Histone methylases MLL1 and MLL3 coordinate with estrogen receptors in estrogen-mediated HOXB9 expression

Abstract

Homeobox gene HOXB9 is a critical player in development of mammary gland and sternum and in regulation of renin which is closely linked with blood pressure control. Our studies demonstrated that HOXB9 gene is transcriptionally regulated by estrogen (E2). HOXB9 promoter contains several estrogen-response elements (ERE). Reporter assay based experiments demonstrated that HOXB9 promoter EREs are estrogen responsive. Estrogen receptors ERα and ERβ are essential for E2-mediated transcriptional activation of HOXB9. Chromatin immunoprecipitation assay demonstrated that ERs bind to HOXB9 EREs as a function of E2. Similarly, histone methylases MLL1 and MLL3 also bind to HOXB9 EREs and play a critical role in E2-mediated transcriptional activation of HOXB9. Overall, our studies demonstrated that HOXB9 is an E2-responsive gene and ERs coordinate with MLL1 and MLL3 in E2-mediated transcriptional regulation of HOXB9.

Figure 1

Effect of E2 on HOXB9 expression. (A) JAR cells (grown in phenol-red free media) were treated with varying concentrations of E2. RNA was isolated, reverse transcribed, and subjected to regular PCR (top panel) and real-time PCR (bottom panel) using primers specific to HOXB9. β-actin was used as a loading control. Each experiment was repeated at least thrice. Bars indicate standard errors (p < 0.05). (B) Effect of E2 on HOXB9 expression in MCF7 cells was analyzed in a similar way as described for JAR cells in figure A. Top panel shows the agarose gel analysis of the PCR products and bottom panel shows the real-time PCR data. (C) JAR cells were treated with 100 nM E2 for varying time periods (0 –12 h) and RNA was reverse transcribed and analyzed by regular PCR (left panel) and real-time PCR (right panel) using HOXB9 and β-actin primers. Each experiment was repeated at least thrice (n = 3).

Figure 2

Analysis of HOXB9 promoter EREs and their E2-response using luciferase based reporter assay. (A) HOXB9 promoter (up to -3000 nt) contains three ERE_1/2 sites named as ERE1 to ERE3 and an imperfect full EREs (ERE4). The promoter segments containing these ERE_1/2 sites along with ~150 nt on both sides were cloned into luciferase based reporter construct pGL3 and used for transfection. (B) E2-response of HOXB9 promoter EREs in JAR cells. The ERE containing pGL3 constructs were transfected into JAR cells for 24 h. Control cells were treated with either none (no construct transfected, control lane), or empty pGL3 vector or non-ERE-pGL3 (non-ERE). Both control as well as plasmid transfected cells were then treated with 100 nM E2 for 6 h and subjected to luciferase assay by using ONE-Glo Luciferase Assay System. The ratio of E2-induced luciferase activities over corresponding E2-untreated samples were plotted. The experiment with four replicate treatments was repeated at least twice. Bars indicate standard errors. (C) E2-response of HOXB9 promoter EREs in MCF7 cells. Experiments were performed in MCF7 cells in the same way as described in figure B.

Figure 3

Roles of ERα and ERβ in E2-induced expression of HOXB9. (A) Effect of ERα knockdown: JAR cells were transfected with ERα and a scramble antisense (9 μg each) for 48 h. Antisense-transfected cells were treated with E2 (100 nM for 6 h). RNA was isolated and subjected to reverse transcriptase-PCR analysis by using primers specific to ERα and HOXB9. PCR with ERβ was done to confirm specificity of ERα antisense and β-actin was used quantitative control. PCR products were analyzed in agarose gel. Lane 1: control cells (no E2 control), lane 2: cells were treated with 100 nM E2. Lane 3: cells were initially transfected with 9 μg of scramble antisense followed by exposure to E2. Lane 4: cells were initially transfected with 9 μg of ERα antisense and then treated with E2. Real-time PCR analysis of ERα, ERβ and HOXB9 relative to β-actin is plotted. Each experiment was repeated at least thrice (n = 3). Bars indicate standard errors. (B) Effect of ERβ knockdown or a combined knockdown of ERα and ERβ on E2-induced HOXB9 expression. Experiments and analysis were performed in the same way as shown in panel A.

Figure 4

E2-dependent recruitment of ERα and ERβ in the ERE regions of HOXB9 promoter. (A-B) JAR cells were treated with 100 nM E2 for 6 h and subjected to ChIP assay using antibodies specific to ERα and ERβ. β-actin antibody was used as control IgG. The immuno-precipitated DNA fragments were PCR-amplified using primers specific to ERE1-4 of HOXB9 promoter. Primers specific to a promoter sequence containing no ERE (non-ERE) were used as control. Lanes 1, 3, 5 and 7 are no-E2 controls. Lanes 2, 4, 6 and 8, were E2-treated samples. ChIP DNA fragments were analyzed by real-time PCR and shown in panel B. Each experiment was repeated at least thrice. Bars indicate standard errors. (C) Dynamics of recruitments of ERα and ERβ onto ERE1, ERE3 and ERE4 of HOXB9 promoter: Cells were treated with 100 nM E2 for varying time periods and then subjected to ChIP assay using antibodies specific to ERα and ERβ. Immuno-precipitated DNA fragments were PCR-amplified using primers specific to ERE1, ERE3 and ERE4 of HOXB9 promoter. Lane 1: control cells (no E2 treatment control). Quantification of recruitment level (% relative to input) is plotted in respective bottom panels. Bars indicate standard errors.

