Hydroxymethylation

 5-hydroxymethylcytosine

5-hydroxymethylcytosine (5hmC) is the primary product of the oxidation of 5-methylcytosine (5mC) by the TET enzyme.

 

What is the function of DNA hydroxymethylation and why is it important?

Though the precise role of 5hmC is the subject of intense research and debate, early studies strongly indicate that it is functionally distinct from 5mC. 5mC is classically associated with processes that silence or down-regulate gene expression while increasing evidence suggests 5hmC serves alternative roles.  

 

Based on current knowledge and publications in the field, we’ve summarized below 5 major reasons why 5hmC is important:

 

  1. 5hmC is a stable modification enriched within gene bodies, concentrated at transcriptional regulatory elements and positively associated with transcriptional activity

DNA base modifications are dynamic marks and their relative concentration at given loci is influenced by multiple factors including the cell and tissue type from which the DNA is derived1, the differentiation state of a cell2, and the balance between cellular homeostasis and stress response3. Hydroxymethylation is most abundant in genomic regions of low to intermediate CpG content where it is associated with promoters, poised and active enhancers, and gene bodies.  Experimental evidence indicates that 5hmC concentration in these regions correlates with gene expression, suggesting a role in transcriptional regulation4,5,6,7. With 5hmC, less is more. Because 5hmC can be highly enriched at specific genomic regions and loci even when global levels are modest, measuring total hydroxymethylation is not sufficient to understand the influence this mark has on biology2,8. For example, TrueMethyl™ oxidative bisulfite (oxBS) analysis of a 1000nt genomic locus in embryonic stem cells demonstrated that the BS-only data set was confounded by 17% 5hmC overall, with several CpG's exhibiting >30% hydroxymethylation  - significantly higher than global 5hmC levels. 

 

  1. 5hmC recruits transcriptional regulators, splicing factors, DNA repair proteins and chromatin regulators that are distinct from those recruited by 5mC

Researchers have identified several DNA modification readers that show preferential binding to 5mC or 5hmC and its oxidized derivatives with little overlap. Proteins that bind 5hmC specifically include DNA repair factors, splicing mediators and transcription regulators. Understanding how these selective complexes regulate chromatin structure and gene expression will open new avenues for epigenetic research9,10.

 

  1. 5hmC plays an essential role in embryonic development, cellular differentiation and stem cell reprogramming

5hmC is the primary product of TET-mediated 5mC oxidation, a process which plays an essential role in normal embryonic development, the maintenance of pluripotency and stem cell reprogramming. Knockout studies have demonstrated that TET family members are essential for embryonic viability and influence cell lineage decisions during later stages of development. These early results indicate that 5hmC plays an important and active role in stem cell maintenance and differentiation11,12,13,14,15,16,17.

 

  1. 5hmC is a strong prognostic indicator in cancer and other diseases

Loss of global hydroxymethylation is associated with malignant human cancers such as melanoma and may serve as a strong diagnostic and prognostic indicator. Moreover, mutations in TET2 are commonly observed in a wide range of hematopoietic malignancies and are associated with poor patient outcomes18,19,20. 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.

 

Leading pharmaceutical companies have recognized the importance of 5hmC as a novel clinical biomarker in disease. For example, researchers at Novartis have identified an important role for hydroxymethylation in Fragile X syndrome (FXS), an inherited genetic condition that causes a wide range of developmental problems. Novartis discovered that quantifying 5mC and 5hmC at novel regions of the FMR1 genomic locus could be used to both diagnose the disease and determine patient responsiveness to mGlur5 antagonist therapy. This groundbreaking discovery would not have been possible using traditional bisulfite sequencing-based techniques, further highlighting the importance of accurately measuring both 5mC and 5hmC to truly understand epigenetic mechanisms in disease. See the patent application for more information.

 

  1. 5hmC is implicated in the regulation of neurological plasticity, immunology and other dynamic biological systems by the TET enzyme family

Global levels of 5hmC are highest in the brain where it may play an important role in neurological development and memory formation21,22,23,24. The TET enzymes and 5hmC have also been implicated in cardiovascular dynamics25 and liver function26, suggesting that this modification has far-reaching impact. Several fundamental questions remain to be answered in the nascent DNA modification field. It’s clear that researchers must determine the impact of both 5hmC and 5mC in their model systems to truly understand the biological functions of DNA modification. 


 

References

1. Journal of Nucleic Acids 2011 Article ID 870726. 

Distribution of 5-Hydroxymethylcytosine in Different Human Tissues

2. Cell Stem Cell. 2013 Apr 4;12(4):470-8. 

 Stage-specific roles for tet1 and tet2 in DNA demethylation in primordial germ cells

3. PLoS One. 2013 Oct 25;8(10):e77859. 

Long-lasting changes in DNA methylation following short-term hypoxic exposure in primary hippocampal neuronal cultures

4. Nat Commun. 2013;4:1995.

 Dynamics of 5-hydroxymethylcytosine during mouse spermatogenesis

5. Epigenetics. 2013 Mar;8(3):317-32. 

Early de novo DNA methylation and prolonged demethylation in the muscle lineage

6. Genome Res. 2012 Mar;22(3):467-77. 

Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes.

7. Neuron. 2013 Sep 18;79(6):1109-22. 

Tet1 is critical for neuronal activity-regulated gene expression and memory extinction.

8. Genome Res. 2012 Mar;22(3):467-77. 

Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes.

9. Genome Biol. 2013 Oct 24;14(10):R119. 

A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation

10. Cell. 2013 Feb 28;152(5):1146-59. 

Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives

11. Nature. 2013 Aug 8;500(7461):222-6. 

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells

12. Genome Biol. 2013 May 26;14(5):R46. 

5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

13. Nat Commun. 2012 May 8;3:818. 

Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster.

14. Cell Stem Cell. 2013 Sep 5;13(3):351-9. 

FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.

15. Cell Stem Cell. 2013 Apr 4;12(4):453-69. 

Replacement of Oct4 by Tet1 during iPSC induction reveals an important role of DNA methylation and hydroxymethylation in reprogramming.

16. Mol Cell. 2013 Mar 28;49(6):1023-33. 

Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion

17. Science. 2013 Jan 25;339(6118):448-52.

 Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine

18. Cell. 2012 Sep 14;150(6):1135-46. 

Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma.

19. Cell. 2013 Jul 18;154(2):311-24. 

MicroRNA-antagonism regulates breast cancer stemness and metastasis via TET-family-dependent chromatin remodeling

20. Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11994-9. 

TET1 plays an essential oncogenic role in MLL-rearranged leukemia.

21. Neuron. 2013 Sep 18;79(6):1109-22. 

Tet1 is critical for neuronal activity-regulated gene expression and memory extinction.

22. Proc Natl Acad Sci U S A. 2013 Sep 3;110(36):14682-7. 

Alteration of genic 5-hydroxymethylcytosine patterning in olfactory neurons correlates with changes in gene expression and cell identity

23. Science. 2013 Aug 9;341(6146):1237905. 

Global epigenomic reconfiguration during mammalian brain development

24. Neuron. 2013 Sep 18;79(6):1086-93. 

TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation

25. Circulation. 2013 Oct 29;128(18):2047-57. 

Ten-Eleven Translocation-2 (TET2) Is a Master Regulator of Smooth Muscle Cell Plasticity

26. Nucleic Acids Res. 2013 Jun;41(11):5639-54. 

Dynamic changes in 5-hydroxymethylation signatures underpin early and late events in drug exposed liver


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