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Oncogene
2011 Nov 24;3047:4731-9. doi: 10.1038/onc.2011.186.
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The exonuclease activity of hPMC2 is required for transcriptional regulation of the QR gene and repair of estrogen-induced abasic sites.
Krishnamurthy N
,
Ngam CR
,
Berdis AJ
,
Montano MM
.
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We have previously reported that the expression of antioxidative stress enzymes is upregulated by trans-hydroxytamoxifen (TOT) in breast epithelial cell lines providing protection against estrogen-induced DNA damage. This regulation involves Estrogen Receptor β (ERβ) recruitment to the Electrophile Response Element (EpRE) and a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). We have also demonstrated that ERβ and hPMC2 are required for TOT-dependent recruitment of poly (ADP-ribose) polymerase 1 (PARP-1) and Topoisomerase IIβ (Topo IIβ) to the EpRE. Sequence analysis reveals that the C-terminus of hPMC2 encodes a putative exonuclease domain. Using in vitro kinetic assays, we found that hPMC2 is a 3'-5' non-processive exonuclease that degrades both single-stranded and double-stranded substrates. Mutation of two conserved carboxylate residues drastically reduced the exonuclease activity of hPMC2, indicating the relative importance of the catalytic residues. Western blot analysis of breast cancer cell lines for Quinone Reductase (QR) levels revealed that the intrinsic exonuclease activity of hPMC2 was required for TOT-induced QR upregulation. Chromatin immunoprecipitation (ChIP) assays also indicated that hPMC2 was involved in the formation of strand breaks observed with TOT treatment and is specific for the EpRE-containing region of the QR gene. We also determined that the transcription factor NF-E2-related factor-2 (Nrf2) is involved in the specificity of hPMC2 for the EpRE. In addition, we determined that the catalytic activity of hPMC2 is required for repair of abasic sites that result from estrogen-induced DNA damage. Thus, our study provides a mechanistic basis for transcriptional regulation by hPMC2 and provides novel insights into its role in cancer prevention.
Figure 2. Representative autoradiograms and graphs of single and double stranded substrates with WT and DM hPMC2Single-turnover experiments, in which enzyme concentration is in excess of DNA, were performed with 20 nM single or double stranded EpRE substrate and increasing concentrations of either WT or DM hPMC2. The DNA was radioactively labeled on the 5'-end and increasing amounts of the enzyme was added and the reaction monitored for a period of one hour. Gel images were obtained with a Molecular Dynamics Storm 840 phosphorimager system and data analysis was performed using ImageQuaNT software. The increase in product formation observed over the time course of the reaction was then plotted on Kaleidagraph. A. Representative autoradiogram of three independent experiments of single stranded EpRE substrate with 500 nM WT or DM hPMC2. B. Representative autoradiogram of three independent experiments of double stranded EpRE substrate with 500 nM WT or DM hPMC2. C. Kaleidagraph of product formed over time with single stranded EpRE substrate at varying concentrations of WT or DM hPMC2: 100 (●), 300 (▲), 500 (■) nM WT hPMC2 and 500 nM (◯) DM hPMC2. The inset shows the same plot over a shorter time period (12 min.) to illustrate the difference in the rate constants at varying concentrations of the enzyme. D. Kaleidagraph of product formed over time with double stranded EpRE substrate at varying concentrations of WT or DM hPMC2: 100 (●), 300 (▲), 500 (■) nM WT hPMC2 and 500 nM (◯) DM hPMC2.
Figure 3. hPMC2 exonuclease activity is required for TOT-induced QR expression and DNA strand breaksMCF7 cells were transfected with either hPMC2 miRNA that targets hPMC2 3'-UTR or hPMC2 miRNA along with plasmid expressing WT or the DM hPMC2. Cells were either untreated or treated with 10−6 M TOT for 3 hours. A. Proteins were extracted from cells and processed for western blot analyses of hPMC2 as described in “Materials and Methods”. Image shown is representative of three independent experiments. B. Proteins were extracted from cells and processed for western blot analyses of QR as described in “Materials and Methods”. Levels of QR were quantitated and normalized to GAPDH. The gel image shown is representative of three independent experiments. Columns in the bar graph represent the fold change in QR levels in control or TOT-treated samples. Error bars indicate standard error of the mean of 3 independent experiments. a, significance (P < 0.05) vs. untreated cells; b, significance (P < 0.05) vs. control transfected cells with the same treatment. C. DNA strand breaks were detected by BrdUTP labeling by terminal deoxynucleotidyl transferase (TdT) and ChIP assays were performed using anti-BrdU antibodies as described in “Materials and Methods”. The EpRE containing region of the QR gene was then amplified and the bars represent the fold change in DNA strand breaks at the EpRE in control or TOT-treated samples. Error bars indicate standard error of the mean of 3 independent experiments. a, significance (P < 0.01) vs. untreated cells; b, significance (P < 0.01) vs. control transfected cells with the same treatment.
Figure 4. Nrf2 is required for hPMC2 recruitment at the EpRE region of the QR geneA. Nrf2 miRNA or control MCF7 cells were treated with 10−6 M TOT for 3 hours. Proteins were extracted from cells and processed for western blot analyses of Nrf2 expression as described in “Materials and Methods”. Levels of Nrf2 were quantitated and normalized to GAPDH. The gel image shown is representative of three independent experiments. Columns in the bar graph represent the fold change in Nrf2 expression levels in control or TOT-treated samples. Error bars indicate standard error of the mean of 3 independent experiments. a, significance (P < 0.05) vs. untreated cells; b, significance (P < 0.01) vs. control transfected cells with the same treatment. B. Nrf2 miRNA or control MCF7 cells were treated with 10−6 M TOT for 3 hours and processed and analyzed using ChIP assays and hPMC2 antibody. The EpRE containing region of the QR gene was then amplified and the bars represent the mean of three replicate experiments for hPMC2 recruitment. Error bars indicate standard error of the mean of 3 independent experiments. a, significance (P < 0.01) vs. untreated cells; b, significance (P < 0.01) vs. control transfected cells with the same treatment.
Figure 5. hPMC2 exonuclease activity is required for repair of estrogen-induced abasic sitesMCF10A cells were transfected with either hPMC2 miRNA that targets hPMC2 3'-UTR or hPMC2 miRNA along with plasmid expressing WT or the DM hPMC2. Cells were either untreated or treated with 50 nM E2 for 24 hours. Genomic DNA was isolated from cells and processed for Aldehyde-Reactive Probe (ARP) labeling as described in “Materials and Methods”. Columns represent the number of AP sites/105 base pair (bp) in control or E2-treated samples. Error bars indicate standard deviation of 2 independent experiments. a, significance (P < 0.05) vs. untreated cells; b, significance (P < 0.05) vs. control transfected cells with the same treatment.
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