Epigenetics articles of the month: July 2015

Want a hand keeping up with the latest Epigenetics literature? We have summarized our favorite papers from July.

Suppression of the alternative lengthening of telomere pathway by the chromatin remodeling factor ATRX

Expression of ATRX suppresses telomere lengthening in cancer cells

For cancer cells to divide indefinitely, they must maintain their telomeres throughout cell division. Whilst telomere maintenance is usually achieved through a mechanism that involves the telomerase enzyme, in 15% of cancers telomere length is maintained by a telomerase-independent pathway called the alternative lengthening of telomeres (ALT) pathway that is dependent on homologous recombination.

Recent research has identified inactivating mutations in the chromatin remodeling ATRX/DAXX/H3.3 complex in tumors displaying the ALT phenotype. David Clynes, Clare Jelinska and colleagues from the University of Oxford have further investigated the relationship between ATRX expression and the ALT phenotype. By studying U-2 OS cells, a well-established model for studying the ALT pathway, they found that:

  • Expression of ATRX in ATRX-null U-2 OS cells reverses the ALT phenotype and results in progressive decrease in telomere length.
  • Ectopic expression of ATRX in this cell line leads to elevated telomeric H3.3. This increase is prevented in the absence of DAXX, suggesting that ATRX-mediated suppression of ALT is dependent on DAXX.
  • The presence of a stabilizing ligand for the non-canoniocal DNA conformation, G4, results in the ALT becoming partially resistant to suppression by ATRX. This indicates that such non-canonical DNA structures may increase ALT activity.
  • ATRX expression lowers the frequency of stalled replication forks and sequesters MRN, a complex that repairs and restarts stalled replication forks.

The authors propose a model whereby loss of ATRX results in defective telomere chromatinization, which promotes non-canonical DNA secondary structure. These in turn present a barrier to DNA replication, leading to replication fork stalling, homologous recombination and subsequent recombination-mediated telomere synthesis. These findings may provide a basis for future development of therapies to target ALT-based cancers.

Read the full paper in Nature Communications, July 2015.

A DNA hypomethylation signature predicts antitumor activity of LSD1 inhibitors in SCLC

An epigenetic signature can predict response to new SCLC therapy

Epigenetic mechanisms have frequently been shown to be dysregulated in cancers. One such epigenetic mechanism is histone demethylation by lysine demethylase 1 (LSD1), a histone modifying enzyme that is overexpressed in some human cancers. Previously research has demonstrated that loss of LSD1 expression reduces cancer growth.

Helai Mohammad and colleagues from GlaxoSmithKline and Johns Hopkins University conducted a screen to identify small molecule inhibitors for LSD1. In this study, the authors describe the discovery of an LSD1 inhibitor called GSK2879552 and characterize its effect on cancer cells. Here is what they found:

  • GSK2879552 inhibits the growth of 9 of 20 small cell lung cancer (SCLC) cell lines tested and 20 of 29 acute myeloid leukemia (AML) cell lines.
  • Tumor growth is also inhibited in SCLC xenograft bearing mice orally administered with GSK2879552.
  • DNA hypomethylation correlates with sensitivity to GSK2879552 and a panel of 45 differentially methylated probes can predict sensitivity.
  • When the probe set and inhibitor were tested on three patient-derived xenograft models, efficacy was observed only when the sensitivity-associated DNA methylation signature was present.

The results presented in this study demonstrate that GSK2879552 might be an effective treatment for SCLC, a form of cancer that currently has limited treatment options. Although this inhibitor was not effective in all cancer cell lines, the authors have demonstrated that a specific DNA methylation signature could be used as a biomarker to stratify patients that might respond to treatment.

Read the full paper in Cancer Cell, July 2015.

Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis

TET proteins maintain the consistency of gene transcription

Ten-eleven translocation (TET) proteins are involved in DNA demethylation by successively oxidizing 5-methyl cytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), 5-formyl cytosine (5-fC) and 5-carboxyl cytosine (5-caC). TET proteins are important during embryogenesis when genome-wide demethylation occurs.

A team led by Anjana Rao from La Jolla Institute for Allergy and Immunology, California investigated the role of Tet1 and Tet3 in early embryonic development. By using Tet1 and Tet3 double knockout (Tet1/3 DKO) mouse embryos, they found that:

  • Single cells of Tet1/3 DKO eight-cell embryos display global loss of 5-hmC and gain of 5-mC.
  • In single blastomeres there is a high level of transcriptome variability in Tet1/3 DKOs compared with controls, both at the eight cell stage and at E3.5.
  • Whilst control blastocycts are substantially demethylated on all chromosomes, levels of 5-mC/5-hmC are increased but highly variable between different Tet1/3 DKOs.
  • Despite this variability in gene expression, a number of genes involved in lipid metabolism and cholesterol biosynthesis are consistently downregulated in Tet1/3 DKOs.

