Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)-a key cell lineage implicated in age-related breast cancers-from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer

Insulin Resistance in Women Correlates with Chromatin Histone Lysine Acetylation, Inflammatory Signaling, and Accelerated Aging

BackgroundEpigenetic changes link medical, social, and environmental factors with cardiovascular and kidney disease and, more recently, with cancer. The mechanistic link between metabolic health and epigenetic changes is only starting to be investigated. In our in vitro and in vivo studies, we performed a broad analysis of the link between hyperinsulinemia and chromatin acetylation; our top "hit" was chromatin opening at H3K9ac.MethodsBuilding on our published preclinical studies, here, we performed a detailed analysis of the link between insulin resistance, chromatin acetylation, and inflammation using an initial test set of 28 women and validation sets of 245, 22, and 53 women.ResultsChIP-seq identified chromatin acetylation and opening at the genes coding for TNFα and IL6 in insulin-resistant women. Pathway analysis identified inflammatory response genes, NFκB/TNFα-signaling, reactome cytokine signaling, innate immunity, and senescence. Consistent with this finding, flow cytometry identified increased senescent circulating peripheral T-cells. DNA methylation analysis identified evidence of accelerated aging in insulin-resistant vs. metabolically healthy women.ConclusionsThis study shows that insulin-resistant women have increased chromatin acetylation/opening, inflammation, and, perhaps, accelerated aging. Given the role that inflammation plays in cancer initiation and progression, these studies provide a potential mechanistic link between insulin resistance and cancer

Insulin Resistance in Women Correlates with Chromatin Histone Lysine Acetylation, Inflammatory Signaling, and Accelerated Aging

BACKGROUND: Epigenetic changes link medical, social, and environmental factors with cardiovascular and kidney disease and, more recently, with cancer. The mechanistic link between metabolic health and epigenetic changes is only starting to be investigated. In our in vitro and in vivo studies, we performed a broad analysis of the link between hyperinsulinemia and chromatin acetylation; our top hit was chromatin opening at H3K9ac. METHODS: Building on our published preclinical studies, here, we performed a detailed analysis of the link between insulin resistance, chromatin acetylation, and inflammation using an initial test set of 28 women and validation sets of 245, 22, and 53 women. RESULTS: ChIP-seq identified chromatin acetylation and opening at the genes coding for TNFα and IL6 in insulin-resistant women. Pathway analysis identified inflammatory response genes, NFκB/TNFα-signaling, reactome cytokine signaling, innate immunity, and senescence. Consistent with this finding, flow cytometry identified increased senescent circulating peripheral T-cells. DNA methylation analysis identified evidence of accelerated aging in insulin-resistant vs. metabolically healthy women. CONCLUSIONS: This study shows that insulin-resistant women have increased chromatin acetylation/opening, inflammation, and, perhaps, accelerated aging. Given the role that inflammation plays in cancer initiation and progression, these studies provide a potential mechanistic link between insulin resistance and cancer

Minor groove binder distamycin remodels chromatin but inhibits transcription.

The condensed structure of chromatin limits access of cellular machinery towards template DNA. This in turn represses essential processes like transcription, replication, repair and recombination. The repression is alleviated by a variety of energy dependent processes, collectively known as "chromatin remodeling". In a eukaryotic cell, a fine balance between condensed and de-condensed states of chromatin helps to maintain an optimum level of gene expression. DNA binding small molecules have the potential to perturb such equilibrium. We present herein the study of an oligopeptide antibiotic distamycin, which binds to the minor groove of B-DNA. Chromatin mobility assays and circular dichroism spectroscopy have been employed to study the effect of distamycin on chromatosomes, isolated from the liver of Sprague-Dawley rats. Our results show that distamycin is capable of remodeling both chromatosomes and reconstituted nucleosomes, and the remodeling takes place in an ATP-independent manner. Binding of distamycin to the linker and nucleosomal DNA culminates in eviction of the linker histone and the formation of a population of off-centered nucleosomes. This hints at a possible corkscrew type motion of the DNA with respect to the histone octamer. Our results indicate that distamycin in spite of remodeling chromatin, inhibits transcription from both DNA and chromatin templates. Therefore, the DNA that is made accessible due to remodeling is either structurally incompetent for transcription, or bound distamycin poses a roadblock for the transcription machinery to advance

Circular Dichroism spectroscopy to study distamycin induced structural changes of chromatosomes and chromatosomal DNA.

