All tags Epigenetics Epigenetics articles of the month: October 2015

Epigenetics articles of the month: October 2015

Need to keep up with the latest epigenetics research? Take a look at our selection of top research articles from the past month.

The menu of features that define primary microRNAs and enable de novo design of microRNA genes

A unified model of what it takes to be a miRNA gene

The first step of miRNA synthesis is to generate primary miRNAs (pri-miRNAs) through transcription of miRNA genes. pri-miRNA transcripts contain stem loop regions that are then cleaved by the Microprocessor complex to form pre-miRNAs. However, the miRNA features that dictate the choice of step-loops for initial processing is currently unclear.

To understand the sequence and structural features that define pri-miRNAs, Wenwen Fang and David Bartel from Massachusetts Institute of Technology assessed the cleavage efficiencies of over 50,000 variants of three human pri-miRNAs. They found that:

  • There is a preference for base pairing across the stem, with the exception of position 8.
  • A mismatched GHG motif at positions 7–9 at the base of the hairpin enhances pri-miRNA processing
  • The preferred Microprocessor substrate is a 35-bp hairpin flanked by single-stranded sequences.
  • Artificial miRNA genes designed with the identified features were successfully processed.

The results presented in this paper provide a simplified and unified model of what it takes to be a miRNA gene, and have demonstrated that these principles can be used to generate artificial pri-miRNAs that can be processed into functional miRNAs.

Read the full paper in Molecular Cell, October 2015. 

Find out about research into miRNA biomarkers.



Dynamic m6A mRNA methylation directs translational control of heat shock response

m6A at the 5’UTR mediates translation during heat shock

N6-methyladenosine (m6A) is the most abundant mRNA modification and is implicated in diverse biological functions. Although the 5’ untranslated region (5’UTR) of miRNAs is relatively less methylated, little is understood about how 5’UTR methylation is regulated. 

To understand both the regulation and function of methylation at the 5’UTR, a team led by Shu-Bing Qian from Cornell University, New York sought to understand the impact of heat shock stress on m6A modification in mouse embryonic fibroblasts (MEFs) and HeLa cells. They found that:

  • Heat shock leads to elevated m6A in the 5’UTR, particularly in upregulated transcripts. 
  • The m6A reader YTHDF2 protects methylation at the 5’UTR by competing with the m6A eraser FTO.  Silencing this reader results in loss of 5’UTR m6A.  
  • YTHDF2 knockdown reduces the increase in Hsp70 synthesis during heat shock and FTO knockdown increases 5’UTR methylation and translation efficiency of Hsp70.
  • m6A modification of Hsp70 5’UTR promotes translation under heat shock stress independent of the N7-methylguanosine (m7G) cap.

These results suggest a role for m6A in mediating mRNA translation independent of the m7G cap. Contrary previous research, this paper indicates that m6A is a dynamic rather than static mark.

Read the full paper in Nature, October 2015.

m6A at the 5’UTR mediates translation during heat shock.
m6A is the most abundant mRNA modification and is implicated in diverse biological functions. Although there is relatively less methylation at the 5’ untranslated region (5’UTR) of miRNAs, little is understood about how 5’UTR methylation is regulated. 
To understand both the regulation and function of methylation at the 5’UTR, a team led by Shu-Bing Qian from Cornell University, New York sought to understand the impact of heat shock stress on m6A modification in mouse embryonic fibroblasts (MEFs) and HeLa cells. They found that:
•    Heat shock leads to elevated m6A in the 5’UTR, particularly in upregulated transcripts. 
•    The m6A reader YTHDF2 protects m6A at the 5’UTR by competing with the m6A eraser FTO.  Silencing this reader results in loss of 5’UTR m6A.  
•    YTHDF2 knockdown reduces the increase in Hsp70 synthesis during heat shock and FTO knockdown increases 5’UTR methylation and translation efficiency of Hsp70.
•    m6A modification of Hsp70 5’UTR promotes translation under heat shock stress independent of the N7-methylguanosine (m7G) cap.
These results suggest a role for m6A in mediating mRNA translation independent of the m7G cap. Contrary to what is commonly believed, this paper also indicates that m6A is a dynamic rather than static mark.
Read the full paper in Nature, October 2015.


