Epigenetics articles of the month: June 2015

Read our selection of the most exciting Epigenetics research papers published this month.

A network comprising short and long noncoding RNAs and RNA helicase controls mouse retina architecture

Retinal cell layer uniformity is conferred by miRNA-lncRNA interaction

The retina consists of a series of nuclear layers: the photoreceptor layer resides on the outer part, followed by the inner nuclear and the ganglion cell layers. The thickness of each layer is remarkably uniform, suggesting that allocation of cells is tightly controlled.

To investigate the molecular mechanisms governing uniformity in retinal cell layers, Jacek Krol and colleagues from the Friedrich Miescher Institute for Biomedical Research, ETH Zurich and the University of Basel looked at the role of non-coding RNAs. By investigating the relationship between long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in photoreceptors during mouse postnatal development, they found that:

  • The lncRNA Rncr4 accumulates in the retina, alongside accumulation of precursor and mature forms of miR-183/96/182.
  • Rncr4 stimulates processing of pri-miR-183/96/182 in developing photoreceptors.
  • Processing of pri-miR-183/96/182 is inhibited by the DEAD-box protein Ddx3x, and this inhibition is antagonized by Rncr4 in late postnatal photoreceptors.
  • In Ddx3x knockdown retinas, premature accumulation of miR-183/96/182 results in increased variation in photoreceptor layer thickness.
  • miR-183/96/182 targets mRNA of the protein Crb1; the outer limiting membrane protein, which is a component of the adhesion belt between glial and photoreceptor cells.

The authors propose a model in which Crb1 is expressed at a high level in early photoreceptors, ensuring strong adhesion between glia and photoreceptors. After retinal layers reach their final thickness, suppressed expression of Crb1 by miR-183/96/182—which accumulates in response to Rncr4—weakens the adhesion belt.

Read the full paper in Nature Communications, June 2015.

Interested in non-coding RNA research? Take a look at our non-coding RNA and miRNA resources.

5-Formylcytosine can be a stable DNA modification in mammals

DNA is stably 5-fC modified in vivo

5-Formylcytosine (5-fC) is DNA modification produced from the TET enzyme-mediated oxidation of 5-hydroxymethylcytosine (5-hmC), which is in turn produced from the oxidation of 5-methylcytosine (5-mC). Although it is thought that a role of oxidized cytosine bases is to serve as intermediates of enzyme-mediated DNA methylation, it has also been shown that 5-hmC can be a stable DNA modification.

To explore the possibility that 5-fC, too, might be a stable rather than active DNA intermediate, a team led by Shankar Balasubramanian from the University of Cambridge studied the dynamics of 5-fC  in genomic DNA in vivo. They found that:

  • Global 5-hmC and 5-fC dynamics differ throughout mouse development, with no correlation between the level of 5-fC and that of its precursors, 5-mC and 5-hmC.
  • Labeling 5-fC by supplying 13C-containing precursors resulted in a slow accumulation of 13C in 5-fC, indicating that 5-fC is a stable DNA modification with a slow rate of turnover.
  • In contrast, labeling in 5-mC and 5-hmC is much higher, indicating a faster rate of turnover of these DNA modifications.

Overall, this research indicates that 5-fC can be a stable DNA modification. As 5-fC has been shown to have a large number of binding proteins and even alter the structure of DNA, the authors argue that stably 5-fC-modified DNA could have profound consequences for gene expression regulation that may be distinct to those caused by 5-mC and 5-hmC.

Read the full paper in Nature Chemical Biology, June 2015.

Learn more about 5-hmC and other DNA modifications.

Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Pseudouridine is a widespread and dynamic mRNA modification

Out of over 100 known RNA modifications, pseudouridine (Ψ) is the most abundant. This modification is already known to have numerous roles, including in translational fidelity and mRNA splicing; however, its prevalence and function in mRNA is poorly understood.

mRNA is present in low amounts, and this creates a technical challenge for detecting transcriptome-wide pseudouridylation events. By developing a new pseudouridylation profiling method (CeU-seq) that uses biotin pulldown to pre-enrich Ψ-containing RNA, Xiaoyu Li and colleagues from Peking University found that:

  • Ψ is more prevalent than previously thought, and is present in mammalian mRNA at a Ψ/U ratio of 0.2–0.6%.
  • mRNA Ψ content alters in response to stresses, including heat shock, poly(I:C) and H2O2 treatment.
  • Ψ profiles show common pseudouridylation sites as well as tissue-specific features in the mouse liver and brain.