These data show that TET proteins have a role a role in maintaining transcriptional consistency. The authors suggest that TET enzymes and DNA cytosine modification could modulate transcriptional noise, and that this would affect certain gene networks more than others.

Read the full paper in PNAS, July 2015.

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Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA

Oct4 is regulated by a pseudogene RNA

Oct4, a member of the POU family of transcription factors, is required for embryonic stem cell (ESC) self-renewal. Many pathways are involved in the regulation of Oct4, including epigenetic silencing, post-translational modification and miRNA expression.

Recent studies have demonstrated the existence of Oct4 pseudogenes that may also contribute to the the regulation of Oct4 expression. To further understand how pseudogenes affect Oct4 expression, a team led by Roberta Benetti from the University of Trieste in Italy investigated the role of Oct4 pseudogenes in controlling mESC pluripotentency and self-renewal. They found that:

  • Four additional novel Oct4 pseudogenes, Oct4P2, Oct4P3, Oct4P4 and Oct4P5 are expressed in mESCs.
  • Oct4P4 overexpression results in reduced potential for mESC self-renewal and causes a 50% reduction in Oct4 protein levels.
  • Sense-orientated Oct4P4-derived lncRNAs localize in trans to the promoter of the Oct4 gene and mediate enrichment of heterochromatin marks H3K9me3, HP1α and SUV39H1 at the Oct4 promoter.
  • Oct4P4 lncRNA knockdown reduces SUV39H1 at the Oct4 promoter leading to a loss of heterochromatin. This reactivates endogenous Oct4 that promotes expression of key transcription factors of mESC self-renewal.

The authors have demonstrated that a novel pseudogene RNA plays an important role in inducing and silencing ancestral Oct4. Taken together, the data suggest that the Oct4P4-SUV39H1 complex is necessary to silence the Oct4 promoter and directs H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene.

Read the full paper in Nature Communications, July 2015.

Obesity-induced DNA hypermethylation of the adiponectin gene mediates insulin resistance

Methylation of the adiponectin promoter by Dnmt1 exacerbates metabolic disease

Expression of the adiponectin protein is negatively correlated with obesity and related metabolic disorders. Although hypoadiponectinemia is frequently assumed to be due to a failure in transcriptional regulation, the underlying mechanism that mediated dysregulation in obesity has not previously been investigated.

To understand whether epigenetic changes contribute to the regulation of adiponectin expression, a team led by Jae Bum Kim from Seoul National University investigated the role of DNA methylation in adiponectin expression and metabolic disease. They found that:

  • Adiponectin mRNA levels are significantly reduced in obese mice whereas mRNA levels of key adiponectin-regulatory transcription factors are not altered.
  • DNA methylation of a region of the adiponectin promoter (referred to as R2) in adipocytes is positively correlated with obesity in humans and mice. Methylation level of this region is inversely correlated with adiponectin mRNA.
  • Expression of the DNA methyltransferase 1 (Dnmt1) gene is elevated in adipocytes from obese mice compared with that of lean mice. Dnmt1 knockout led to a reduction in methylation of the R2 region and an increase in adiponectin mRNA and protein.
  • Pro-inflammatory cytokines induce Dnmt1 expression and activity, and increase R2 methylation.
  • DNA methyltransferase inhibition in obese db/db mice increases adiponectin expression and improves insulin resistance.

Taken together, these results demonstrate that Dnmt1-mediated methylation of the adiponectin promoter is a regulator of adiponectin expression and exacerbates metabolic disease in obesity. The authors suggest that stimulation of adiponectin expression by inhibition of DNA methyltransferase is a potential future therapeutic approach for obesity-related disease.

Read the full paper in Nature Communications, July 2015.

If you are interested in finding out more about this topic, register for our Epigenetics, Obesity and Metabolism conference, October 11–14, 2015, Cambridge UK.

Uridylation of RNA hairpins by Tailor confines the emergence of microRNAs in Drosophila

Tailor-mediated uridylation regulates miRNA abundance

Uridylation is an RNA modification that is important for regulating miRNA biosynthesis and turnover. Although uridylation is shown to occur in flies as well as in mammals, no miRNA-modifying terminal uridylyl transferase enzymes (TUTases) have previously been identified in flies.

