (Fig. ), and two key mediators of cell cycle progression, CCND1 and E2F1, lost most or all of their estrogen stimulation when cells were depleted of ERK2 but not ERK1 (Fig. ).
To examine the possibility of a direct nuclear role for ERK2 in estrogen-stimulated expression of CCND1, we monitored recruitment of ERK2 to the ERα binding site at the 3′ enhancer of the CCND1 gene. We observed a hormone-stimulated recruitment, which was abrogated by inhibition of MAPK activation by the MEK inhibitor U0126 and also by ERα knock-down (Fig. ). Thus, our data show that ERK2 is a major regulator of proliferation and of the expression and E2 stimulation of genes promoting cell cycle progression. Further, estrogen stimulated the recruitment of ERK2 to the ERα binding site in the estrogen-regulated gene CCND1, and this recruitment required ERα and active MAPK, indicating a convergence of ERK2 and ERα at the level of chromatin. We explored this aspect further in genome-wide analyses described below.
Genome-wide analysis of ERα and ERK2 binding sites: overlapping binding sites and conservancy of the binding sites.
The marked effects of ERK2 depletion on the ability of hormone to regulate gene expression and cell proliferation, and the observed estrogen-stimulated interaction between ERK2 and ERα, suggested the possibility that ERK2 might in fact be recruited by the nuclear receptor to ER binding sites in chromatin. To investigate this, we first performed a limited ChIP-qPCR analysis in MCF-7 cells after E2 treatment at regions of a number of genes that we previously identified as being ERα binding sites. Notably, we detected estrogen-stimulated recruitment of ERK2 and ERK1 to the ER binding sites of all of these estrogen-regulated genes (see Fig. S2 at www.life.illinois.edu/bkatzlab/supplementalfigure2.eps
). Since we obtained a stronger recruitment with ERK2 and observed more major effects of this kinase on estrogen-regulated gene expression and cell proliferation, we undertook ChIP-chip analyses to examine genome-wide ERα and ERK2 binding sites (Fig. ).
FIG. 3. ChIP-on-Chip analysis of genome-wide ERα and ERK2 binding sites. (A) UCSC Genome Browser view of ERα and ERK2 binding sites identified by our ChIP-chip studies. MCF-7 cells were treated with vehicle or 10 nM E2 for 45 min. After formaldehyde (more ...)
MCF-7 cells were treated with vehicle or 10 nM E2 for 45 min and cross-linked with formaldehyde, and ERα and ERK2 containing chromatin complexes were immunoprecipitated with ERα or ERK2 specific antibodies. After DNA amplification, genome-wide microarray analysis was performed using Affymetrix GeneChip Human Tiling 2.0R arrays. We identified only background levels of ERα and ERK2 binding sites (less than 50) in cells in the absence of E2 treatment, but upon E2 treatment we obtained a mean number of 4547 ERα binding sites, similar to the number of ERα binding sites previously reported (6
), and 1,303 ERK2 binding sites. Many genes, such as the well-known ERα target genes pS2 (also known as TFF1) and LRRC54 (also known as TSKU), harbored overlapping ERα and ERK2 binding sites (Fig. ).
Of note, 63% of ERK2 binding sites overlapped with ERα binding sites, suggesting that ERα might be the major transcription factor tethering ERK2 to chromatin after treatment of cells with E2. We then used the cis
-regulatory element annotation system (CEAS) to further analyze the binding sites (21
). The location of ERα and ERK2 binding sites were mapped to the nearest gene in both upstream and downstream directions on both strands, within 300 kb (Fig. ). About 50% of ERα binding sites and ERK2 binding sites were localized to distal enhancers (intergenic regions), followed by intronic regions (ca. 30%), proximal promoters (20% for ERK2 versus only 5% for ERα), and exon regions (3 to 7%). Interestingly, when we analyzed overlapping ERα and ERK2 binding sites relative to annotated genes, their distribution very much resembled the distribution of ERα binding sites. Further, using Oncomine concept maps, genes associated with ERK2 binding sites (see Fig. S3A at www.life.illinois.edu/bkatzlab/supplementalfigure3.eps
) and with ERK2 and ERα overlapping binding sites after estrogen (see Fig. S3B at the same URL) were found to be those showing a strong positive correlation with ERα expression in breast tumors. These data imply that ERα is likely a major determinant of ERK2 binding to chromatin in estrogen-treated cells. Of interest, ERα and ERK2 cobound regions were biased two times more toward E2-regulated genes compared to ERα but not ERK2 regions. In addition, when we compared chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) data (15
) to our ERα and ERK2 overlapping binding sites, we observed that 85% of these sites mapped to ChIA-PETs, implying their association with chromatin looped regions.
