Repression of ESR1 through Actions of Estrogen Receptor Alpha and Sin3A at the Proximal Promoter▿

Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERα has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERα represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERα gene, ESR1. Upon estrogen treatment, ERα is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERα-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERα is identified. These data support a model of repression wherein actions of ERα and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression

Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription

The Proteasome Inhibitor Bortezomib Induces an Inhibitory Chromatin Environment at a Distal Enhancer of the Estrogen Receptor-α Gene

<div><p>Expression of the estrogen receptor-α (ERα) gene, <i>ESR1</i>, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating <i>ESR1</i> gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of <i>ESR1</i> gene expression in breast cancer cells. Expression of <i>ESR1</i> mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ∼400 base pair (bp) of the transcription start site (TSS). Here, we show that loss of <i>ESR1</i> gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb) from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2γ. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of <i>ESR1</i> mRNA expression that highlights the importance of the chromatin environment at the −150 kb distal enhancer in regulation of basal expression of <i>ESR1</i> in breast cancer cells.</p></div

Proteasome inhibition decreases transcription factor occupancy on the <i>ESR1</i> enhancer and promoter.

<p>MCF7 cells were treated with vehicle (nt) or 30 nM bortezomib (B) for 24 hours and ChIP analyses were performed to examine occupancy at regions of <i>ESR1</i> depicted in the diagram in Fig. 2A. Data representing a minimum of three independent experiments are presented as percent of input sample for <i>A</i>) GATA3, <i>B</i>) FOXA1, <i>C</i>) AP2γ, <i>D</i>) RNA PolII, and <i>E</i>) IgG (control) on the indicated regions. Data are shown as mean ± SEM. Statistical analysis comparing untreated and bortezomib-treated groups were performed using a Student’s paired t-test. Statistically significant differences of p<0.05 are indicated with an asterisk (*).</p

DNase sensitivity is reduced in the <i>ESR1</i> enhancer region with proteasome inhibition.

<p><i>A</i>) Schematic of <i>ESR1</i> promoter structure depicting the location of primers used for detecting DNase sensitivity and ChIP analysis <i>B)</i> Nuclei were isolated from MCF7 cells treated for 24 hours with vehicle or 30 nM bortezomib. Isolated nuclei were either left undigested or digested with DNase for 5 minutes, followed by incubation with Proteinase K to stop the DNase reaction, as described in Materials and Methods. DNA was purified and q-PCR was performed with primers to the indicated regions. Data are shown as the 2^{∧(Ct cut–Ct uncut)} and presented as mean ± SEM. Statistical analysis was performed using a Student’s t-test. No statistically significant differences were observed (p>0.05).</p