Epigenetics articles of the month: February 2016

Keep up to date with the latest epigenetics research.

Genetic variants near MLST8 and DHX57 affect the epigenetic age of the cerebellum

Identification of genes implicated in epigenetic age

DNA methylation changes over time, reflecting the age of an individual. CpG DNA methylation levels can give a measure of epigenetic age of a tissue, a factor that has been shown to correlate with mortality; for example, the epigenetic age of blood has been found to be associated with all-cause mortality.

In this paper, a team led by Steve Horvath from the University of California, Los Angeles investigated whether cerebellar epigenetic age acceleration – DNA methylation age adjusted for chronological age – could be used as an endophenotype for biological age. Using a genome-wide association study, they found the following:

  • The epigenetic age of the cerebellum correlates with chronological age.
  • Cerebellar age acceleration is significantly associated with five SNPs inside DHX57 and near MLST8 and PGP.
  • Cerebellar DHX57 expression levels are positively correlated with chronological age, but not epigenetic age.
  • MLST8 expression is significantly correlated with epigenetic age acceleration and chronological age.
  • Genes related to cerebellar age acceleration overlap with genes related to age-related macular degeneration, Alzheimer’s disease and Parkinson’s disease.

This is the first paper that presents SNPs associated with epigenetic age acceleration and shows correlation with expression of an implicated gene. By doing this, the authors have shown the utility of using epigenetic tissue age as an endophenotype, rather than linking SNPs directly to clinical outcomes.

Read the full paper at Nature Communications, February 2016.

5-Hydroxymethylcytosine marks sites of DNA damage and promotes genome stability

5-hmC and TET are required for DNA repair

During DNA demethylation, ten-eleven translocation (TET) enzymes convert 5-methylcytosine (5-mC) to 5-hydroxylmethylcytosine (5-hmC). Epigenetic 5-hmC DNA modification impacts gene expression and 5-hmC-containing regions have a more open chromatin confirmation.

Upon DNA damage, cells attempt to repair DNA by the DNA damage response (DDR) pathway. This mechanism is dependent of the relaxation of chromatin to allow access for protein complexes. In this paper, a team led by Peter Mark Carlton form Kyoto University in Japan aimed to find out how 5-hmC contributes to the DDR.

  • 5-hmC co-localizes with DNA damage markers, 53BP1 and γH2AX.
  • Chemical or microirradiation-induced DNA damage in HeLa cells results in 5-hmC accumulation at sites of DNA damage.
  • 5-hmC accumulation is not present in human embryonic stem cells (ESCs) upon DNA damage – these cells have constitutively high γH2AX and 5-hmC levels.
  • TET knockout mouse ESCs are unable to effectively repair induced DNA damage.

The results presented in this paper show that 5-hmC and TET enzymes are essential for DNA repair and genome integrity.

Read the full paper in Cell Reports, February 2016.

Shelterin protects chromosome ends by compacting telomeric chromatin

Telomere chromatin compaction reduces accessibility to DNA damage response components

Chromosome ends are often misrecognized as DNA breaks. The shelterin protein complex protects telomeres from the DNA repair mechanism. Dysregulation of this process is implicated in cancer and aging; however, it is not clear how shelterin exerts its protective function.

To find out how shelterin protects telomere ends, a team led by Ahmet Yildiz from the University of California, Berkeley investigated the structure of telomeric chromatin using super-resolution microscopy on human cells.

  • Telomeres form compact chromatin structures, that are not dependent on DNA methylation or histone modification.
  • Depleting shelterin subunits results in telomere decompaction, with deletion of TRF1, TRF2 or TIM2 having the greatest impact.
  • As telomere volume increases, cells have a larger number of telomere-dysfunction-induced loci, suggesting that telomere compaction acts as a mechanism for telomere protection.
  • Recompaction of telomeric chromatin displaces DNA damage response (DDR) signals from telomeres.

The results presented in this paper suggest that shelterin-mediated telomere compaction reduce telomere-dysfunction-induced loci and protects chromosome ends from the DDR machinery.

Read the full paper in Cell, February 2016.

The histone variant H2A.X is a regulator of the epithelial-mesenchymal transition

H2A.X regulates the epithelial-mesenchymal transition

For cancer cells to spread throughout the body by metastasis, they must undergo epithelial-mesenchymal transition (EMT). EMT-related transcription factors are regulated by changes to chromatin configuration, and there is evidence that loss of the histone variant H2A.X results in increased cell migration and invasion.