The data presented in this paper have highlighted that Ψ is widespread and dynamically regulated on mRNA as well as other forms of RNA. By developing a new technique for the detection of pseudouridylation on mRNA, the authors have paved the way for future studies to further understand the biological relevance of this widespread RNA modification.  

Read the full paper in Nature Chemical Biology, June 2015.

Structural basis for retroviral integration into nucleosomes

Intasome lifts DNA from the histone surface to allow retroviral integration

Retroviruses have the ability to integrate into nucleosomal DNA, despite the limited availability of nucleosomes and constraints to DNA conformation. Retroviral integration is catalyzed by the intasome complex, which is associated with the ends of viral DNA. However, how the intasome interfaces with chromosomal DNA to allow retroviral integration into nucleosomes is currently unknown.

Using single-particle cryo-electron microscopy, a team led by Peter Cherepanov from the Francis Crick Institute and Imperial College London determined the structure of the prototype foamy virus (PFV) intasome-nucleosome interface. They found that:

  • An extensive interface is formed between the intasome and nucleosome, which involves three integrase subunits, both gyres of nucleosomal DNA and a H2A-H2B heterodimer.
  • The path of target DNA through the DNA binding site of the intasome deviates from the path taken through the nucleosome, having the effect of deforming DNA and lifting it from the surface of the H2A-H2B heterodimer.
  • Substitutions in certain intasome amino acids in the vicinity of contacts with nucleosomal DNA and the C-terminal helix of H2B—in particular K168E or P135E/T240E—disrupt intasome-nucleosome interactions.
  • PFV integration into DNA was less frequent at transcription sites, favoring condensed perinuclear chromatin.
  • K168E or P135E/T240E substitutions reduce PFV vector ability to stably transduce HT1080 cells and reduced the ability of the virus to discriminate against highly expressed regions.

This research suggests a molecular basis for the capture of nucleosomal DNA by the intasome, and demonstrates that by lifting DNA from the histone surface, retroviral integration can be achieved within relatively inaccessible nucleosomes.

Read the full paper in Nature, June 2015.

A unique gene regulatory network resets the human germline epigenome for development

Extensive epigenetic reprogramming of human primordial germ cells

Epigenetic reprogramming of mammalian primordial germ cells (PGCs) restores full germline potency and erases genomic imprints and epimutations, making it essential for development.

To investigate epigenetic reprogramming in the human germline, a team led by Azim Surani studied transcriptome transitions and epigenetic reprogramming in week 4–9 human PCGs (hPCGs) in vivo, and in an in vitro model for hPGC-like cells (hPGCLCs). They found that:

  • The transcriptome of hPGCs is distinct from those of mice. However, human and mouse share a core transcriptome consisting of key germ cell genes and pluripotency genes.
  • SOX17 and BLIMP1 activate a gene regulatory network that results in the repression of DNA methylation pathways in hPGCLCs.
  • During weeks 4–9, hPGCs undergo progressive demethylation as well as chromatin reorganization that results in X reactivation.
  • A notable fraction of potentially hazardous L1 long interspersed elements (LINEs) and SVA loci remain methylated, as do loci associated with certain metabolic and neurological disorders.

Overall, this paper has established that a distinct network of gene regulation results in extensive epigenetic reprogramming in hPGCs. The finding that certain loci can escape DNA demethylation suggests that these might be candidates for transgenerational epigenetic inheritance.

Read the full paper in Cell, June 2015.

Interested in epigenetics and development? Watch our webinar on epigenetic mechanisms in early mammalian development.

Spaciotemporal expression and transcriptional perturbations by long noncoding RNAs in the mouse brain

Long non-coding RNAs are differentially expressed in the developing and adult brain

The mammalian brain is enormously complex, and brain development relies on specification and differentiation of an array of different neuronal and glial cell types. Long non-coding RNAs (lncRNAs) have been implicated in this process; however, the in vivo expression dynamics and molecular pathways regulated by these loci are unclear.