<p>(A) Chromatosome or (B) chromatosomal DNA (50 µM nucleotide concentration) is treated with distamycin in drug to DNA base ratios of 0.08 (­­­), 0.16 (·····), and 0.25 (-·-·-).Chromatosome, chromatosomal DNA and distamycin solutions are prepared in 5 mM Tris HCl (pH 7.4), 15 mM NaCl and titrations are performed at 25°C.</p

Interaction of distamycin with histones.

<p>ITC profiles for the interaction of distamycin with (A) core histones and (B) linker histone in 5 mM Tris HCl (pH 7.4), 100 mM NaCl at 25°C.</p

ATP independence of distamycin induced remodeling.

<p>(A) Agarose gel electrophoresis to study the effect of distamycin on chromatosomes, with and without prior treatment of apyrase. For apyrase treatment, chromatosomes (300 µM DNA base) were treated with apyrase at 2 U/ml for 30 minutes at 30°C. Chromatosomes were then incubated with distamycin in the drug to DNA base ratios indicated, and electrophoresed on 1.5% agarose gel. (B) Agarose gel electrophoresis to study the effect of distamycin on mononucleosomes, reconstituted on a 200 bp DNA fragment, containing a centrally positioned 601 positioning sequence. Distamycin treatment was performed as indicated.</p

Analysis of remodeled structures.

<p>Bands 1–3 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057693#pone-0057693-g001" target="_blank">Figure 1A</a> were electroeluted and analyzed separately for their histone and DNA components. (A) SDS-PAGE analysis of histones isolated from the electroeluted samples. Lanes 1–3 contain histones isolated from the corresponding bands in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057693#pone-0057693-g001" target="_blank">Figure 1A</a>. (B) Western blot analysis of histones present in the SDS-PAGE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057693#pone-0057693-g002" target="_blank">Figure 2A</a>). (C) DNA component of the bands 1–3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057693#pone-0057693-g001" target="_blank">Figure 1A</a>. In all the cases, bands 1–3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057693#pone-0057693-g001" target="_blank">Figure 1A</a> correspond to lanes 1–3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057693#pone-0057693-g002" target="_blank">Figure 2</a>.</p

Remodeling of chromatosomes by distamycin A.

<p>(A) Agarose gel electrophoresis to study the effect of distamycin on chromatosomes. Chromatosome samples were incubated with distamycin at room temperature for 90 minutes at the drug concentrations indicated and analyzed on 1.5% agarose gel. Chromatosomes incubated with buffer (lanes 1 and 2) served as negative controls. Arrows numbered 1–3 indicate the bands excised and electroeluted for further characterization. (B) Effect of distamycin on chromatosomes, monitored as a function of time. Chromatosome samples (300 µM) were treated with distamycin (50 µM), at room temperature, for varying time intervals, and analyzed on 1.5% agarose gel. (C) Agarose gel electrophoresis to study the effect of distamycin on chromatosomal DNA.l.</p

Loss of epigenetic suppression of retrotransposons with oncogenic potential in aging mammary luminal epithelial cells

A primary function of DNA methylation in mammalian genomes is to repress transposable elements (TEs). The widespread methylation loss that is commonly observed in cancer cells results in the loss of epigenetic repression of TEs. The aging process is similarly characterized by changes to the methylome. However, the impact of these epigenomic alterations on TE silencing and the functional consequences of this have remained unclear. To assess the epigenetic regulation of TEs in aging, we profiled DNA methylation in human mammary luminal epithelial cells (LEps)—a key cell lineage implicated in age-related breast cancers—from younger and older women. We report here that several TE subfamilies function as regulatory elements in normal LEps, and a subset of these display consistent methylation changes with age. Methylation changes at these TEs occurred at lineage-specific transcription factor binding sites, consistent with loss of lineage specificity. Whereas TEs mainly showed methylation loss, CpG islands (CGIs) that are targets of the Polycomb repressive complex 2 (PRC2) show a gain of methylation in aging cells. Many TEs with methylation loss in aging LEps have evidence of regulatory activity in breast cancer samples. We furthermore show that methylation changes at TEs impact the regulation of genes associated with luminal breast cancers. These results indicate that aging leads to DNA methylation changes at TEs that undermine the maintenance of lineage specificity, potentially increasing susceptibility to breast cancer.publishedVersio