Poly (ADP-ribose) polymerase 1 transcriptional regulation: a novel crosstalk between histone modification H3K9ac and ETS1 motif hypomethylation in BRCA1-mutated ovarian cancer

Abstract

Poly (ADP-ribose) polymerase 1 (PARP1) plays a critical role in ovarian cancer progression. However, the epigenetic mechanism regulating PARP1 transcription remains largely unknown. Here, we show that the hypomethylated ETS1 motif is a key regulatory element for the PARP1 gene in BRCA1-mutated ovarian cancer. Mechanistically, the ETS1 motif hypomethylation-mediated increase of active histone marker H3K9ac and transcription factor ETS1 enrichment synergistically activates PARP1 transcription. Clinicopathological data indicate that a hypomethylated ETS1 motif was associated with high-grade tumors (P = 0.026) and pN1 (P = 0.002). Univariate survival analysis demonstrated an association between the hypomethylated ETS1 motif and an increased risk of death in BRCA1-mutated ovarian cancer patients. Our findings imply that the genetic (such as BRCA1 mutation) and epigenetic mechanisms (such as hypomethylated ETS1 motif, and histone modification H3K9ac and transcription factor ETS1 binding) are jointly involved in the malignant progression of PARP1-related ovarian cancer.

Figure 1. ETS1 motif methylation and PARP1 transcriptional activity

(A) the schematic represents the cytosine located at the ETS1 and ETS2 motif that were point mutated to generate the thymine. (Bi-Biv) 293T cells, SKOV3 cells and primary non-mutated and BRCA1-mutated ovarian cancer cells were transfected with mutant plasmids. At 24 hours after transfection, whole-cell extracts were analyzed for luciferase activity. Bar graphs show mean ± SD. *P < 0.05 vs. Control. Mut., Mutation. (Ci) the schematic represents the selected nucleotide sequence with or without a methyl group at the fifth position of the cytosine pyrimidine ring at the ETS1 motif. (Cii) the CD spectra of the selected nucleotide sequence in the presence of 100 mM Na+ or 100 mM K+ are shown.

Figure 2. Characteristic histone modification and ETS1 factors enrichment patterns around the hypomethylated ETS1 motif in BRCA1-mutated ovarian cancer

(Ai) chromatin immunoprecipitation was performed using antibodies to H3K9ac, H3K18ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, H3K36me3, H3K79me, H3K9me, H3K9me2, H3K9me3, H3K27me, H3K27me2, and H3K27me3. PCR was performed for regions around the ETS1 motif. A negative control without antibodies was included for comparison. (Aii) representative results of three independent experiments are shown. (B) expression levels of the GCN5, PCAF and ETS1 factors in BRCA1-mutated ovarian cancer. Bar graphs show mean ± SD, *P < 0.05 vs. Control.

Figure 3. H3K9ac and ETS1-mediated transcriptional regulation of PARP1

(Ai) RT-PCR showing GCN5, PCAF and ETS1 factors levels before and after knockdown by SHRNA, and normalized to β-actin expression. (Aii) representative results of three independent experiments are shown. (Bi) EdU labeling showing proliferation of GCN5, PCAF and ETS1-silenced and control cells. Blue, Hoechst 33342 labeling of cell nuclei; Red, EdU labeling of nuclei of proliferative cells. (Bii) the EdU incorporation rate was expressed as the ratio of EdU positive cells to total Hoechst33342 positive cells. (Ci and Cii) analysis of histone modification H3K9ac and transcription factor ETS1 enrichment around the ETS1 motif after the deletion of GCN5, PCAF or ETS1 factors. (D) The interactions of ETS1 and GCN5 or PCAF were examined by the immunoprecipitation of cell extracts with an antibody to ETS1, and the co-immunoprecipitation of ETS1, GCN5 and PCAF by western blot analysis. Results of Fig.3 A-D were obtained in BRCA1-mutated ovarian cancer cells, and the same results were also obtained in 293T cells, SKOV3 cells, and non-BRCA1 mutated ovarian cancer cells (data not shown). (Ei-Ev) the PARP1 expression levels after deletion of H3K9ac and ETS1 enrichment around the ETS1 motif in 293T cells, SKOV3 cells, and primary non-mutated and BRCA1-mutated ovarian cancer cells. Bar graphs show mean ± SD. *P < 0.05 vs. Control.

Figure 4. Kaplan-Meier analysis of overall survival for 69 BRCA1-mutated ovarian cancer patients

The following variables were analyzed: age at diagnosis, menstruation, grade, pT, pN, pM, FIGO, CA125, ascites, residual tumor, and p53 and ETS1 methylation. Pre, premenopausal; Post, postmenopausal; FIGO, International Federation of Gynecology and Obstetrics; Pos, positive; Neg, negative; M, methylated; UM, unmethylated.