All tags Epigenetics Top Epigenetics articles: December 2014

Top Epigenetics articles: December 2014

​​Want to know what you missed over the holiday period? We've summarized our favorite epigenetics papers from December.

Paternal diet defines offspring chromatin state and intergenerational obesity

Epigenetic marks mediate intergenerational obesity

Obesity is one of the most pressing epidemics facing western culture. There is evidence that parental weight and nutritional state influence offspring body mass. The precise mechanism of these effects is unclear, but epigenetic phenomena are believed to be involved. Paternal models of intergenerational effects have recently become popular, as they remove difficult to control oocyte and gestational effects.

Andrew Pospisilik and colleagues from the Max Planck Institute developed a fruit fly model of paternal-diet-induced intergenerational metabolic reprogramming (IGMR). They used dietary sugar to induce increased triglyceride levels in male parental flies. In their model, body weight of F1 offspring increased with paternal sugar and F1 triglyceride levels increased with low and high parental sugar.

Using this model the authors found that:

  • Chromatin domains are de-silenced in offspring and sperm of sugar-exposed males. 
  • The repressive H3K9/K27me3 marks and regulating proteins are required for IGMR.
  • The IGMR chromatin state signature is conserved in mice and humans.

This paper provides evidence for intergenerational control of obesity and chromatin states. There are relatively few examples of intergenerational inheritance of traits with mechanistic explanations. This work adds to a growing body of evidence that epigenetic states mediate transgenerational inheritance of complex traits. 

Read the full report in Cell, December 2014.

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An epigenomic roadmap to induced pluripotency reveals DNA methylation as a reprogramming modulator

DNA methylation as a driver of reprogramming to pluripotency

​Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSC) using defined transcription pluripotency factors. This process requires global reshaping of the epigenetic landscape including dynamic changes in the methylation levels and chromatin modifications of specific genes. These changes can silence genes necessary for maintaining the differentiated state and reactivate pluripotency genes. However, these global changes in the epigenetic landscape during different stages of reprogramming remain poorly understood. 

Jeong-Sun Seo and colleagues at the Seoul National University and Life Science Institute employed whole-genome bisulfite sequencing, ChIP-seq for histone 3 lysine 4 tri-methylation (H3K4me3), histone 3 lysine 27 tri-methylation (H3K27me3) and histone 3 lysine 36 tri-methylation (H3K36me3), and RNA-seq to obtain a single-base pair resolution map of the epigenetic changes during the reprogramming process in mouse cells.

The researchers found that during reprogramming:

  • There are dynamic changes in DNA methylation, such that increases in DNA methylation happen gradually, while decreases in methylation are observed only at the embryotic stem cell-like state.
  • During early reprogramming, binding sites for activated or overexpressed transcription factors are marked by focal methylation depletion and changes in chromatin that are permissive to gene expression.
  • During later reprogramming to embryonic stem cell-like pluripotent cells, hypermethylation is extended to a broader range of up to 20 kb from the binding sites.
  • Genes with CpG-rich promoters show low methylation levels and their expression is driven by histone modifications.
  • Regulation of genes with CpG-poor promoters is driven by DNA methylation.

These findings provide a deeper understanding of how DNA methylation shapes the epigenetic landscape in reprogramming to pluripotency and could shed light on the mechanisms that underlie not only iPCS reprogramming but also cell lineage commitment and stem cell-based therapies.

Read the full report in Nature Communications, December 2014.

Abcam products used: Anti-histone H3 tri-methyl K4 (ab8580) and  anti-histone H3 tri-methyl K36 (ab9050) ChIP-grade antibodies.



Deconstructing transcriptional heterogeneity in pluripotent stem cells

Stem cell-to-cell variation in gene expression

Pluripotent stem cells (PSCs) are able to differentiate into all cell types and can self-perpetuate in culture. They are key to understanding organismal development at a basic level, and hold promise for regenerative medicine. Distinct PSC subtypes exist that vary in self-renewal and differentiation capacity, permitting study of the transcriptional underpinning of these processes. 

James Collins and colleagues from Harvard University set out to characterize the transcriptional heterogeneity in mouse PSCs. To do this, they used recently developed single-cell RNA-seq and ChIP-seq technologies to examine PSCs after various genetic and chemical perturbations.