Panoramix enforces piRNA-dependent cotranscriptional silencing

Panoramix is a Piwi component that is essential for piRNA-dependent silencing

Nuclear Piwi proteins transcriptionally silence transposons in germ cells. This is thought to be dependent on Piwi-interacting RNA (piRNA)-guided recognition of nascent transposon transcripts. However, the components of Piwi effector complexes at target loci is currently unknown.

A team led by Gregory Hannon from the University of Cambridge sought to further understand the precise mechanisms by which nuclear Piwi transcriptionally silences transposons in Drosophila ovaries. They found that:

  • The CG9754 protein is critical for transposon silencing in both germ and follicle cells.
  • Knockdown of CG9754 or Piwi resulted in an increase in transposon transcripts, suggesting that CG9754 is specifically required for transcriptional silencing of Piwi-targeted transposons.
  • Transcriptional gene silencing by Piwi correlates with H3K9me3 at silenced transposons. CG9754 mutation results in a corresponding loss of H3K9me3.
  • Tethering CG9754 to nascent mRNA transcripts causes deposition of H3K9me3 and silencing of the locus.

These results show that CG9754 is a component of Piwi complexes that is essential for piRNA-dependent silencing. The authors name this protein Panoramix after the mentor who empowers the comic book character Asterix and suggest that this protein may act as an adaptor between the piRNA pathway and the general silencing machinery.

Read the full paper in Science, October 2015



Single-cell ChIP-seq reveals cell populations defined by chromatin state

New technique for single-cell ChIP analysis​​

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used method for mapping genome-wide protein-DNA interactions. However, a limitation of this technique is that it provides averaged profiles that are insensitive to variability between individual cells. Consequently, the extent of chromatin-state heterogeneity is currently unexplored.

By combining microfluidics, DNA barcoding and next-generation sequencing, a team led by Bradley Bernstein from Massachusetts General Hospital and the Broad Institute of MIT and Harvard developed a new technique to collect chromatin data at single-cell resolution. The group used this technique to profile lysine methylation in a mixed population of cells. They found that:

  • Using this technique, single-cell ES profiles could be distinguished from single-cell MEF profiles.
  • Single-cell data identified three ES cell subpopulations with notable differences in H3K4me2 distribution over pluripotency-related signatures.

This new method with permit the analysis of chromatin states between individual cells and overcomes a major limitation of existing ChIP technologies. Techniques such as this will improve our understanding of the chromatin states of cell populations.

Read the full paper in Nature Biotechnology, October 2015.

Find out more about advanced ChIP techniques.  



Sleep disruption impairs haematopoietic stem cell transplantation in mice

Lack of sleep in donors may affect success of hematopoietic stem cell transplantation  

Hematopoietic cell transplantation is commonly carried out for various malignant and non-malignant diseases. There is growing evidence of circadian regulation of hematopoietic stem cells (HSCs); however the effect of sleep deprivation on transplantation has not previously been investigated.

A team led by Luis de Lecea from Stanford University, California investigated the effects of sleep deprivation on HSCs. They found that:

  • Four hours of sleep deprivation in mice halved transplantation efficiency into an irradiated recipient.
  • Homing potential to the bone marrow, a crucial component of successful hematopoietic transplantation, was reduced in HSCs from sleep deprived mice due to a change in responsiveness to the migration-guiding cue SDF-1α.
  • Sleep deprivation downregulates expression of miR-19b and subsequent upregulation of its target; the suppressor of cytokine signaling 3 (SOCS3) gene that inhibits HSC migration and homing.
  • Growth hormone is secreted during sleep. HSC treatment with growth hormone results in elevated miR-19b and has a dose-dependent effect on the migration capacity of HCSs.

The authors have demonstrated that sleep deprivation reduces the success rate of haematopoietic stem cell transplantation in mice and suggests molecular mechanisms underpinning this. The study highlights the potential importance of adequate donor sleep for successful hematopoietic cell transplantation.

Read the full text in Nature Communications, October 2015. 



Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2

New PRC2 crystal structure determined

Polycomb repressive complex 2 (PRC2) is an important epigenetic regulator that catalyzes histone trimethylation at H3K27. Activity of this complex is stimulated in a positive feedback loop by H3K27me3. Misregulation of PRC2 is implicated in human disease, including some cancers. For example PRC2 activity is inhibited by a histone missense mutation—H3K27M— that is found in certain pediatric brain cancers.

Despite the importance of PRC2 in epigenetic regulation and human disease, very little is known about the structure of this complex. In this paper, Lianying Jiao and Xin Liu from the University of Texas Southwestern Medical Center determined the crystal structure of PRC2 in complex with a stimulating H3K27me3 peptide and a H3K27M cancer mutant. Their structure provided the following mechanistic insights:

  • PRC2 components Ezh2, Eed and the VEFS domain of Suz12 closely assemble. Eed is engulfed by a belt-like structure on Ezh2, and Suz12 contacts both subunits to confer enzyme activity. 
  • Two separate regions of Ezh2 are required together to form the active catalytic subunit.
  • Binding of the inhibitory cancer mutant H3K27M directly competes with the H3R26 residue and blocks the lysine access channel of the active site, preventing substrate binding.
  • Upon binding of the stimulatory H3K27me3 peptide, the stimulation-responsive motif (SRM) of Ezh2 undergoes a disordered to ordered conformational transition, initiating an allosteric change that communicates with the active site.

This research has uncovered the structural basis for PRC2 activity and regulation. The authors show that regulatory signals are integrated at the active site to enable complex regulation by a large numbers of factors.

Read the full paper and a research summary in Science, October 2015

Watch our webinar to find out about the role of Polycomb group proteins in cancer.  



An LSC epigenetic signature is largely mutation independent and implicated the HOXA cluster in AML pathogenesis

Differential DNA methylation defines leukemia stem cell subgroups

Acute myeloid leukaemia (AML) is a malignancy of the bone marrow and is initiated by leukemia stem cells (LSCs). Although LSCs are able to engraft in immunodeficient mice, the blast cells LSCs give rise to have lost these stem cell properties. LSCs and blasts are clonally related, suggesting that the differences in properties between these cell types may be conferred by epigenetic variations.

To investigate this possibility, a team led by Andrew Feinberg from John Hopkins University School of Medicine, Maryland looked at the DNA methylation signatures of LSCs and blasts. They found that:

  • AML LSCs and blasts exhibit epigenetic differences with significant hypomethylation in LSCs. In particular, REC8 and HOXA genes were hypomethylated and highly expressed in LSCs.
  • DNA hypomethylation-dependent overexpression of HOXA genes is a core mechanism for LSC activity.
  • The LSC epigenetic and gene expression signatures are associated with overall survival in human AML.
  • Individual hematopoietic stem/progenitor cell populations are tightly clustered in terms of their genome-scale methylation profile. These epigenetic distinct subgroups may reflect the cell of origin.

In this paper, the researchers have identified an epigenetic signature that may represent a therapeutic target to improve patient survival in AML.

Read the full paper in Nature Communications, October 2015.




Identification of and molecular basis for SIRT6 loss-of-function point mutations in cancer

Point mutations in SIRT6 can lead to cancer development

SIRT6 is a sirtuin that has histone deacetylation activity and also acts as a tumor suppressor. Although this protein is often downregulated or deleted in human cancers, no point mutations have previously been identified. This has hindered progress in our understanding of the tumor-suppressive role of SIRT6.

In this paper, a team led by Raul Mostoslavsky from Harvard Medical School, Massachusetts characterize eight tumor-associated point mutations in SIRT6. They found that:

  • Point mutations in SIRT6 occur in a variety of human cancers. The mutations affect highly conserved residues at different locations within the protein and affect protein stability or localization.
  • All of the point mutations are likely to affect catalytic activity either directly or through structural rearrangements.
  • In vitro deacetylase activity of the SIRT6 mutants is reduced compared with wild-type. Histones isolated from SIRT6 mutant cells had higher levels of histone acetylation than those from wild-type cells.
  • The SIRT6 mutants are unable to suppress transformation to tumor cells in vivo.

The research presented in this paper provides strong evidence for a tumor-suppressing role of SIRT6 and identifies specific cancer-associated mutations.

Read the full text in Cell Reports, October 2015.


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