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, 22 (23), 6299-309

Role of Acetylated Human AP-endonuclease (APE1/Ref-1) in Regulation of the Parathyroid Hormone Gene

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Role of Acetylated Human AP-endonuclease (APE1/Ref-1) in Regulation of the Parathyroid Hormone Gene

Kishor K Bhakat et al. EMBO J.

Abstract

The human AP-endonuclease (APE1/Ref-1), a multifunctional protein central to repairing abasic sites and single-strand breaks in DNA, also plays a role in transcriptional regulation. Besides activating some transcription factors, APE1 is directly involved in Ca2+-dependent downregulation of parathyroid hormone (PTH) expression by binding to negative calcium response elements (nCaREs) present in the PTH promoter. Here we show that APE1 is acetylated both in vivo and in vitro by the transcriptional co-activator p300 which is activated by Ca2+. Acetylation at Lys6 or Lys7 enhances binding of APE1 to nCaRE. APE1 stably interacts with class I histone deacetylases (HDACs) in vivo. An increase in extracellular calcium enhances the level of acetylated APE1 which acts as a repressor for the PTH promoter. Moreover, chromatin immunoprecipitation (ChIP) assay revealed that acetylation of APE1 enhanced binding of the APE1-HDACs complex to the PTH promoter. These results indicate that acetylation of APE1 plays an important role in this key repair protein's action in transcriptional regulation.

