Top epigenetics articles: January 2015

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Reduced expression of MYC increases longevity and enhances healthspan

Transgenic mice show reduced MYC is key to longevity

​MYC is a transcription factor that promotes cancer when it is deregulated, but knocking down the dosage has a prolonging effect on lifespan in mice. This indicates that MYC is a potent regulator of senescence, as that is one of the major anti-proliferation obstacles that cancer overcomes.

John M. Sedivy and team at Brown University created a mouse model of MYC haploinsufficiency, where only one functional copy of the transcription factor is present, which allowed for an examination of gene dosage effects.

The researchers found that haploinsufficiency mutants:

• Have resistance to age-related problems, with transcriptomics showing enrichments for alterations to metabolism and immune function.
• Appear to be more active, with higher metabolic rates and healthier lipid metabolisms.
• Have changes in nutrient and energy sensing pathways, including reduced serum IGF-1, which is a textbook marker of longevity.

When it comes to evolutionary past, MYC is a conserved regulator of ribosome biogenesis that is reflected in this model by a decrease in translation, which is inversely correlated to longevity. Overall, this research attests to the power of manipulating genetic dosage to increase longevity and quality of life.

Read the full report in Cell, January 2015.

Abcam products used: Mouse IGF-1 ELISA kit (ab100695)

m⁶A mRNA methylation facilitates resolution of naï​ve pluripotency toward differentiation

m⁶A mRNA methylation promotes stem cell differentiation and priming

Pluripotent stem cells can exist in a naï​ve state that resembles pre-implantation cells found in the inner cell mass (ICM) or in a primed state, known as epiblast stem cells (EpiSCs), which resemble post-implantation epiblast cells. Currently, little is known about molecular regulators that modulate transition to primed pluripotency.

Jacob Hanna and colleagues at Weizmann Institute of Science and Tel Aviv University performed a siRNA screen in primed EpiSCs. The screen targeted transcriptional and epigenetic regulators known to be involved in naï​ve pluripotency.

The authors were able to identify Mettl3, an N6-methyladenosine (m⁶A) transferase that is a component of the m⁶A mRNA methylating complex, as a regulator that terminates murine naï​ve pluripotency. The
m⁶A RNA modification is known to be involved in regulating gene expression through RNA splicing and stability as well as modulating the binding capacity to reader proteins.

The research revealed that:

• When Mettl3 is knocked out, both naï​ve embryonic stem cells and pre-implantation epiblasts remain viable, but show an almost complete loss of m⁶A modification in mRNAs.
• Depletion of m⁶A in mRNAs decreases stem cell priming and differentiation competence, leading to a hyper-naï​ve pluripotency state.
• Of naï​ve pluripotency promoting genes, 80% were methylated at m⁶A in Mettl3 expressing stem cells, whereas mRNA levels of pluripotency regulators increased in Mettl3 knock-out cells during differentiation.

This study reveals a critical regulator role for m⁶A mRNA methylation that acts as a molecular switch during murine naï​ve pluripotency by directly reducing the stability of m⁶A mRNA transcripts, including key naï​ve​ pluripotency promoting factors. Thus, m⁶A methylation can down-regulate the levels of pluripotency factors in a timely fashion to promote priming and differentiation. 

Read the full report in Science, January 2015.

Abcam products used: FITC conjugated anti-BrdU antibody (ab74545) 

​EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumors to TopoII inhibitors

Epigenetic inhibitors as therapeutic tools to fight lung cancer

Pharmacological targeting of specific epigenetic modifiers is increasingly used to treat cancers. Epigenetic modifiers that elicit gene silencing, such as those targeting H3K27me3, are particularly promising and may be effective as anti-cancer agents.

In this paper, Carla Kim et al. studied the therapeutic potential of drugs that specifically target EHZ2, an enzyme that methylates H3K27me3 and promotes gene repression, on non-small cell lung cancers (NSCLCs). They investigated whether EHZ2 inhibition can increase sensitivity to a TopoII inhibitor, a current chemotherapy that is only successful in a minority of patients.

The authors:

• Identified genetic biomarkers (mutations in BRG1 or EGFR genes) present in a number of NSCLC cell lines that were predictive of increased sensitivity to a Topoll inhibitor during EZH2 inhibition.
• Xenografted these sensitized cell lines in vivo and observed reduced tumor growth with dual Topoll and EZH2 pharmacological inhibition. In addition, mice carrying mutations in EGFR exhibited reduced tumor formation in response to dual treatment. In contrast, the same treatment in wild-type mice resulted in continued tumor growth.
• Demonstrated genetic antagonism of BRG1 and EGFR​ by showing that BRG1 associates with the EGFR regulatory element and disrupts its function.

Developing tailor-made treatments for lung cancer is vital for effective treatment. These authors have provided important evidence for the use of EHZ2 inhibitors in specific sensitized cohorts, rather than a one-treatment-fits-all approach. 

Read the full report in Nature, January 2015.

Abcam products used: anti-histone H3 antibody (ab1791)

Site- and allele-specific polycomb dysregulation in T-cell leukemia 

Epigenetic de-silencing by insertions in T cell leukemia

T cell acute lymphoblastic leukemias (T-ALLs) are an aggressive group of cancers, which differ greatly from case to case in their underling genetic cause. The cause is often a complex interaction of mutations, many of which remain unknown. TAL1 oncogene activation is frequently altered in T-ALLs; however, many cases that are TAL⁺ lack characteristic TAL1 lesions, suggesting deregulation of epigenetic pathways. In these cases, one or both TAL1 alleles can be inappropriately activated but the mechanism is unclear.

Vahid Asnafi and Bertrand Nadel from Aix-Marseille University in France sought to understand the epigenetic basis of these unsolved cases. They used ChIP-seq to identify epigenetic marks and developed an allelic-ChIP assay to distinguish between histone marks on both chromosomes. 

Using these techniques in T-ALL cell lines, the authors found that:

• Microinsertions in the TAL1​ promoter occur in a repressive H3K27me3 region.
• One insertion occurred at a looping-region bordering the H3K27me3 region on one allele. This permitted allelic-ChIP using primers specific to the insertion and the normal chromosomes.
• There was a decrease in H3K27me3 and increase in H3K27ac on the inserted allele.
• The first in vivo example of oncogenic activation by recombination activating gene (RAG)-mediated episomal reinsertion, accounts for over 20% of unsolved cases of monoallelic TAL1 expression level.

This work provides a better understanding for the complex epigenetic mechanisms at play in cancer. Further it demonstrates several firsts, which are likely to be applicable beyond this specific cancer to others. It may also provide a framework to understand similar epigenetic processes in other biological phenomena. 

Read the full report in Nature Communications, January 2015.

Abcam products used: anti-histone H3K27me3 antibody (ab6002) and anti-histone H3K27ac antibody (ab4729)

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​​Single-cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming

Non-coding RNAs play functional role in cellular reprogramming 

Cellular reprogramming during development is perhaps the most important role of epigenetic processes. Complex regulatory networks are necessary to determine the developmental fate of each cell. Long non-coding RNAs (lncRNAs) have emerged as key players in many processes, but most lncRNAs still have unknown function. Specifically, the role of lncRNAs in cellular reprogramming remains unclear.

Daniel Kim and Barbara Wold from Caltech, California, chose to investigate the role of lncRNAs in early reprogramming. To do so, they used emerging single-cell RNA sequencing analysis. Single-cell analysis are particularly useful for transcriptomic studies, since each cell represents a possibly unique expression state with particular regulators and target gene behavior. 

The authors found that:

• Some of the earliest molecular events in reprogramming are the activation of the Ras signaling pathways and transcription of pluripotency factors and many lncRNAs.
• Functional studies confirmed that lncRNAs repress linage-specific genes during reprogramming.
• lncRNAs appear to specifically target metabolic genes.

These results show that lncRNAs have a functional and crucial role in cellular reprogramming. Studies such as these further demonstrate that much of the non-protein coding "junk" DNA is actually necessary for fundamental biological processes. 

Read the full report in Cell Stem Cell, January 2015.

If you would like more on non-coding RNA, find events through our miRNA and non-coding RNA conference calendar.

Lagging​-strand replication shapes the mutational landscape of the genome

DNA replication induces novel mutation signature

Mutations accumulate throughout the genome in various non-random patterns, but the mechanisms underlying these patterns remain unclear. It is known that nucleosome and transcription factor binding impact nucleotide substitution rates. Okazaki fragments are a consequence of bidirectional DNA amplification, and Okazaki junctions are where these fragments are ligated. Nucleosome and TFs create a partial block on DNA, resulting in increased Okazaki junctions at their binding sites.