We also performed a conservancy analysis of the different groups of binding sites, which compares the conservation of the binding site area across different species from zebrafish to human (and includes human, chimp, mouse, rat, dog, chicken, fugu, and zebrafish). This analysis (Fig. ) revealed that the three groups of binding sites (i.e., ERα, ERK2, and overlapping ERα and ERK2) showed high conservation across species, but, of note, the overlapping ERα and ERK2 binding sites showed the highest conservation. This might suggest an evolutionarily conserved function for these cooccupied genomic locations of ERα and ERK2.
Characterization of ERK2 recruitment to ERα binding sites.
To characterize the kinetics of ERK2 recruitment to ERα binding sites, we performed an E2 treatment time course. ERK2 recruitment, monitored at the ER binding sites of two estrogen-stimulated genes, increased by 5 min of E2 treatment, reached maximum levels at 30 to 45 min, and decreased somewhat by 1 h (Fig. ). Interestingly, this temporal profile of ERK2 recruitment after E2 is virtually identical to that which we have observed for ERα recruitment after hormone (40
FIG. 4. Characterization of ERK2 recruitment to ERα binding sites upon E2 treatment. (A) Time course of ERK2 recruitment to ERα binding sites in the estrogen-responsive LRRC54 and pS2 genes. MCF-7 cells were treated with 10 nM E2 for the indicated (more ...)
To investigate the ERα dependency of ERK2 recruitment to genomic binding sites, we utilized siRNA-mediated knockdown of ERα. Knockdown of ERα (Fig. ) or treatment with the ER antagonist ICI182,780 (data not shown) completely abolished E2 stimulated recruitment of ERK2 to ER binding sites, indicating that functionally active ERα is required for recruitment of ERK2. Moreover, ChIP-reChIP experiments confirmed that ERα and ERK2 were present together at these binding sites (Fig. ). In contrast, depletion of ERK2 with siRNA did not impact ERα recruitment to binding sites of these estrogen-regulated genes (Fig. ).
Next, we queried the importance of ERK2 activation by MEK1 for the observed recruitment to chromatin of ERK2 and ERα. The MEK inhibitor U0126 nearly completely prevented ERK2 recruitment to ER binding sites, implying that activated ERK2 is required for recruitment (Fig. ). In contrast, U0126 did not affect recruitment of ERα to the regions studied (Fig. ). Hence, ERα is recruited upon E2 treatment to ER binding sites independent of ERK2, whereas ERK2 recruitment requires ERα and active ERK2 is required for its own chromatin localization.
Identification of CREB1 as a transcription factor regulating ERK2 chromatin binding and hormone-stimulated cell proliferation.
Based on our genome-wide mapping of ERα and ERK2 binding sites, we performed bioinformatic analysis to identify enriched transcription factor binding motifs in these binding sites. For this purpose, we used two programs: CEAS, which analyzes the full length of the binding site, and SeqPos, which analyzes enrichment around the center of the binding site. Both approaches revealed the response element for CREB1 to be highly enriched. We further assessed involvement of CREB1 with ERK2 and ERα actions, because CREB1 is a known MAPK target and is also highly expressed in these breast cancer cells.
As shown in Fig. , ERK2 and CREB1 showed increased recruitment to overlapping binding sites for ERα and ERK2 in regulated genes after E2 treatment of cells. E2 also elicited a rapid increase in phosphoCREB1 (Fig. ). We confirmed by ChIP an E2-stimulated rapid recruitment of CREB1 to overlapping ERα and ERK2 binding sites in the E2-regulated genes LRRC54/TSKU and pS2/TFF1 (Fig. ), with the time course paralleling the recruitment of ERK2 and ERα after E2. The co-presence of CREB1 with ERK2 was also observed by ChIP-reChIP experiments (Fig. ). To establish whether this transcription factor is a putative tethering factor for ERK2, we examined the effect of knockdown of CREB1 on the recruitment of ERK2 to the estrogen-stimulated genes. As shown in Fig. , knockdown of CREB1 with siRNA reduced the estrogen-stimulated recruitment of ERK2 to these estrogen-regulated genes while having no impact on recruitment of ERα. Furthermore, knockdown of CREB1 markedly reduced cell proliferation and prevented estrogen stimulation of proliferation (Fig. ) and the estrogen-stimulated expression of S-phase and proliferation-associated genes (Fig. ). Thus, the findings in Fig. and indicate that ERα and CREB1 are involved in ERK2 recruitment to chromatin upon estrogen treatment and that ERK2 and CREB1 greatly impact hormone-stimulated cell proliferation.
FIG. 5. CREB1 is a cooperating transcription factor in ERK2 recruitment to chromatin binding sites and in E2 regulation of cell proliferation. (A) Box plots showing that E2 treatment stimulates recruitment of ERK2 and CREB1 to ERα binding sites (n = (more ...)