Hydroxymethylation

Epigenetics is more than silencing

What is hydroxymethylation?

Modifications on the DNA regulate every part of normal cellular programming: which genes will be transcribed and form proteins, and which regions will be silenced. The most well-studied modification is DNA Methylation (5mC), which typically down-regulates or silences transcription at specific DNA loci.

Hydroxymethylation (5’hydroxy methylcytosine or 5hmC) is functionally different from conventional Methylation (5mC). The function of 5hmC is still subject of intense research, but research has shown that it is functionally distinct from 5mC. 5hmC recruits transcriptional regulators, splicing factors, DNA repair proteins and chromatin regulators that are distinct from those recruited by 5mC. It may be part of the process of active demethylation and could therefore lead to transcriptional activation.

5hmC is involved in every part of normal cellular programming. These residues are especially frequent during embryonic development, stem cell reprogramming and cellular differentiation. It has also been shown to serve as a strong biomarker in the development and progression of cancer and other diseases.

Loss of global hydroxymethylation is associated with malignant human cancers such as melanoma. Next to an important diagnostic indicator, it may serve as a prognostic marker. In many hematopoietic malignancies, mutations in TET2 are commonly observed an are associated with poor patient outcomes. Dramatic changes in total 5hmC levels have also been implicated in the progression of major neurological disorders including Alzheimer’s disease, Friedrich’s ataxia and Huntington’s disease.

Hydroxymethylation at GenomeScan

  • Hydroxymethylation profiling on all types of DNA, from freshly isolated to FFPE
  • Multiple QC steps to ascertain that high-quality data is achieved
  • All experiments are performed under ISO 17025 (L518)
  • Guaranteed quality: 95% of the CpGs on the BeadArray detected with (p≤0.01), adding up to over 805,000 detected loci
  • Fast turnaround time
  • Free scientific advice before the start of the project to achieve the best experimental setup
  • One project manager that guides you through the whole project
  • Competitive pricing: processing of large sample numbers allows us to acquire BeadChips and reagents for the best price.
    Subtraction of oxBS from BS reveals methylation at single-base resolution
    Adapted from CEGX

How do we measure hydroxymethylated DNA?

Standard methods based on bisulfite-conversion lead to equal conversion of both 5mC and 5hmC residues. Either of the two modifications are therefore reported back as 5mC, which makes the data difficult to interpret since solely 5mC has a gene-silencing function.

To measure hydroxymethylation, the samples are split into two aliquots. One of the aliquots undergoes the standard bisufite treatment and is eventually hybridized to the MethylationEPIC BeadChip. The other aliquot undergoes an oxidative treatment, before the same workflow is applied. The sample is then analysed using a second BeadArray. By comparison of the two data-sets, 5mC and 5hmC can be accurately quantified in parallel at single-base resolution.

With this superior detection method, it is evident that the 850,000 probes on the array do not strictly represent methylated residues. For certain compartments such as brain tissue, a large fraction of the loci is in reality hydroxymethylated (blue striped box) and therefore can recruit for example transcriptional activators.

Extent of hydroxymethylation in various samples (without normalization)
The Average Beta Value (Avβ) measures the percentage of methylation per methylation locus on the array and ranges from 0 to 1. For every methylation locus, multiple probes are present on Illumina’s BeadArrays. Avβ is the ratio of the methylated probe intensities and the overall intensities.
X-axis: Avβ of oxidated and then BS-treated duplicate sample. Y-axis: Avβ of solely BS treated aliquot of identical sample. Blue square: loci that give a different fluorescent intensity on each of the micro-arrays and thus represent the hydroxymethylated residues.

Achieve the compulsory quality for hydroxymethylation measurements

We are proud to be a preferred partner of Cambridge Epigenetics (CEGX) – the developer and manufacturer of the 5hmC kit -. Our highly-perfected performance of the workflow with very low signal-to-noise ratios leads to the top-quality data required for the subtraction of fluorescent signals. This is a result of our 15 years of experience in array-analyses, our expert technicians and of the strict workflow under ISO/IEC 17025 (L518). This leads to highly reproducible results, with correlations between experiments generally ≥0.99.

Reduced Representation & Whole Genome Bisulfite Sequencing

Hydroxymethylation

Targeted methylation

MethylationEPIC ‘850K’ BeadChip

EpiRestore: restore all low integrity DNA