The proteasome inhibitor bortezomib induces an inhibitory chromatin environment at a distal enhancer of the estrogen receptor-α gene

Abstract

Expression of the estrogen receptor-α (ERα) gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 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 ESR1 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 ESR1 mRNA expression that highlights the importance of the chromatin environment at the -150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells.

Figure 1. The effects of proteasome inhibition on ERα expression are reversible.

A) MCF7 cells were treated with bortezomib for 24 hours and harvested immediately (t = 0) or at the indicated time from 4 to 48 hours post bortezomib removal. ESR1 mRNA levels were measured by qRT-PCR. Data are shown relative to an untreated control before bortezomib-treatment, which was set at 1. Results are representative of three independent experiments and are shown as mean ± SEM. For statistical analysis, ANOVA was performed using the ΔCt values followed by a post hoc Tukey’s test to compare each point to the 0 hour. Statistically significant values (p<0.05) are indicated with an *. B) Western blots were performed on whole cell lysates from cells that were treated with vehicle (−) or bortezomib (B) for 24 hours (24 h). After 24 hours, bortezomib was removed by washing cells twice with PBS and replacing media. Cells were then harvested at the indicated times between 4 and 48 hours after bortezomib removal. Blots were probed with antibodies for ERα, with actin serving as a loading control.

Figure 2. DNase sensitivity is reduced in the ESR1 enhancer region with proteasome inhibition.

A) Schematic of ESR1 promoter structure depicting the location of primers used for detecting DNase sensitivity and ChIP analysis B) 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).

Figure 3. Proteasome inhibition decreases transcription factor occupancy on the ESR1 enhancer and promoter.

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 ESR1 depicted in the diagram in Fig. 2A. Data representing a minimum of three independent experiments are presented as percent of input sample for A) GATA3, B) FOXA1, C) AP2γ, D) RNA PolII, and E) 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 (*).

Figure 4. Proteasome inhibition decreases p300 occupancy on the ESR1 enhancer.

MCF7 cells treated with vehicle (nt) or 30 nM bortezomib (B) for 24 hours and ChIP assays were performed to assess occupancy of p300 at the indicated region. Immunoprecipitation was performed using antibodies for A) p300 and B) IgG, as a control. Data are presented as percentage of the input sample. The data represent the average of a six independent experiments ± SEM. Statistical analysis was performed to compare untreated and bortezomib-treated samples using a Student’s paired t-test. Statistically significant differences of p<.05 are indicated with an asterisk (*).

Figure 5. Proteasome inhibition decreases histone acetylation and increases H4K20me3 on the distal ESR1 enhancer.

MCF7 cells were treated for 24 hours with vehicle (nt) or 30 nM bortezomib (B), and ChIP was performed as described in methods using antibodies to A) pan acetylated histone 3 (AcH3), B) pan acetylated histone 4 (AcH4), C) histone 4 lysine 20 tri methylation (H4K20me3), and D) IgG as control. Data are presented as percentage of input sample and represent a minimum of three independent experiments. Error bars indicate the SEM, and an asterisk (*) indicates a statistically significant difference between vehicle and bortezomib-treated samples using a Student’s paired t-test. * p<0.05, ** p<.01.

Figure 6. Model of ESR1 regulation by proteasome inhibitors.

Under basal conditions in the absence of estrogen, the ESR1 distal enhancer is acetylated on histones H3 and H4, and is occupied by FOXA1, AP2γ, GATA3, p300 and RNA PolII. Histone H3 and H4 are also acetylated on the proximal promoter which is occupied by RNA PolII and AP2γ. After the addition of bortezomib, the distal enhancer exhibits decreased histone acetylation and increased histone methylation. Occupancy of FOXA1, AP2γ, GATA3, p300 and RNA PolII decreases on the distal enhancer. In contrast, histone acetylation, methylation, and AP2γ occupancy on the proximal promoter are unchanged, but RNA PolII occupancy decreases. These data support a model where bortezomib-induced changes in the chromatin environment around the distal enhancer regulate ESR1 expression in ER+ breast cancer cells.