Fig 1.
IL-20 is up-regulated by estradiol treatment in MCF-7 cells and is highly expressed in ER-positive breast cancer.
(A) Heat map representation of the gene expression profiles of 39 interleukin (IL) genes in MCF-7 cells with or without E2 treatment for 4 h. (B) Expression of IL-20 in MCF-7, MAD-MB-231, and MCF-10A cells with or without E2-treatment for 4 h and normalized against 18s rRNA. (C) Quantitative RT-PCR of IL-20 in seven normal breast tissues, four triple negative breast carcinoma, six ER-negative breast carcinoma, and twenty-five ER-positive breast carcinoma cDNA samples. (Human breast cancer qPCR Array, OriGene). (D) Gluck and TCGA Breast gene expression datasets of IL-20 mRNA. Data and statistics were obtained from the Oncomine database. (E) IHC analyses of IL-20 protein expression in human breast carcinomas in tissue microarrays (TMAs). The staining results show that ER-positive breast cancer expresses significantly more IL-20 than ER-negative breast cancer, and cancer adjacent normal tissues. Hematoxylin was used as counterstain. Representative images of normal adjacent breast tissue (1), ER-negative malignant breast tissue (2), and ER-positive malignant breast tissue (3) are shown.
Fig 2.
ERα is required for the E2-mediated induction of IL-20.
(A) Western blot shows the protein level of ERα in cells transfected with the siESR1 following E2-stimulation for 30 min. (B) Expression of IL-20 in MCF-7 cells is dependent on the presence and activity of ERα and E2, and normalized against 18s rRNA. (C) Binding of ERα to the IL-20 promoter region determined by ChIP assays. Upper panel: Schematic of the IL-20 locus (exons as open boxes) and the six amplicons (black segments) used in ChIP assays. The specific anti-ERα antibody, HC-20X, was used in the ChIP experiments. Lower panel: Bar chart of the relative levels of ERα at each of the six regions. The mean and SD were calculated from at least two independent experiments. (D) Suppressed ERα binding to the IL-20 promoter (segment 2) by ICI treatment. The ChIP experiment was carried out in the absence or presence of E2 for 30 min. (E) Inhibition of RNA Pol II binding to the IL-20 promoter (segment 2) by ICI treatment or over-expression of truncated ERα (ERα264–595) determined by ChIP. The experiment was carried out in the presence or absence of E2 for 30 min.
Fig 3.
KMT2B regulates E2-mediated induction of IL-20 gene through the modification of H3K4.
(A) Enrichment analysis of the three H3K4 methylations (H3K4me1, H3K4me2, and H3K4me3) at the IL-20 promoter in MCF-7 cells using ChIP. The experiment was performed with or without E2. The mean and SD were calculated from at least three independent experiments. (B) Expression of IL-20 as determined by RT-qPCR in KMT2A, KMT2B, KMT2C, KMT2D, or KMT2E-depleted MCF-7 cells in the presence or absence of E2 and normalized against 18s rRNA. (C) Binding of KMT2B to the IL-20 promoter region as determined by ChIP assays. Upper panel: Schematic of the IL-20 locus (exons as open boxes) and the six amplicons (black segments). The specific anti-KMT2B antibody, ab104444, was used for the ChIP experiments. DNA isolated from immunoprecipitated chromatin was amplified by qPCR using designed primers. Lower panel: Bar chart showing the relative levels of KMT2B at each of the six IL-20 gene regions. The mean and SD were calculated from at least two independent experiments. (D) ChIP assays showing the depletion of H3K4me1 at the IL-20 promoter in KMT2B knockdown cells in the presence or absence of E2. The mean and SD were calculated from at least three independent experiments. (E) Western blotting of nuclear extracts prepared from MCF-7 cells transfected with control siRNA or KMT2B-siRNA in the presence or absence of E2. The antibodies used are shown in the right panel.
Fig 4.
ERα and KMT2B interdependently co-localized at IL-20 chromatin.
(A-B) ChIP analysis of KMT2B recruitment (A) and H3K4me1 enrichment (B) with or without siESR1(ERα knockdown) for 3 days or ICI treatment for 24 h, followed by E2 for 30 min. (C-D) ChIP assay showing the effect of KMT2B depletion on the recruitment of ERα (C) and RNA Pol II (D). The primers that amplify the ERα binding region of IL-20, i.e. segment2, were used in these experiments.
Fig 5.
KMT2B and ERα form a complex at the promoter of the IL-20 gene.
(A) Kinetic ChIP experiments were performed using KMT2B, H3K4me1, and ERα specific antibodies. A single chromatin was prepared for ChIP assay at each time point. (B) ChIP-ReChIP to determine the KMT2B and ERα co-occupancy at the IL-20 promoter. Chromatin was prepared from MCF-7 cells treated with E2 for 30 minutes and then subjected to the ChIP procedure using the antibodies labeled as "1st ChIP.” The second immunoprecipitation was carried out using the antibodies labeled as "2nd ChIP.” (C) Co-immunoprecipitation of endogenous KMT2B and ERα. MCF-7 cells were treated with E2 for 24 h, and whole-cell lysates were immunoprecipitated using KMT2B or ERα antibodies. Western blotting was performed on the immunoprecipitated proteins using anti-KMT2B or anti-ERα. (D) Upper panel: Schematic of the ERα functional domains. Lower panel: Immunoprecipitation analysis of the ERα functional domains that interact with KMT2B. Interactions between the endogenous KMT2B and the in vivo transcribed/translated Flag-tagged ERα fragment were confirmed by an immunoprecipitation assay using anti-KMT2B antibody followed by western blotting with anti-FLAG antibody. (E) KMT2B and IL-20 protein expression in ER-positive and ER-negative breast cancer assayed by IHC.
Fig 6.
The effect of KMT2B and IL-20 knockdown on cell proliferation in E2-induced MCF-7 cells.
(A) Cell proliferation assays and (B) colony formation assays of KMT2B or IL-20 depleted MCF-7 cells. Cells were transfected with control siRNA, siKMT2B, or siIL-20 in the absence or presence of E2. (C) Cell-cycle analysis of KMT2B or IL-20 depleted MCF-7 cells using propidium iodide staining and flow cytometry. MCF-7 cells were transfected with control siRNA, siKMT2B, and siIL-20 in the absence or presence of E2.