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Background: A missense mutation in the Forkhead domain-containing FOXL2 (Foxl2 p.C134W) transcription factor is found in nearly all adult-type granulosa cell tumors, but the oncogenic mechanism of this mutation is not known. Other Forkhead family transcription factors have well-described “pioneer” activity, binding to compacted, nucleosome-bound DNA and increasing accessibility for other regulatory proteins. Objectives: To develop novel cell culture model systems and determine whether oncogenic Foxl2-C134W has pioneering activity. Methods: CRISPR/Cas9 editing was used to generate isogenic granulosa cell tumor cells lacking either the FOXL2 wild-type allele (single knock-out; SKO) or both the mutant and wild-type FOXL2 alleles (double knock-out; DKO). ATAC-Seq and endogenous Foxl2 ChIP-Seq were performed on these isogenic lines to determine the differential chromatin accessibility at Foxl2-bound regulatory regions across genotypes. ENCODE pipelines and data standards were used for analysis, and the irreproducible discovery rate was used to identify high-reliability ATAC-seq and ChIP-seq peaks. De novo motifs were identified with STREME. Promoter capture HiC was performed using a bespoke genome-wide hybrid capture probe set, and CHiCAGO was used to identify significant HiC interactions. Results: Endogenous ChIP-seq of Foxl2-C134W from the SKO cell line identified 1147 high-confidence peaks. De novo motif discovery identified the canonical Foxl2 binding motif (TGTTTACATT, P = 1.4 x 10-7) in 44.9% of peaks, as well as a novel variant motif (TGTTTTGTCT, P = 6.7 x 10-15) in 68.5% of peaks. Median chromatin accessibility at Foxl2-C134W peak regions, as measured by ATAC-seq, was significantly decreased in the DKO cells compared to either SKO or parental cell lines (P < 2 x 10-16 for both comparisons). No difference was observed between SKO and parental cell lines (P = 0.19). Decreased chromatin accessibility at Foxl2-C134W ChIP-seq peaks in the DKO cells was driven by peaks containing the novel variant Foxl2 binding motif. Promoter capture HiC identified promoter-enhancer interactions lost in the DKO cell line involving several genes, including HUNK, NOVA1, andSFRP2 (adjusted P < 0.05). Conclusion: Foxl2-C134W exhibits “pioneering” activity, increasing chromatin accessibility at key gene regulatory elements with associated re-programming of promoter-enhancer interactions. The oncogenic mechanism of Foxl2-C134W in granulosa cell tumors may involve changes in DNA binding specificity, re-directing this pioneering function to sites containing a novel variant Foxl2 binding motif. Citation Format: Veena Vuttaradhi, Eleonora Khlebus, Thomas Welte, Namrata Khurana, Barrett Lawson, Robert Tyler Hillman. Oncogenic Foxl2 is a chromatin-remodeling pioneer transcription factor in adult-type ovarian granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1462. |
