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, 45 (11), 1573-80

Epigenetic Silencing of SOD2 by Histone Modifications in Human Breast Cancer Cells

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Epigenetic Silencing of SOD2 by Histone Modifications in Human Breast Cancer Cells

Michael J Hitchler et al. Free Radic Biol Med.

Abstract

Many breast cancer cells typically exhibit lower expression of manganese superoxide dismutase (MnSOD) compared to the normal cells from which they arise. This decrease can often be attributed to a defect in the transcription of SOD2, the gene encoding MnSOD; however, the mechanism responsible for this change remains unclear. Here, we describe how altered histone modifications and a repressive chromatin structure constitute an epigenetic process to down regulate SOD2 in human breast carcinoma cell lines. Utilizing chromatin immunoprecipitation (ChIP) we observed decreased levels of dimethyl H3K4 and acetylated H3K9 at key regulatory elements of the SOD2 gene. Consistent with these results, we show that loss of these histone modifications creates a repressive chromatin structure at SOD2. Transcription factor ChIP experiments revealed that this repressive chromatin structure influences the binding of SP-1, AP-1, and NFkappaB to SOD2 regulatory cis-elements in vivo. Lastly, we show that treatment with the histone deacetylase inhibitors trichostatin A and sodium butyrate can reactivate SOD2 expression in breast cancer cell lines. Taken together, these results indicate that epigenetic silencing of SOD2 could be facilitated by changes in histone modifications and represent one mechanism leading to the altered expression of MnSOD observed in many breast cancers.

Figures

Fig. 1
Fig. 1
Levels of MnSOD mRNA, protein, and activity are lower in breast cancer cell lines than immortalized breast epithelial cells. (a) Relative SOD2 mRNA levels in breast cell lines determined by SYBR green real-time PCR. The steady state levels of SOD2 mRNA in the cell lines were normalized to 18S control, then compared to MCF-10A using the ΔΔCt method. Values shown are the average of three independent experiments ± standard error of mean (* p < 0.05). (b) The levels of MnSOD protein and activity in the model cell lines used during this study.
Fig. 2
Fig. 2
Locations of the seven real time PCR amplicons (A1-A7) used to analyze SOD2 by chromatin immunoprecipitation (ChIP) and chromatin accessibility. Binding sites for AP-1, NFκB and SP-1 within SOD2 are indicated by black lines. (A) The seven real time PCR amplicons used during histone and transcription factor ChIP as well as chromatin accessibility represented by black boxes below the line. Exon 1 of the SOD2 gene are designated by grey boxes. Numbering scheme was derived from build 17 of the human genome project. (B) Oligonucleotide primer sequences used to analyze the seven regions of SOD2 during chromatin immunoprecipitation and chromatin accessibility experiments.
Fig. 3
Fig. 3
Breast cancer cells have decreased levels of dimethyl H3K4 at SOD2 compared to immortalized breast epithelial. Methylation of H3K4 histones at seven regions (A1-A7) of the SOD2 promoter and at GAPDH in breast cell lines was determined using chromatin immunoprecipitation (ChIP). Fold enrichments of dimethyl H3K4 at these regions were calculated as described in methods and materials. Values given are the averages of three independent experiments ± the standard error. (* p < 0.05 between MCF-10A and breast cancer cell lines at respective amplicons. For individual statistical analysis between MCF-10A and breast cancer cell lines see Supplemental Table S1).
Fig. 4
Fig. 4
The SOD2 promoter of breast cancer cells is associated with hypoacetylated histones. The association of acetylated H3K9 with SOD2 and GAPDH in breast cell lines was determined using chromatin immunoprecipitation (ChIP). The level of acetylated H3K9 at seven regions (A1-A7) of the SOD2 and GAPDH promoters in breast cell lines were calculated as described in methods and materials. Values given are the averages of three independent experiments ± the standard error. (* p < 0.05 between MCF-10A and breast cancer cell lines at respective amplicons. For individual statistical analysis between MCF-10A and breast cancer cell lines see Supplemental Table S2).
Fig. 5
Fig. 5
The SOD2 gene of breast cancer cell lines is less accessible than immortalized breast epithelial cells. Chromatin accessibility of the seven regions (A1-A7) of the SOD2 promoter and GAPDH was determined by DNAse I hypersensitivity. Accessibility index of each region was determined using SYBR green real-time PCR. Accessibility index for each amplicon was then as described in methods and materials. Values given are the averages of three independent experiments ± the standard error. (* p < 0.05 between MCF-10A and breast cancer cell lines at respective amplicons. For individual statistical analysis between MCF-10A and breast cancer cell lines see Supplemental Table S3).
Fig. 6
Fig. 6
Binding of AP-1, SP-1 and NFκB are decreased at SOD2 in breast cancer cell lines. The placement and level of SP-1, NFκB, and AP-1 binding at SOD2 was determined using (ChIP). Fold enrichments of these transcription factors SOD2 was calculated as described under methods and materials.
Fig. 7
Fig. 7
The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate increased SOD2 expression in breast cancer cell lines. MB-231, T47D, MCF7 and MCF7-5C breast cancer cell lines were treated 200 ng/ml trichostatin A or 5 mM sodium butyrate for 12 hours. Total RNA was then harvest and subjected to quantitative real time RT-PCR analysis to determine changes in SOD2 expression. The levels of SOD2 mRNA were normalized using 18S rRNA. The relative level of SOD2 mRNA in each sample were determined by comparing TSA and sodium butyrate treated cells with their matched DMSO controls using the ΔΔCt method. Values shown are the average of three independent experiments ± stand are error of mean (* p < 0.5 versus untreated control). N.D.; not determined.
Fig. 8
Fig. 8
Changes in MnSOD expression correlate with chromatin function at SOD2. The relationship between SOD2 expression and the chromatin parameters in breast cells represented as radar graphs. The average values of relative SOD2 mRNA (north axis) were plotted against enrichment of H3K4 methylation at amplicon 4 (east axis), enrichment of acetylated H3K9 at amplicon 4 (south axis) and chromatin accessibility (west axis). These graphs contrast the differences in chromatin biology at SOD2 to clearly relate of the changes between MCF-10A and MB-231, T47D, MCF7, and MC7-5C breast cancer cell lines.
Fig. 9
Fig. 9
Proposed model for how changes in histone modifications alter SOD2 expression. The epigenetic state of SOD2 when MnSOD levels are high. Hyperacetylated histones create an accessible chromatin structure at SOD2 that allows transcription factors to bind. (A) When MnSOD is epigenetically repressed by histone hypoacetylation, an inaccessible chromatin structure forms at SOD2. This change in chromatin structure inhibits SP-1, NFκB, and AP-1 binding at SOD2 which decreases MnSOD transcription, protein and activity. This process is reversible and increased SOD2 expression in metastatic disease may be effected by further epigenetic alterations during progression leading to the epigenetic reactivation depicted in part (B).

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