Genomic imprinting mediated by DNA methylation restricts gene expression to a single allele determined by parental origin and is not generally considered to be under genetic or environmental influence. Here, we focused on a differentially methylated region (DMR) of approximately 1.9 kb that includes a 101-bp noncoding RNA gene (nc886/VTRNA2-1), which is maternally imprinted in ∼75% of humans. This is unlike other imprinted genes, which demonstrate monoallelic methylation in 100% of individuals. The DMR includes a CTCF binding site on the centromeric side defining the DMR boundary and is flanked by a CTCF binding site on the telomeric side. The centromeric CTCF binding site contains an A/C polymorphism (rs2346018); the C allele is associated with less imprinting. The frequency of imprinting of the nc886 DMR in infants was linked to at least two nongenetic factors, maternal age at delivery and season of conception. In a separate cohort, nc886 imprinting was associated with lower body mass index in children at 5 y of age. Thus, we propose that the imprinting status of the nc886 DMR is “tunable” in that it is associated with maternal haplotype and prenatal environment. This provides a potential mechanism for transmitting information, with phenotypic consequences, from mother to child.

Ovarian cancer ranks as the most deadly gynecologic cancer, and there is an urgent need to develop more effective therapies. Previous studies have shown that G9A, a histone methyltransferase that catalyzes mono- and dimethylation of histone H3 lysine9, is highly expressed in ovarian cancer tumors, and its overexpression is associated with poor prognosis. Here we report that pharmacologic inhibition of G9A in ovarian cancer cell lines with high levels of expression induces synergistic antitumor effects when combined with the DNA methylation inhibitor (DNMTi) 5-aza-2'-deoxycytidine (5-aza-CdR). These antitumor effects included upregulation of endogenous retroviruses (ERV), activation of the viral defense response, and induction of cell death, which have been termed "viral mimicry" effects induced by DNMTi. G9Ai treatment further reduced H3K9me2 levels within the long terminal repeat regions of ERV, resulting in further increases of ERV expression and enhancing "viral mimicry" effects. In contrast, G9Ai and 5-aza-CdR were not synergistic in cell lines with low basal levels. Taken together, our results suggest that the synergistic effects of combination treatment with DNMTi and G9Ai may serve as a novel therapeutic strategy for patients with ovarian cancer with high levels of G9A expression. Dual inhibition of DNA methylation and histone H3 lysine 9 dimethylation by 5-aza-CdR and G9Ai results in synergistic upregulation of ERV and induces an antiviral response, serving as a basis for exploring this novel combination treatment in patients with ovarian cancer. .

Graphical Abstract Highlights d Anillin-like Mid1 is a substrate of the SIN kinase Sid2 d SIN signaling controls Mid1's membrane localization d Sid2 temporally regulates Cdr2-Mid1 association d Cdr2 and Mid1 are key SIN substrates for resetting cell division site placement

<div>Abstract<p>Ovarian cancer ranks as the most deadly gynecologic cancer, and there is an urgent need to develop more effective therapies. Previous studies have shown that G9A, a histone methyltransferase that catalyzes mono- and dimethylation of histone H3 lysine9, is highly expressed in ovarian cancer tumors, and its overexpression is associated with poor prognosis. Here we report that pharmacologic inhibition of G9A in ovarian cancer cell lines with high levels of <i>G9A</i> expression induces synergistic antitumor effects when combined with the DNA methylation inhibitor (DNMTi) 5-aza-2′-deoxycytidine (5-aza-CdR). These antitumor effects included upregulation of endogenous retroviruses (ERV), activation of the viral defense response, and induction of cell death, which have been termed "viral mimicry" effects induced by DNMTi. G9Ai treatment further reduced H3K9me2 levels within the long terminal repeat regions of ERV, resulting in further increases of ERV expression and enhancing "viral mimicry" effects. In contrast, G9Ai and 5-aza-CdR were not synergistic in cell lines with low basal <i>G9A</i> levels. Taken together, our results suggest that the synergistic effects of combination treatment with DNMTi and G9Ai may serve as a novel therapeutic strategy for patients with ovarian cancer with high levels of G9A expression.</p><p><b>Significance:</b> Dual inhibition of DNA methylation and histone H3 lysine 9 dimethylation by 5-aza-CdR and G9Ai results in synergistic upregulation of ERV and induces an antiviral response, serving as a basis for exploring this novel combination treatment in patients with ovarian cancer. <i>Cancer Res; 78(20); 5754–66. ©2018 AACR</i>.</p></div>

<p>Includes one supplementary table, 13 supplementary figures and figure legends for supplementary figures</p>