The authors found:

  • Expression of signaling and developmental factors is highly variable, groups together and persists through multiple cell divisions.
  • Housekeeping and metabolic genes showed consistent expression across cells, while signaling pathway genes varied greatly.
  • Removal of miRNAs or blockage of signaling pathways drives cells toward a low-variation ground state capable of enhanced self-renewal with a distinct chromatin state.
  • Specific miRNAs acting on traditional pluripotency promoting mRNAs (Myc, Lin28a) encourage transitional transcriptional states.

The analysis of the dynamic transcriptional landscape presented here provides several new insights into variation in PSCs. It demonstrates that variation in regulation of gene expression directly impacts the transition between pluripotency and differentiation.

See the full report in Nature, December 2014.

Abcam products used: anti-histone H3 tri-methyl K36 (ab9050), anti-histone H3 tri-methyl K9 (ab8898), anti-histone H3 acetyl K27 (ab4729), anti-Oct4 (ab19857) and anti-Nanog (ab80892) antibodies.



Divergent reprogramming routes lead to alternative stem-cell states

Induced pluripotent stem cells are not all created equal

Pluripotent cells are cells capable of differentiating into all three germ layers, and hold much therapeutic promise. These cells come from one of two sources: embryonic stem cells or via somatic cell reprogramming.

Somatic cell reprogramming is done by artificially expressing key transcription factors. These induced pluripotent stem cells (iPSC) are known to be true stem cells by their ability to generate all cell types and generate iPSC mice. However, it is currently unknown if all iPSCs are the same or not.

Peter Tonge and colleagues sought to characterize the range of possible iPSCs. To do this they reprogrammed embryonic fibroblasts with the doxycycline-inducible PiggyBac transposon system to establish 28 cell lines. They then characterized gene expression in these cell lines.

They found that: 

  • Elevated reprogramming factor expression causes embryonic fibroblasts to go through unique epigenetic modifications until thy arrive at a stable, Nanog-positive, alternative pluripotent cells.
  • Pluripotent cells are made of numerous cell states.

Peter Tonge and colleagues identified previously unknown induced pluripotent stem cell populations. It is thought that unique pluripotent cell lines, like those identified here, may prove useful in developing tailored therapeutic cell lines. 

Read the full report in Nature, December 2014. 

Abcam products used: Anti-GFP antibody (ab6673).



DNA hydroxymethylation profiling reveals that WT1 mutations result in loss of TET2 function in acute myeloid leukemia

Tumor suppressor protein WT1 regulates DNA hydroxymethylation

Mutations in epigenetic modifiers are common in cancers such as acute myeloid leukemia (AML). One class of modifiers often mutated are the TET2 and IDH1/IDH2 proteins, which demethylate 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). It is unknown if mutations in such proteins lead to genome-wide changes in 5-mC or 5-hmC in AML, or how this contributes to disease progression.

Ross L. Levine and colleagues from Cornell University sought to characterize the role of these 5-hmC regulators in AML. They previously showed that TET2 and IDH1/IDH2 mutations rarely co-occur with mutations in the tumor suppressor gene WT1 in AML. The authors therefore hypothesized that WT1 independently affects TET2 demethylation function and a mutation in any of these genes would be sufficient to alter 5-hmC.

The authors showed that:

  • There is a global reduction in 5-hmC in AML samples.
  • WT1, TET2​ and IDH1/IDH2 ​mutations are associated with similar patterns of 5-mC alteration.
  • WT1, TET2​ and IDH1/IDH2 mutant AML cells show 5-hmC alteration at enhancers.
  • WT1 loss resembles TET2 knockout indicating WT1 forms a complex with TET2 that directly regulates 5-hmC.

The data presented here provides a better understanding of the role of epigenetic regulators in AML. More importantly, they show the involvement of the tumor suppressor protein WT1 in regulation of 5-hmC. These results provide further insight into the regulation of epigenetic marks in cancerous and normal tissue.

Read the full report in Cell Reports, ​December 2014.

Abcam products used: anti-SC35 antibody (ab28428).



​Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex

Developing CRISPR-Cas9 technology for improved insight into genomic gain of function

The ability to study the effect of genome-wide gain-of-function alterations in gene expression is currently limited to laborious and costly technologies such as cDNA library over-expression systems. The CRISPR-Cas9 complex offer a promising technique for gene up-regulation, yet the current system yields only moderate increases in gene transcription.