Figure 5

Effect of knockdown of MLL1, MLL2, MLL3, and MLL4 on E2-induced expression of HOXB9. JAR cells were transfected with 5 μg of MLL1, MLL2, MLL3, and MLL4 specific antisense oligonucleotides separately. Control cells were treated with a scramble antisense with no homology with MLLs. The antisense-treated cells were incubated for 48 h followed by treatment with 100 nM E2 for 6 h. RNA was isolated from treated and control cells and subjected to reverse transcriptase-PCR by using primers specific to HOXB9 along with MLL1, MLL2, MLL3, and MLL4. β-actin was used as control. The PCR products were analyzed by agarose gel. Quantification of transcript accumulation was done by using real-time PCR (shown in respective bottom panel). (A) Effect of MLL1 knockdown. (Top) Lane 1: control cells (no E2-treatment); lane 2: cells that were initially transfected with scramble antisense followed by exposure to E2. Lanes 3: cells were initially transfected with MLL1 antisense and then treated with E2. Real-time PCR analysis of the expression profiles of MLL1 and HOXB9 (relative to β-actin, average of three replicate experiments, n = 3) were quantified and plotted in the bottom panel. MLL2 was used as control to determine target specificity of MLL1 antisense. (B-D) These figures show the effects of knockdown of MLL2 (MLL1 as control), MLL3 (MLL4 as control), and MLL4 (MLL3 as control), respectively, in the similar manner as shown for MLL1 in panel A. (E-F) Effect of combined knockdown of MLL1 and MLL3 in E2-induced HOXB9 expression. The knockdown experiments were performed in the same way as shown in panels A-D. For combined knockdown (lane 5), MLL1 and MLL3 antisenses were mixed together in equimolar amounts and then transfected. Real-time PCR analysis of the expression profiles of MLL1, MLL3 and HOXB9 were plotted in panel F.

Figure 6

E2-dependent recruitment of MLLs (MLL1-4) in ERE1, ERE3, and ERE4 of HOXB9 promoter. (A-B) JAR cells were treated with 100 nM E2 for 6 h and subjected to ChIP assay using antibodies specific to MLL1, MLL2, MLL3 and MLL4. β-actin antibody was used as control IgG. The immuno-precipitated DNA fragments were PCR-amplified using primers specific to ERE1, ERE3 and ERE4 of HOXB9 promoter. Lanes 1, 3, and 5 are no-E2 controls. Lanes 2, 4, and 6 were E2-treated samples. ChIP DNA fragments were analyzed by real-time PCR and shown in panel B. Each experiment was repeated at least thrice. Bars indicate standard errors. (C) Dynamics of recruitment of MLL1, MLL2, MLL3 and MLL4 onto HOXB9 promoter: Cells were treated with 100 nM E2 for varying time periods and then subjected to ChIP assay using antibodies specific to MLL1, MLL2, MLL3 and MLL4. Immuno-precipitated DNA fragments were PCR-amplified using primers specific to ERE1, ERE3 and ERE4 of HOXB9 promoter. Lane 1: control cells (no E2 control). Quantifications of recruitment level (% relative to input) are plotted in respective bottom panels. Bars indicate standard errors.

Figure 7

E2-dependent enrichment of H3K4-trimethylation level and recruitment of RNA polymerase II (RNAP II) in the ERE1, ERE3 and ERE4 of HOXB9 promoter. (A-C) JAR cells were treated with 100 nM E2 for varying time periods (0 – 6 h) and then subjected to ChIP assay using antibodies specific to H3K4-trimethylation and RNAPII. Immuno-precipitated DNA fragments were PCR amplified using primers specific to ERE1, ERE3 and ERE4 of HOXB9 promoter respectively. Lane 1: control cells (no E2 control). Lanes 2–7 are E2-treated samples. Quantifications of recruitment level (% relative to input) are plotted in respective right panels. Bars indicate standard errors.

Figure 8

Effects of ERα and ERβ knockdown on recruitment of MLL1 and MLL3 on ERE1, ERE3 and ERE4 of HOXB9 promoter. JAR cells were transfected with ERα and ERβ antisenses separately for 48 h followed by exposure to E2 (100 nM for additional 6 h). Cells were harvested and subjected to ChIP assay using anti-MLL1 and anti-MLL3 antibodies. The immuno-precipitated DNA fragments were PCR-amplified using primer specific to ERE1, ERE3, and ERE4 regions of HOXB9 promoter. Lane 1: control cells (no E2 control). Lanes 2–4 are E2-treated samples. Real-time quantification of recruitment level (% relative to input) is plotted in respective bottom panels. Bars indicate standard errors.