Aims: Studies of DNA methylomes hold enormous promise for biomedicine but are hampered by the technological challenges of analyzing many samples cost-effectively. Recently, a major extension of the previous Infinium HumanMethylation27 BeadChip ® (Illumina, Inc. CA, USA), called Infinium HumanMethylation450 (Infinium Methylation 450K; Illumina, Inc. CA, USA) was developed. This upgraded technology is a hybrid of two different chemical assays, the Infinium I and Infinium II assays, allowing (for 12 samples in parallel) assessment of the methylation status of more than 480,000 cytosines distributed over the whole genome. In this article, we evaluate Infinium Methylation 450K on cell lines and tissue samples, highlighting some of its advantages but also some of its limitations. In particular, we compare the methylation values of the Infinium I and Infinium II assays. Materials & methods: We used Infinium Methylation 450K to profile: first, the well-characterized HCT116 wild-type and double-knockout cell lines and then, 16 breast tissue samples (including eight normal and eight primary tumor samples). Absolute methylation values (b-values) were extracted with the GenomeStudio TM software and then subjected to detailed analysis. Results: While this technology appeared highly robust as previously shown, we noticed a divergence between the b-values retrieved from the type I and type II Infinium assays. Specifically, the b-values obtained from Infinium II probes were less accurate and reproducible than those obtained from Infinium I probes. This suggests that data from the type I and type II assays should be considered separately in any downstream bioinformatic analysis. To be able to deal with the Infinium I and Infinium II data together, we developed and tested a new correction technique, which we called 'peak-based correction'. The idea was to rescale the Infinium II data on the basis of the Infinium I data. While this technique should be viewed as an approximation method, it significantly improves the quality of Infinium II data. Conclusion: Infinium 450K is a powerful technique in terms of reagent costs, time of labor, sample throughput and coverage. It holds great promise for the better understanding of the epigenetic component in health and disease. Yet, due to the nature of its design comprising two different chemical assays, analysis of the whole set of data is not as easy as initially anticipated. Correction strategies, such as the peak-based approach proposed here, are a step towards adequate output data analysis. keywoRds: bisulfite-based method n dNA methylation n dNA methylome n epigenetics n epigenomics n Infinium I n Infinium II n Infinium Methylation 450k n peak-based correction

EMBo reports Vol 12 | no 7 | 2011 647 review review DNA methyltransferases (DNMTs) establish and maintain DNA methylation patterns at specific regions of the genome, thereby contributing to gene regulation. It is becoming evident that an intricate web of pathways target DNMTs to these genomic regions. Here, we review the understanding of these regulatory mechanisms and provide an overview of the new findings, emphasizing the emerging scenario in which several levels of regulation are coordinated to control DNMTs. The mechanisms involved include the dynamic interplay between interdependent post-translational modifications that regulate DNMTs, post-transcriptional regulation by miRNAs and the emerging role of non-coding RNA in targeting mammalian DNMTs. The analysis of these mechanisms is imperative to the understanding of the role of DNA methylation in regulating gene expression during development and in disease.

Histone methylation plays key roles in regulating chromatin structure and function. The recent identification of enzymes that antagonize or remove histone methylation offers new opportunities to appreciate histone methylation plasticity in the regulation of epigenetic pathways. Peptidylarginine deiminase 4 (PADI4; also known as PAD4) was the first enzyme shown to antagonize histone methylation. PADI4 functions as a histone deiminase converting a methylarginine residue to citrulline at specific sites on the tails of histones H3 and H4. This activity is linked to repression of the estrogen-regulated pS2 promoter. Very little is known as to how PADI4 silences gene expression. We show here that PADI4 associates with the histone deacetylase 1 (HDAC1). Kinetic chromatin immunoprecipitation assays revealed that PADI4 and HDAC1, and the corresponding activities, associate cyclically and coordinately with the pS2 promoter during repression phases. Knockdown of HDAC1 led to decreased H3 citrullination, concomitantly with increased histone arginine methylation. In cells with a reduced HDAC1 and a slightly decreased PADI4 level, these effects were more pronounced. Our data thus suggest that PADI4 and HDAC1 collaborate to generate a repressive chromatin environment on the pS2 promoter. These findings further substantiate the "transcriptional clock" concept, highlighting the dynamic connection between deimination and deacetylation of histones.Until recently, it was unclear whether enzymes capable of antagonizing histone methylation existed. However, recent studies have revealed a growing number of lysine demethylases that can reverse histone lysine methylation, such as LSD1/ KDM1 and the JmjC-domain-containing proteins (16,21,31,37,43,45,47).In addition to the methylation of lysine, histones can also be methylated on arginine (6, 34). Recent work has identified the JMJD6 protein as an H3R2 and H4R3 demethylase (5). An alternative pathway for the reversal of arginine methylation has been identified in mammals. In this pathway, a methyl group is removed from a methylarginine residue by conversion of this residue to citrulline. The reaction is termed deimination because the methyl group is removed along with the imine group of arginine (7, 42). The enzyme that catalyzes this reaction is peptidylarginine deiminase 4 (PADI4; also known as PAD4) (7, 42). PADI4 has a relatively broad substrate specificity, since the enzyme can deiminate multiple arginine sites on histones H3 (R2, R8, R17, and R26) and H4 (R3) (42). PADI4 is a Ca 2ϩ -dependent enzyme (1). Functionally, the induction of PADI4-mediated deimination has been best studied as part of the estrogen signaling pathway, particularly in the context of the estrogen-regulated pS2 promoter.Chromatin immunoprecipitation (ChIP)-based kinetic analyses performed on human breast cancer cell lines have shown that in the presence of estrogen (E2), pS2 expression is controlled by the estrogen receptor alpha (ER␣), which cycles on its promoter. Not only does the receptor cycle, it also foll...

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DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns.