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. 2012 Jan;32(2):569-75.
doi: 10.1128/MCB.05869-11. Epub 2011 Nov 14.

CBP mediates NF-κB-dependent histone acetylation and estrogen receptor recruitment to an estrogen response element in the BIRC3 promoter

Madhumita Pradhan  1 , Sarah C Baumgarten, Leslie A Bembinster, Jonna Frasor
Affiliations

CBP mediates NF-κB-dependent histone acetylation and estrogen receptor recruitment to an estrogen response element in the BIRC3 promoter

Madhumita Pradhan et al. Mol Cell Biol. 2012 Jan.

Abstract

Estrogen receptor (ER) and NF-κB are transcription factors with profound effects on breast cancer cell proliferation and survival. While many studies demonstrate that ER and NF-κB can repress each other, we previously identified a gene signature that is synergistically upregulated by these two factors in more aggressive luminal B breast tumors. Herein, we examine a novel mechanism of cross talk between ER and NF-κB that results in the upregulation of the antiapoptotic gene BIRC3 (also known as cIAP2). We demonstrate that NF-κB, acting through two response elements, is required for ER recruitment to an adjacent estrogen response element (ERE) in the BIRC3 promoter. This effect is accompanied by a major increase in NF-κB-dependent histone acetylation around the ERE. Interestingly, CBP, a histone acetyltransferase previously implicated in repressive interactions between ER and NF-κB, plays a permissive role by promoting histone acetylation and ER recruitment, as well as enhanced expression of BIRC3. These findings suggest a new gene regulatory mechanism by which inflammation and NF-κB activation can influence ER recruitment to inherently inactive ER binding sites. This fine-tuning mechanism may explain how two factors that generally repress each other's activity may work together on certain genes to promote breast cancer cell survival and tumor progression.

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Figures

Fig 1
Fig 1
Estradiol (E2) cannot induce expression of BIRC3 but potentiates regulation by proinflammatory cytokines. MCF-7 cells were treated for 2 h with increasing doses of TNF-α in the absence or presence of 10 nM E2 (A), 10 ng/ml IL-1β or IL-6 in the absence or presence of 10 nM E2 (B), increasing doses of E2 in the absence or presence of 10 ng/ml TNF-α (C), or 10 nM E2 and/or 10 ng/ml TNF-α in the absence or presence of 1 μM 4-hydroxytamoxifen (TAM) (D). BIRC3 mRNA was measured by qPCR, and fold change was calculated using the ΔΔCT method with 36B4 as an internal control. The data represent the means ± SEM for three independent replicates. *, P < 0.05 compared to treatment with either E2 or cytokine alone. nd, not detectable; ns, not significant.
Fig 2
Fig 2
The NF-κB pathway is required for enhanced BIRC3 expression by the combination of E2 and TNF-α. (A) BIRC3 mRNA expression was examined in MCF-7 cells following 24 h of exposure to adenoviral vectors for GFP (control) or a dominant-negative form of IκBα (IκBα-DN) and an additional 2 h of treatment with E2, TNF-α, or both. (B) MCF-7 cells were transiently transfected with a BIRC3 promoter reporter construct spanning bp −527 to +55 of the promoter, in which the NF-κB response elements (NF1 and NF3) were intact (−527) or mutated (mNF1/3). Dual-luciferase assays were carried out following 4 h of treatment with E2, TNF-α, or both. (C) ChIP assays were carried out for p65 following treatment of MCF-7 cells with E2, TNF-α, or both for various lengths of time. The fold-increase in p65 occupancy at the BIRC3 promoter was calculated from the percent input of each pulldown and then comparing each treatment to untreated controls from four independent experiments. *, P < 0.05 compared to treatment with TNF-α alone. nd, not detectable.
Fig 3
Fig 3
The ability of estrogen to enhance BIRC3 expression requires an intact estrogen response element (ERE) and ER binding to DNA. (A) Schematic representation of the BIRC3 promoter showing the relative position of a putative ERE upstream of two functional NF-κB response elements (NF1 and NF3) (44). (B) MCF-7 cells were transfected with the BIRC3 promoter reporter construct in which the ERE was intact (−527), a region of the promoter containing the ERE was deleted (−247), or the ERE was mutated (mERE). Reporter activity was measured by dual-luciferase assay following 4 h of treatment with E2, TNF-α, or both. (C) Luciferase activity of the −527 promoter fragment, in which the ERE was intact (wild type [WT-ERE]) or mutated to a consensus ERE (Cons-ERE), was measured after 4 h of treatment with E2, TNF-α, or both. (D) BIRC3 mRNA levels were measured in MCF-7 cells treated with E2, TNF-α, or both in the absence or presence of an ER-DNA binding inhibitor, TPBM (20 μM). *, P < 0.05 compared to treatment with TNF-α alone. ns, not significant.
Fig 4
Fig 4
ER recruitment to the BIRC3 promoter requires E2, TNF-α and the NF-κB pathway. (A) ChIP assays for ER were carried out following treatment of MCF-7 cells with E2, TNF-α, or both for up to 60 min, and recruitment to the BIRC3 promoter was examined. (B) ER recruitment to BIRC3 was carried out by ChIP assay following 24 h of exposure to IκBα-DN and 45 min of treatment with E2, TNF-α, or both. *, P < 0.05 compared to treatment with E2 or TNF-α alone.
Fig 5
Fig 5
TNF-α and p65 cause enrichment of acetylated histones around the BIRC3 ERE. (A) AcH3 and AcH4 were examined by ChIP assay in MCF-7 cells treated with TNF-α for 15 min. Enrichment was calculated by first dividing the percentage of input for each AcH3 or AcH4 pulldown by the percentage of input for the total amount of H3 or H4, respectively, and second by comparing the ratio of acetylated to total histones in TNF-α-treated cells to that in vehicle-treated control cells. PCR was carried out using small amplicons that tile along the BIRC3 promoter. *, P < 0.05 compared to an untreated control. (B) Acetylation of H3 and H4 at the ERE was assessed following transfection with siNeg (control) or sip65 and treatment with or without TNF-α for 15 min. Fold change was calculated from the percentage of input for each pulldown and comparison to untreated siNeg controls for 3 independent experiments. **, P < 0.01 compared to untreated siNeg control. (C) p65 protein levels were examined by Western blotting in MCF-7 cells transfected with siNeg or sip65 for 48 h.
Fig 6
Fig 6
Role of CBP in BIRC3 expression, ER recruitment, and histone acetylation around the ERE. (A) MCF-7 cells were transfected with siRNA for CBP, and regulation of BIRC3 mRNA by E2, TNF-α, or both was assessed by qPCR. (B) ER recruitment to the BIRC3 promoter was assessed by ChIP assays following transfection with siNeg, siER, or siCBP and treatment with E2 plus TNF-α for 15 min. Data are represented as a percentage of ER recruitment relative to the siNeg control. (C) Acetylated H3 and H4 at the ERE were examined following transfection with siNeg or CBP. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, compared to the siNeg control. (D) ChIP assays for CBP or IgG were carried out after 15 min of treatment with E2, TNF-α, or both.
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