Andrew Jackson and Martin Taylor from the University of Edinburgh set out to understand the overlap between yeast Okazaki junctions and nucleotide substitutions. They developed a novel method called embedded ribonucleotide sequencing (emRiboSeq) to track different polymerases such as the error-prone Pol-α​ DNA. This was accomplished by mapping ribonucleotides incorporated into DNA which are normally removed. 

Using this and other methods, the authors found that: 

• Mutations occur more often at the core of nucleosome binding sites, representing the 5' ends of Okazaki fragments. 
• Pol-α-synthesized DNA is retained an accounts for about 1.5% of the yeast genome.
• Similar Okazaki fragment mutational signatures around nucleosome and transcription factor binding events were conserved in humans.

The authors propose a model wherein DNA-binding proteins re-associate with post-replication DNA and act as barriers to Pol-α-synthesized DNA, resulting in its incorporation. This in turn leads to increased mutation hotspots at TF binding sites in both yeast and humans.

Read the full report in Nature​, January 2015.

Genomic profiling of DNA methyltransferases reveals a role for DNMT3b in genic methylation

Histones guide recruitment of DNMT3b to transcribed gene bodies

DNA methylation is mediated by the de novo methyltransferases DNMT3A and DNMT3b, whereas maintenance of these epigenetic tags is predominantly the task of DNMT1. However, the mechanisms that govern the recruitment and activity of these DNA methyltransferases remain unclear.

To address this issue, Tuncay Baubec and colleagues at the Swiss based Institute for Biomedical Research used streptavidin ChIP-sequencing of biotin tagged functional proteins to determine genomic binding and site specific activity of DNMT3A and DNMT3b in embryonic stem cells (ESCs).

The researchers found that:

• Both de novo enzymes localize to methylated, CpG rich areas within nucleosome linker regions, but are excluded from active promoters and enhancers. 
• The recruitment of DNMT3b to transcribed genes occurs through SETD2 mediated H3K36me3, which guides DNMT3b binding and de novo DNA methylation to transcribed gene sites. 
• DNMT3A2 shows the opposite behavior, with decreased methylation at transcribed genes.
• Reintroduction of DNMT3A2 and DNMT3B1 to DNA methylation deficient DNMT triple knockout cells leads to reproducible levels of re-methylation, even in the absence of DNMT1, with DNMT3A2 more processive than DNMT3B1.

The authors provide insight into the complex genomic binding that translates into site-specific de novo methylation activity. They further demonstrate that it is possible to detect regulated targeting of DNA methylation in organisms that have genome-wide methylation, such as mammals.

Read the full report in Nature, January 2015.

Abcam products used: Anti-Histone H3 (tri methyl K36) antibody (ab9050),  Anti-Histone H3 antibody (ab1791), Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody [H5] (ab24758).

A vlincRNA participates in senescence maintenance by relieving H2AZ-mediated repression at the INK4 locus

A very long intergenic ncRNA controls senescence

​Long non-coding RNAs (lncRNAs) are a diverse class of ncRNA that generally regulate chromatin structure and function. Long intergenic ncRNAs (lincRNAs) are one class of lncRNA, with a more recently discovered very long variety, termed vlincRNA.

Estelle Nicolas and research team from Université​ de Toulouse in France profiled strand-specific transcriptomic changes in their human stem cell model of accelerated senescence, which is achieved by oncogene activation.

The researchers found that:

• While most differentially expressed (sense and antisense) RNAs are repressed, vlincRNAs tend to be activated. 
• A novel antisense vlincRNA termed VAD is strongly induced during senescence and is required for the ongoing maintenance of senescence. 
• VAD regulates chromatin confirmation in cis by promoting the decrease of histone H4 acetylation and H3K4 trimethylation, both of which are transcriptional activators. This allows VAD to contribute to the repression of cell proliferation genes that are antisense to it.
• VAD also acts in trans on the INK4 locus where it activates the transcription of cell cycle inhibitors, which act as tumor suppressors. It accomplishes this by inhibiting the addition of repressive H2A.Z at the promoters of the INK4 locus in senescent cells.

Ultimately, this research highlights the dual role of vlincRNAs in senescence-related pathways, both as a sensor of a change in cellular environment and the subsequent mediator of transcriptional profiles to maintain cell cycle arrest.

Read the full report in Nature Communications, January 2015.

Abcam products used: Anti-HMGA1a/HMGA1b antibody (ab4078),  anti-histone H2A.Z antibody (ab4174),
anti-p400 antibody (ab5201), anti-RNA polymerase II CTD repeat YSPTSPS phospho S2 antibody (ab5095), and anti-DDAH1 antibody (ab82908).