Figures

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Fig. 1. In vivo acetylation of APE1. (A) Two-dimensional gel electrophoresis of (I) recombinant APE1 (100 ng) and (II) HeLa nuclear extract (50 µg). After isoelectric focusing on an Immobiline dry strip (pH gradient 3–10) in an IPGphor electrophoresis system (Amersham), the proteins were separated by SDS–PAGE, and APE1 was visualized by western blotting. The asterisk indicates modified APE1. (B) Extracts of HCT116 cells transfected with FLAG-tagged APE1 expression plasmid (lane 2) or empty vector (lane 1), after labeling with [3H]Na- acetate, were immunoprecipated with FLAG antibody and analyzed by SDS–PAGE and fluorography. Lane 3: in vitro acetylated [3H]APE1 marker. (C) Western analysis with APE1 antibody of extracts of HCT116 immunoprecepitated with either AcLys antibody (α-AcLys; lane 2) or pre-immune control IgG (lane 1); total cell extract (lane 3) was used as a reference. (D) Panel I: western analysis with AcLys antibody of extracts of HCT116 cells transfected with FLAG-APE1 (lane 5), FLAG-APE1 plus p300 expression plasmids (lane 4), or empty vector (lane 3) followed by immunoprecipitation with FLAG antibody. The specificity of AcLys antibody for AcAPE1 was shown by western analysis of 0.1 µg of APE (lane 1) or AcAPE1 (lane 2). Western analysis of the same blot with FLAG antibody (panel III) and Coomassie staining (panel II) of duplicate immunoprecipitate in the same experiment to show comparable amounts of APE1 in samples with different levels of AcAPE1.
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Fig. 2. In vitro acetylation of APE1. (A) Immunoprecipitated p300 from HCT116 extract was used for in vitro acetylation of wild-type (WT) (lane 2) or NΔ40 APE1 (lane 3) with [3H]acetyl-CoA, followed by SDS–PAGE and fluorography. Lane 1: non-specific IgG control with full-length APE1. Lower panel: Coomassie staining after SDS–PAGE. (BIn vitro acetylation with purified p300 HAT domain of wild-type APE1 (lane 1) or NΔ40 APE1 (lane 2) and [3H]acetyl-CoA followed by SDS–PAGE and fluorography. The asterisk indicates autoacetylated p300 HAT domain.
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Fig. 3. Identification of AcLys residues in APE1. (A) Acetylation of N1 and N11 peptides (sequences in the lower panel) with immunoprecipitated p300 and [3H]acetyl-CoA as described in Materials and methods. (B) Mass spectroscopic analysis of in vitro acetylated N1 peptide. Peak corresponds to the unmodified peptide (m/z 2186.9) and peak Y to the monoacetylated form (m/z 2228.8). (C) Wild-type APE1 (10 µg) was incubated with 0.4 µg of p300 (HAT domain) and 1 mM acetyl-CoA for 1 h at 30°C. N-terminal sequencing in cycle 6 identified AcLys (AcK) residues as PTH-AcLys, which elutes just before PTH-Ala. (D) Acetylation of wild-type APE1 and K7R (KR), K6R/K7R (RR) or K6L/K7L (LL) mutants as described in Materials and methods. Lower panel: Coomassie staining after SDS–PAGE.
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Fig. 4. AcAPE1 enhances binding of HCT116 nuclear extract to 5-32P-labeled nCaRE-B oligonucleotide. (A) EMSA with extract alone (lane 2), supplemented with 50 or 100 ng of APE1 (lanes 3 and 4) or AcAPE1 (lanes 5 and 6). Lane 1: free probe. The arrow indicates specific complex. (B) EMSA with nuclear extracts of HCT116 cells transfected with plasmids for wild-type APE1 (lane 3), K6R/K7R (RR) APE1 (lane 4), p300 (lane 5), p300 HAT mutant (p300 HM; lane 6), wild-type APE1 + p300 (lane 7) and wild-type APE1 + p300 mutant (lane 8), and no extract in lane 1. (C) EMSA with nuclear extracts of HCT116 cells transfected with K6R/K7R (RR) APE1 (lane 2), RR + p300 (lane 3), RR + p300 HM (lane 4) and HDAC1 (lane 5). (D) Western analysis for the APE1 level in the extracts used in (B).
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Fig. 5. Repression of the PTH gene by AcAPE1. (A) BHK-21 cells were co-transfected with a PTH-luciferase reporter plasmid (1 µg) and 1 µg of expression plasmid for APE1 (wild-type or K6R/K7R mutant), p300 (wild-type or a HAT mutant) or equivalent amounts of empty vector. At 8 h after transfection, the cells were incubated in fresh medium containing no or 2 mM Ca2+ for 36 h. Luciferase activity was normalized with co-expressed β-galactosidase. The basal promoter activity of PTH-luc reporter in the presence of empty expression vector was normalized to 10. The bar graph shows the average of three independent experiments performed in duplicate. Cells were treated with TSA (100 ng/ml) in two sets. Western analysis for the APE1 level (lower panel) in the transfected cell extracts used in (A). (B) HCT116 cells transfected with FLAG-APE1 were incubated in medium containing 100 ng/ml TSA (lane 2) for 8 h. Immunoprecipitate of extracts with FLAG antibody were analyzed by western blotting using AcLys antibody. (C) BHK-21 cells were transfected with 5 nM (lane 2) and 10 nM (lane 3) p300 duplex siRNA oligo, and cell extracts were analyzed by western blotting using p300 (upper panel) and APE1 antibody (lower panel). (D) BHK-21 cells were co-transfected with a PTH-luciferase reporter plasmid (1 µg) and 10 nM p300 siRNA oligo along with either 1 µg of APE1 expression plasmid or an equivalent amount of empty expression vector. Luciferase activity was measured as described in (A). (E) BHK-21 cells were co-transfected with SV40-luciferase reporter plasmid (1 µg) containing PTH nCaRE-B upstream of the promoter, and 1 µg of the indicated expression plasmid for wild-type APE1 or K6R/K7R mutant, or p300 or equivalent amounts of empty vector. Luciferase activity was assayed after incubating cells in the presence of Ca2+ as described before.
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Fig. 6. Enhancement of acetylated APE1 and HAT activity of p300. (A) HCT116 cells transfected with FLAG-APE1 were incubated in medium containing no Ca2+ for 36 h and then treated with no (lane 1) or 2 mM Ca2+ (lane 2) for 4 h. Immunoprecipitates of extracts with FLAG antibody were analyzed by western blotting using AcLys antibody. Lower panel: western analysis for APE1. (B) BHK-21 cells were grown in Ca2+-free medium for 36 h, and then treated with 2 mM Ca2+ for 0, 2, 4, 6, 8 and 12 h. Immunoprecipitated endogenous p300 with [3H]acetyl-CoA was used for in vitro acetylation of core histones, followed by SDS–PAGE and fluorography. Lower panel: Coomassie staining after SDS–PAGE. (C) Western analysis for p300 level.
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Fig. 7. In vivo interaction of APE1 and HDACs. (A) HCT116 cells were co-transfected with expression plasmid for wild-type APE1 and individually for FLAG-tagged HDACs 1–6. The immunoprecipitates of cell extracts with FLAG antibody were analyzed for APE1. Panel I: lane 1, cell extract; lane 8, recombinant APE1 as a marker. Panel II: western analysis of cell extracts to show similar levels of APE1 in different transfected cells. Panel III: western analysis for the expression levels of HDACs. (B) Panel I: HCT116 cells were co-transfected with expression plasmid for wild-type APE1, or K6R/K7R or NΔ40 APE1 mutant and FLAG-tagged HDAC1. The FLAG immunoprecipitates of cell extracts were analyzed for APE1. Lane 1: immunoprecipitates from only APE1-transfected cell extracts. Panel II: western analysis of whole-cell extracts showing similar expression of APE1 in different transfected cells. Panel III: western analysis with FLAG antibody indicating significant expression of HDAC1.
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Fig. 8. (A) ChIP assay for in vivo association of APE1 and HDAC1 with PTH promoter in transfected plasmids. The amount of immuno precipitated PTH promoter sequence was reported as pg/unit volume, using an absolute standard curve (0.001–1000 pg) of PTH-luc standards. Fold enrichment was calculated after normalizing with the absolute amount of input chromatin. (B) Western analysis with AcLys antibody of FLAG immunoprecipitates from cell extracts used in the ChIP assay.

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