To further understand mechanisms regulating the EMT, a team led by William Bonner from the National Cancer Institute, Maryland investigated whether H2A.X downregulation induces changes in cancer gene expression that result in EMT. Using human colon carcinoma cell lines, they found the following:

  • Loss of H2A.X results in activation of colorectal cancer signaling pathways, but these cells do not display enhanced metastatic potential in vivo.
  • Reintroduction of H2A.X partially reverses EMT and enhances proliferation.
  • Transcription factors SLUG and ZEB1 mediate the EMT upon H2A.X loss, and H2A.X expression is significantly correlated to SLUG and ZEB1 expression across colorectal cancer samples.

The results presented in this paper demonstrate that restoration of H2A.X expression results in partial EMT reversal, that is necessary for metastasis. These results suggest that H2A.X is a regulator of EMT.

Read the full paper in Nature Communications, February 2016.

Epigenetic modulation of a miR-296-5p:HMGA1 axis regulates Sox2 expression and glioblatoma stem cells

Regulation of stem cell phenotype by DNA methylation, miRNAs and HMGA1

Multipotent cancer stem cells are important contributors to tumor growth, therapeutic resistance and recurrence. The extreme plasticity of these cells means that epigenetic modification of gene networks allows them to move between a stem-like state that propagates tumor growth and more differentiated non-tumor-propagating states.

A team led by John Laterra from the John Hopkins School of Medicine in Maryland sought to understand how cross-talk between DNA methylation, miRNA expression and transcription factors regulate stem cells in glioblastoma. They found the following:

  • Transcription factors OCT4 and SOX2 repress miR-296-5p expression in the presence of DNA methylation activity.
  • Inhibition of DNA methylation induces miR-296-5p expression and reduces stem cell phenotype in glioblastoma.
  • miR-296-5p targets HMGA1; expression of this gene significantly enhances glioblastoma self-renewal.
  • HMGA1 stimulates SOX2 expression by binding DNA and displacing histone H1.

This work uncovers how an interaction between DNA methylation, miRNAs and HMGA1 regulation of SOX2 contributes to stem cell phenotype.  The authors suggest that miR-269-5p might be a potential therapeutic tool to inhibit stem cell populations in glioblastoma.

Read the full paper in Oncogene, February 2016.

Structural basis for activity regulation of MLL family methyltransferases

Two-step process for MLL activation

Mixed lineage leukemia (MLL) proteins methylate histone H3 at lysine 4 (H3K4). MLL1 protein on its own has poor methyltransferase activity; its activity is enhanced by additional factors including WDR5, RBBP5 and ASH2L. However, how these factors act to regulate MLL1 activity is still unsolved.

To understand this further, a team led by Ming Lei from the Chinese Academy of Sciences used structural, biochemical and computational analyses to look at the structure of MLL proteins in complex with WDR5, RBBP5 and ASHL2. They found the following:

  • The RBBP5-ASH2L heterodimer binds and activates all MLL family members.
  • The SET domain is highly mobile in the apo structure, association with RBBP5-ASH2L suppresses this mobility to lock it in an active state and improve cofactor binding and substrate recognition.
  • Biding of the substrate H3 peptide induces a local structural rearrangement that orients the H3K4 side chain for catalysis.

The authors have shown that a two-step process occurs for MLL activation composed of stabilization of the MLL SET domain by RBBP5-ASH2L binding, followed by further activation by binding to the H3 substrate itself.   

Read the full paper in Nature, February 2016.

epiGBS: reference-free reduced representation bisulfite sequencing

A new methods for studying DNA methylation in non-model species

DNA methylation is analyzed by bisulfite sequencing, in which unmethylated cytosines are converted to uracil. Currently available reduced representation bisulfite sequencing (RRBS) methods are limiting in terms of enzyme selection and multiplex ability, and are dependent on availability of a reference genome.

In this paper, a team led by Koen Verhoeven from the Netherlands Institute of Ecology present epiGBS – a method that enables reference-free RRBS of highly multiplexed libraries by extending genotying by sequencing (GBS) with bisulfite treatment. Here is what the technique involves:

  • DNA digestion with restriction enzymes and ligation of Illumina-compatible barcoded adaptors.
  • Pooling and subjection of fragments to solid phase reversible immobilization (SPRI)-based size selection and nick translation.
  • Bisulfite treatment, amplification by PCR and paired-end sequencing.
  • Use of custom software to cluster Watson and Crick reads derived from the same genomic location.

This method will be useful for examining DNA methylation in non-model organisms for which a reference genome is not available.

Read the full paper in Nature Methods, February 2016.