To shed some light on the role of lncRNAs in brain development, a team led by John Rinn from Harvard University developed a map of lncRNA expression dynamics and regulatory effects in the developing and mature mouse brain. By investigating 13 null mutant mouse models, the team found that:

  • Out of the lncRNAs investigated, ten are expressed in the adult mouse brain, five of these are also expressed in the developing brain.
  • Expression of lncRNAs Lincenc1 and Eldr is established during development and maintained in the corresponding structures of the adult brain, whereas Peril is expressed in both the developing and adult brain and Tug1 in only in the developing choroid plexus.
  • lncRNAs that are expressed in the adult brain have precise and restricted expression patterns.
  • Deletion of lncRNA loci affects expression of gene sets associated with neuronal differentiation, cell fate commitment and cell cycle regulation. Altered expression of adjacent protein-coding genes is observed in 5 of 13 mutant stains.  

These results show that lncRNAs are differentially expressed in time and space and in certain cases can affect the expression of neighboring protein coding genes. These data pave the way for future research into the functional relevance of these genes in neural development and disease.

Read the full paper in PNAS, June 2015.

Interested in finding out more about John Rinn's research? Read our interview.

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape and phenotypic features of mouse ESCs

Link between histone modifications and DNA methylation in embryonic stem cells

DNA methylation and histone modification are two prominent forms of epigenetic regulation. There is evidence that these two distinct modifications are linked, but detailed mechanistic and functional links between the two have not previously been established.

To investigate the relationship between histone modifications and DNA methylation, a team led by David Allis from the Rockefeller University solved a 2.4 crystal structure of the ATRX-DNMT3-DNMT3L  domain of Dnmt3a (ADD3a) in complex with the histone H3 N-terminus. They found that:

  • The unmodified, but not modified, forms of H3K4, H3T3 and H3T6 interact with ADD3a. Modification of these residues disrupts binding of the H3 N-terminus to ADD3a.
  • ADD3a can be modified to enhance binding ability to H3K4me3-, H3T3ph- or H3T6ph-containing H3, rendering the ADD3a domain insensitive to histone modifications.
  • Histone modification-insensitive ADD3a alters Dnmt3a binding specificity and decreases expression of a subset of developmental genes.
  • A Dnmt3a mutants insensitive to H3K4 methylation and H3T3 phosphorylation result in major defects in embryonic stem cell differentiation and chromosome instability, respectively.

The structure-based approach taken by the authors in this paper has highlighted the important link between histone modifications and DNA methylation, and has given insights into the role of histone modifications in establishing DNA methylation patterns. 

View the full paper in Molecular Cell, June 2015.

Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma

A new role for H2A.Z.2 in malignant melanoma

Histone H2A.Z is a highly conserved variant of H2A, and has two separate isoforms; H2A.Z.1 and H2A.Z.2. Isoform-specific functions remain unclear and previous investigations into these isoforms have either focused on H2A.Z.1 or not differentiated between isoforms.

A team led by Emily Bernstein from Icahn School of Medicine at Mount Sinai investigated the role of H2A.Z.1 in malignant melanoma. They found that:

  • Both isoforms of H2A.Z are overexpressed in melanoma.
  • Depletion of H2A.Z.2, but not H2A.Z.1 reduces proliferation in cell lines and induces a G1/S cell cycle arrest.
  • H2A.Z.2 knockdown results in reduced expression of cell cycle regulators that are targets of the E2F family of transcription factors.
  • BRD2 is enriched in H2A.Z.1 and H2A.Z.2 containing nucleosomes and is overexpressed in melanoma.
  • BRD2 and E2F bind H2A.Z.2 to promote high levels of transcription at class I genes in melanoma.
  • H2A.Z.2 knockdown results in reductions of BRD2 and E2F1, loss of histone acetylation, and sensitizes melanoma cells to the effect of BET inhibitors.

The research presented in this study shows H2A.Z.1 and H2A.Z.2 to have different roles in melanoma. Melanoma is a notoriously hard to treat form of cancer, and these results suggest that BET inhibition in combination with H2A.Z.2 depletion may form the basis of a future therapy effective melanoma therapy.

Read the full paper in Molecular Cell, June 2015.