Methylation-deficient Cells Research Articles

LSD1 inhibitor hinders the demethylation of FOXA1 to inhibit prostate cancer progression

Abnormal activation of androgen receptor (AR) occurs in prostate cancer (PC) progression and metastasis. Lysine-specific demethylase 1 (LSD1), the first demethylating enzyme, regulates AR-mediated transcriptional activity. Considering the effect of Forkhead box protein A1 (FOXA1) on the expression of AR, estrogen receptor (ER) and tumor suppressor genes, this study investigated the demethylation of FOXA1 upon treatment with LSD1 inhibitors and assessed the biological behaviors of PC cells. PC cells were cultured and infected with viruses. After transient transfection, CWR22-RV1-Cas9 cells were selected by puromycin with expression of LSD1 detected by Western blot. Apart from measurement of formaldehyde production, immunoprecipitation and chromatin immunoprecipitation (ChIP) were performed, followed by ATAC-seq detection, and Western blot. The data indicated the association between LSD1-binding sites and high levels of FOXA1. LSD1 inhibitor treatment resulted in a dramatic decline in overall FOXA1 binding, significantly reducing chromosomal accessibility and also increasing lysine-methylated FOXA1 level, but it failed to affect H3K4me2 levels at LSD1-FOXA1 occupied sites. Overexpression of LSD1-WT obtained reverse outcome. Besides, LSD1 inhibition diminished binding of FOXA1 and restored lysine-methylation of FOXA1 in methylation-deficient cells with mutant K270R. Moreover, silencing of LSD1 suppressed CWR22-RV1 tumor growth, resulting in increased H3K4me2 and decreased AR-FL/V7 gene expression. K270me is demethylated by LSD1. LSD1 inhibitor disrupts FOXA1 chromatin association, blocks FOXA1 K270-demethylation and hinders AR binding, thereby suppressing PC cell growth.

Abstract 1081: DNA methylation at the intersect of chromatin structure and transcriptome diversity

Abstract Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer, but they have been studied as independent processes. We discovered a DNA methylation regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which in turn, promotes proximal polyadenylation site usage. In this DNA de-methylated context, depletion of the RAD21 cohesin complex component can recover distal polyadenylation site usage. We also confirmed, using chromosomal conformation capture (3C), that CTCF and the cohesin complex mediate chromatin loops in the absence of DNA methylation to modulate polyadenylation site usage. Leveraging data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment. Citation Format: Angela H. Ting, Vishal Nanavaty, Elizabeth Abrash, Byron Lee, Emily Fink, Tae Hyun Hwang, Changjin Hong. DNA methylation at the intersect of chromatin structure and transcriptome diversity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1081.

DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression invivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Abstract IA04: How DNA methylation organizes the cancer epigenome

Abstract The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. We performed a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG Island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome depleted regions, respectively. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multi-scale organization of cancer epigenomes. Citation Format: Peter A. Jones. How DNA methylation organizes the cancer epigenome. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA04.

The role of DNA methylation in directing the functional organization of the cancer epigenome.

The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes.

Methylarsonous acid causes oxidative DNA damage in cells independent of the ability to biomethylate inorganic arsenic.

Inorganic arsenic (iAs) and its toxic methylated metabolite, methylarsonous acid (MMA(III)), both have carcinogenic potential. Prior study shows iAs-induced malignant transformation in both arsenic methylation-proficient (liver) and methylation-deficient (prostate) cells, but only methylation-proficient cells show oxidative DNA damage (ODD) during this transformation. To further define whether arsenic methylation is necessary for transformation or ODD induction, here we chronically exposed these same liver or prostate cell lines to MMA(III) (0.25-1.0 μM) and tested for acquired malignant phenotype. Various metrics of oncogenic transformation were periodically assessed along with ODD during chronic MMA(III) exposure. Methylation-deficient and methylation-proficient cells both acquired a cancer phenotype with MMA(III) exposure at about 20 weeks, based on increased matrix metalloproteinase secretion, colony formation, and invasion. In contrast, prior work showed iAs-induced transformation took longer in biomethylation-deficient cells (~30 weeks) than in biomethylation-proficient cells (~18 weeks). In the present study, MMA(III) caused similar peak ODD levels at similar concentrations and at similar exposure times (18-22 weeks) in both cell types. At the approximate peak of ODD production, both cell types showed similar alterations in arsenic and oxidative stress adaptation factors (i.e., ABCC1, ABCC2, GST-π, SOD-1). Thus, MMA(III) causes oncogenic transformation associated with ODD in methylation-deficient cells, indicating that further methylation is not required to induce ODD. Together, these results show that MMA(III) and iAs cause an acquired malignant phenotype in methylation-deficient cells, yet iAs does not induce ODD. This indicates iAs likely has both genotoxic and non-genotoxic mechanisms dictated by the target cell's ability to methylate arsenic.