Feng Zhang and colleagues performed a 'structure-guided' approach using the crystallographic structure of a Cas9 complex to engineer a system that optimally enhances endogenous transcriptional activity at both coding and non-coding RNAs. 

The authors found that:

  • Examination of the Cas9 atomic structure in complex with a single-guide (sg) RNA and target DNA, identified two exposed anchoring positions: the sgRNA tetra-loop and stem loop 2 (sgRNA 2.0).
  • Appending the RNA aptamer VP64 to these positions enhanced transcriptional activity of a number of specific gene targets. 
  • Combining the sgRNA 2.0 system with other activating domains, including p65 and heat shock factor 1, led to the most effective transcription activation system. 
  • Synthesis of an sg-library targeting > 70,000 genes in the human genome and the application to an in vitro melanoma model, allowed the identification of numerous candidate genes and gene pathways involved in conferring drug resistance to a melonoma targeting drug.

The further development of CRISPR-Cas9 technology will pave the way for future studies to enhance our understanding of geneomic organization and gene regulation in numerous biological systems and disease states.

Read the full report in Nature, ​December 2014.



Genome-wide map of regulatory interactions in the human genome

Mapping long-range chromatin interactions

Over the last three years, the ENCODE project has sought to map the distribution of local chromatin states, epigenetic marks, and regulatory networks across the genome. However, these local interactions fail to explain the full complexity of the genome. Data suggests that long-range 3D chromatin interactions are also vital for cellular function. Technologies to map these interactions at sufficient genome scale and resolution have only recently become feasible.

Michael P. Snyder and colleagues from Stanford University sought to generate a genome-wide map of regulatory interactions in human cells using Chromatin Interaction Analysis by Paired-End Tag sequencing (ChIA-PET). This antibody-based technique identifies long-range DNA interactions that occur via a protein of interest. The authors used six widely distributed DNA binding proteins and histone marks in separate experiments to generate 3D genomic maps.

The authors found: 

  • There was strong enrichment of CTCF, cohesin and ZNF143 transcription factors at long-range interacting loci, indicating that they mediate these interactions.
  • Enhancer-promoter interactions are highly cell-type specific and correlate with gene expression patterns.
  • Proximal regulatory interactions bind distinct proteins versus distal ones: proximal events were associated with regulation of housekeeping genes whereas distal events were associated with dynamic regulation.

This study provides a novel dataset for long-range chromatin interactions occurring in the nucleus. It provides new data that puts forth new models of transcriptional regulation. These maps will create better resources for understanding the still mysterious role of long-range interactions in gene expression.

Read the full report in Genome Research, December 2014.

Abcam products used: anti-histone H3 mono-methyl K4 (ab8895), anti-histone H3 di-methyl K4 (ab7766), anti-histone H3 tri-methyl K4 (ab8580), anti-histone H3 acetyl K27 (ab4729) and anti-Rad21 (ab992) antibodies.



Towards a therapy for Angelman syndrome by targeting a long non-coding RNA

Targeting non-coding RNA to treat Angelman syndrome

Angelman syndrome is caused by a deficiency in the maternally imprinted UBE3A​ gene. This is because the other copy, the paternal copy, of the UBE3A gene is normally silenced. Therefore, an absence of the maternal copy results in a severe deficiency of UBE3A product, an E3 ubiquitin ligase, resulting in intellectual disability and developmental delays. While the cause of Angelman syndrome has been well known for many years, an effective therapy has not been found.

Linyan Meng, Amanda Ward and colleagues sought to correct Angelman syndrome by reactivating the intact paternal allele to restore UBE3A expression. To do this they used a high-throughput imaging screen to identify antisense oligonucleotides that could target the long non-coding RNA UBE3A antisense transcript (UBE3A-ATS) responsible for silencing the paternal UBE3A gene.

Doing this they found that: 

  • Antisense oligonucleotides could specifically reduce UBE3A-ATS.
  • Reduction in UBE3A-ATS unsilenced the paternal UBE3A​ in neurons, in both in vitro and in vivo studies.
  • In an Angelman syndrome mouse model, partial restoration of UBE3A protein via injection of antisense oligonucelotides could restore some cognitive deficits.

This paper presents a specific and feasible method to reactivate silenced paternal UBE3A gene. They present a promising new method for treating Angelman syndrome that paves the way for the treatment of other diseases that require gene therapy.

Read the full report in Nature, December 2014.

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