In this paper, Madalena Reimão-Pinto and colleagues from the Institute of Molecular Biotechnology of the the Austrian Academy of Sciences sought to find out the origin, molecular mechanism and consequences of miRNA uridylation in Drosophila. By studying Drosophila S2 cells, they found that:

  • Selected miRNAs are frequently uridylated in Drosophila. In particular, mitrons are frequently modified with uridine.
  • Depletion of the cytoplasmic TNTase CG1091/Tailor results in a reduction of uridine-containing extensions, indicating that this enzyme is required for the majority of miRNA uridylation.
  • In cells depleted of Tailor, 40% of miRNAs change in abundance compared with control, with the majority increasing in abundance.
  • Uridylation of pre-miRNAs by Tailor changes the accuracy of the 3' overhangs, which affects the efficiency of miRNA production by Dicer.
  • Tailor preferentially targets miRNAs that end in 3' guanosine or uridine. Such miRNAs tend to be newly emerging, rather than conserved.

This research indicates that Tailor-mediated uridylation regulates the abundance of miRNAs by altering the rate of Dicer-mediated miRNA production. Tailor preferentially targets non-conserved miRNAs with 3' guanosine ends, supporting the hypothesis that evolutionary adaptation to pre-miRNA uridylation directs the nucleotide composition of pre-miRNA 3' ends.

Read the full paper in Molecular Cell, July 2015.

To find out more about this research, this paper has been highlighted along with a similar piece of research in a recent Molecular Cell editorial.

The H3K4-methyl epigenome regulates leukemia stem cell oncogenic potential

Global H3K4 trimethylation regulates LSC fate and oncogenic potential

It is well established that epigenetic perturbations cause aberrant gene expression that leads to oncogenesis. However, there is much less known about how the global epigenetic landscape contributes to the function of cancer stem cells.

Stephen Wong and colleagues from Stanford University School of Medicine sought to understand the role of the epigenome in leukemia stem cells (LSCs). By interrogating the epigenetic landscape of subpopulations of acute myeloid leukemia cells in which LSCs were either enriched or depleted, they found that:

  • There are quantitative differences in the levels of H3K4 and H3K79 between populations enriched and depleted for LSCs, with LSCs having a H3K4 hypermethylated and H3K79 hypomethylated state.
  • Global H3K4 hypermethylation of LSCs sustains the expression of the genes Hoxa and Meis1, which are required for acute myeloid leukemia (AML) pathogenesis.
  • Ectopic overexpression of the H3K4 histone demethylase, KDM5B, reduces Hoxa9 and Meis1 transcript levels and suppresses the in vitro growth of leukemia cells.

The results presented in this paper demonstrate that the global state of H3K4 trimethylation has a major role in regulating LSC fate and oncogenic potential. These findings provide a rationale for future research into targeting H3K4me3 deposition as a potential leukemia therapy.

Read the full paper in Cancer Cell, July 2015.

MicroRNA-431 accelerates muscle regeneration and ameliorates musclar dystrophy by targeting Pax7 in mice

miR-431 targets Pax7 and accelerates muscle regeneration

To aid muscle regeneration in response to injury or stress, skeletal muscle stem cells (satellite cells) proliferate and either differentiate or self-renew. Satellite cells are heterogeneous and exist in two populations: a Pax7Hi population that has a higher level of Pax7 expression and a Pax7Lo population.

Although these two populations have distinct biological features, the mechanism that governs Pax7 expression in satellite cells during differentiation is unclear. Rimao Wu and colleagues from the Institute of Basic Medical Sciences in Beijing investigated the possibility that Pax7 levels are modulated by miRNAs. By looking at the role of miR-431 in mouse skeletal cells, the team found that:

  • miR-431 directly targets Pax7 in skeletal muscle and miR-431 expression is inversely correlated with Pax7 expression in differentiating satellite cells and in myoblasts isolated from 3-week and 12-week mice.
  • Overexpression of miR-431 stimulates myogenic differentiation and this effect is abolished by Pax7 overexpression, indicating that miRNA promotes myogenic differentiation by targeting Pax7.
  • miR-431 transgenic mice express Pax7 in skeletal muscle at a lower level than wild type and are enriched for the Pax7Lo subpopulation.
  • Pax7Lo satellite cells in miR-431 transgenic mice are primed more for differentiation and less for self-renewal, and muscle regeneration is accelerated in these mice.
  • Introduction of the miR-431 transgene into mdx mice—a mouse model for muscular dystrophy—reduces the dystrophic phenotype in these mice.

Overall the data in this paper shows that miR-431 targets Pax7 to mediate satellite cell heterogeneity during muscle development and regeneration. The finding that miR-431 expression can reduce the phenotype of mdx mice raises the possibility that miR-431 mimics may be beneficial for patients with muscular dystrophy.

Read the full paper in Nature Communications, July 2015.