DNA methylation (5mC) is an important epigenetic modification that plays critical roles in cellular differentiation, development, and disease. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC), which are generated by the TET family of dioxygenases through oxidation of 5-methylcytosine (5mC) (Ito et al., 2010; Iyer et al., 2009; Ko et al., 2010; Kriaucionis and Heintz, 2009; Loenarz and Schofield, 2009; Tahiliani et al., 2009), have been detected in diverse cell types and tissues in mammals (Ito et al., 2010; Ko et al., 2010; Kriaucionis and Heintz, 2009; Szwagierczak et al., 2010; Tahiliani et al., 2009).

 

Studies have suggested that 5hmC may contribute to DNA demethylation and gene regulation. One possibility is that hydroxylation of mC by TET1 might interfere with DNMT1 activity, leading to a subsequent passive loss of methylation following DNA replication. Alternatively, hmC may be converted to 5-carboxycytosine (5CaC) by Tet dioxygenase. Conversion of 5mC to 5hmC and 5CaC by Tet proteins followed by TDG mediated base excision of 5CaC constitutes a pathway for active DNA demethylation (He et al., 2011). In addition, hydroxylation of mC may promote transcriptional de-repression by dissociation of mC-binding proteins and/or recruitment of effector proteins. The high abundance of hmC in ES cells and in neuronal Purkinje cells and its contribution to DNA demethylation and gene regulation suggests that this modification is important in stem cell biology and cancer (Delhommeau et al., 2009; Ito et al., 2010; Ko et al., 2010; Koh et al., 2011; Tahiliani et al., 2009).

 

To further understand the role of 5hmC, it is necessary to understand where 5hmC localizes in the genome. By combining hMeDIP (hydroxymethylated DNA immunoprecipitation) with the methylation arrays, Arraystar provide services for methylation arrays designed by Arraystar, Roche-Nimblegen and Agilent. This service can identify the genomic location of 5hmC within lncRNA & mRNA promoter regions and other biologically significant genomic regions quickly and cost effectively.

 

LncRNA Promoter Microarrays are designed to investigate the epigenetic modifications and transcription factor binding sites within LncRNA promoter regions, and uncover other biologically significant genomic regions. These regions include, but are not limited to, mRNA promoters, miRNA promoters, H3K4-K27 bivalent domains, CpG islands and CpG island shores.

 

microRNA Genome DNA Methylation microarrays are designed to profile miRNA associated DNA methylation patterns.

 

CpG Island and Promoter microarrays are designed to analyze specific regulatory elements of the genome, such as gene promoters, microRNA promoters and CpG islands. The ultra-high density 2.1 million arrays can even detect the genome-wide methylation status of regulatory elements (such as enhancers) located far from promoters and CpG islands.

 

 

 

Arraystar's scientists are specialized in performing hMeDIP-chip service from genomic DNA extraction to data analysis (figure 1). Just send us your samples, and we'll do the rest. (Please refer to the Sample Submission Guidelines to help you start your project).

	Option A (DNA submitted)	Option B (sample submitted)
DNA Isolation		v
gDNA QC	v	v
Genomic DNA digestion with MseI	v	v
Digested genomic DNA QC	v	v
hMeDIP	v	v
Quality assessment of hMeDIP	v	v
Amplification	v	v
Labeling	v	v
Array hybridization	v	v
Standard data analysis	v	v

* For quotations and inquiries, please specify the type of service that you need: Option A or Option B.

Figure 1. Flowchart of hMeDIP-chip, Genomic DNA is digested by Mse I and denatured. Hydroxymethylated DNA is enriched using an antibody against 5-hmC. Purified hydroxymethylated DNA (IP) and Input DNA (Input) are amplified by the Whole Genome Amplification (WGA) method and labeled with Cy5 and Cy3, respectively, to be subsequently co-hybridized on the Arraystar promoter array. Array images are then extracted and analyzed by Arraystar¡¯s bioinformatics team.

 

For detailed description of the data analysis, please refer to hMeDIP Data Analysis Overview

Delhommeau, F., Dupont, S., Della Valle, V., James, C., Trannoy, S., Masse, A., Kosmider, O., Le Couedic, J.P., Robert, F., Alberdi, A., et al. (2009). Mutation in TET2 in myeloid cancers. N Engl J Med 360, 2289-2301.

He, Y.F., Li, B.Z., Li, Z., Liu, P., Wang, Y., Tang, Q., Ding, J., Jia, Y., Chen, Z., Li, L., et al. (2011). Tet-Mediated Formation of 5-Carboxylcytosine and Its Excision by TDG in Mammalian DNA. Science.

Ito, S., D'Alessio, A.C., Taranova, O.V., Hong, K., Sowers, L.C., and Zhang, Y. (2010). Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129-1133.

Iyer, L.M., Tahiliani, M., Rao, A., and Aravind, L. (2009). Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids. Cell Cycle 8, 1698-1710.

Ko, M., Huang, Y., Jankowska, A.M., Pape, U.J., Tahiliani, M., Bandukwala, H.S., An, J., Lamperti, E.D., Koh, K.P., Ganetzky, R., et al. (2010). Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 468, 839-843.

Koh, K.P., Yabuuchi, A., Rao, S., Huang, Y., Cunniff, K., Nardone, J., Laiho, A., Tahiliani, M., Sommer, C.A., Mostoslavsky, G., et al. (2011). Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200-213.

Kriaucionis, S., and Heintz, N. (2009). The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929-930.

Loenarz, C., and Schofield, C.J. (2009). Oxygenase catalyzed 5-methylcytosine hydroxylation. Chem Biol 16, 580-583.

Szwagierczak, A., Bultmann, S., Schmidt, C.S., Spada, F., and Leonhardt, H. (2010). Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA. Nucleic Acids Res 38, e181.

Tahiliani, M., Koh, K.P., Shen, Y., Pastor, W.A., Bandukwala, H., Brudno, Y., Agarwal, S., Iyer, L.M., Liu, D.R., Aravind, L., et al. (2009). Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930-935.