American Association for Cancer Research
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Figure S1. Overexpression and knockdown of H1.3. from Histone H1.3 Suppresses H19 Noncoding RNA Expression and Cell Growth of Ovarian Cancer Cells

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posted on 2023-03-30, 22:30 authored by Magdalena Medrzycki, Yunzhe Zhang, Weijia Zhang, Kaixiang Cao, Chenyi Pan, Nathalie Lailler, John F. McDonald, Eric E. Bouhassira, Yuhong Fan

Figure S1. Overexpression and knockdown of H1.3. A) Generation of OV-3/H1.3 stable cell lines. Western blotting of H1.3 overexpressing clones. OVCAR-3 cells were transfected with a pcDNA-FLAG-H1.3 construct. G418 resistant clones were screened by Western blotting using an anti-FLAG antibody. Coomassie staining shows equal loading of the cell lysates. Lane 1: OVCAR-3; Lane 2: OVCAR-3 transfected with pcDNA3 vector only (designated as OV-3/V.O); Lanes 3-9: OVCAR-3/H1.3 clones. Clones marked with "*" and "#" were used as representative high and low FLAG-H1.3 expressing clones (OV-3/H1.3(H) and OV-3/H1.3(L)). B) Knockdown of H1.3 levels by shRNA in OV-3/H1.3(H) cells. Whole cell lysates were analyzed by Western blotting using an anti-H1.3 antibody. Western blots using an anti-ACTIN antibody were included as loading controls.

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ARTICLE ABSTRACT

Ovarian cancer is a deadly gynecologic malignancy for which novel biomarkers and therapeutic targets are imperative for improving survival. Previous studies have suggested the expression pattern of linker histone variants as potential biomarkers for ovarian cancer. To investigate the role of histone H1 in ovarian cancer cells, we characterize individual H1 variants and overexpress one of the major somatic H1 variants, H1.3, in the OVCAR-3 epithelial ovarian cancer cell line. We find that overexpression of H1.3 decreases the growth rate and colony formation of OVCAR-3 cells. We identify histone H1.3 as a specific repressor for the noncoding oncogene H19. Overexpression of H1.3 suppresses H19 expression, and knockdown of H1.3 increases its expression in multiple ovarian epithelial cancer cell lines. Furthermore, we demonstrate that histone H1.3 overexpression leads to increased occupancy of H1.3 at the H19 regulator region encompassing the imprinting control region (ICR), concomitant with increased DNA methylation and reduced occupancy of the insulator protein CTCF at the ICR. Finally, we demonstrate that H1.3 overexpression and H19 knockdown synergistically decrease the growth rate of ovarian cancer cells. Our findings suggest that H1.3 dramatically inhibits H19 expression, which contributes to the suppression of epithelial ovarian carcinogenesis. Cancer Res; 74(22); 6463–73. ©2014 AACR.