Global DNA Methylation Analysis Using the Luminometric Methylation Assay

Karimi, Mohsen; Luttropp, Karin; Ekström, Tomas J.

doi:10.1007/978-1-61779-316-5_11

Cited by 22 publications

(17 citation statements)

References 23 publications

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“…S1C), expression of which is unaffected by H1 depletion (21). Global DNA methylation also is not changed in the H1-depleted cells, as determined by the luminometric methylation assay (27) (Fig. S1D).…”

Section: Resultsmentioning

confidence: 81%

H1 linker histone promotes epigenetic silencing by regulating both DNA methylation and histone H3 methylation

Yang

Kim

Toro

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

104

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Epigenetic silencing in mammals involves DNA methylation and posttranslational modifications of core histones. Here we show that the H1 linker histone plays a key role in regulating both DNA methylation and histone H3 methylation at the H19 and Gtl2 loci in mouse ES cells. Some, but not all, murine H1 subtypes interact with DNA methyltransferases DNMT1 and DNMT3B. The interactions are direct and require a portion of the H1 C-terminal domain. Expression of an H1 subtype that interacts with DNMT1 and DNMT3B in ES cells leads to their recruitment and DNA methylation of the H19 and Gtl2 imprinting control regions. H1 also interferes with binding of the SET7/9 histone methyltransferase to the imprinting control regions, inhibiting production of an activating methylation mark on histone H3 lysine 4. H1-dependent recruitment of DNMT1 and DNMT3B and interference with the binding of SET7/9 also were observed with chromatin reconstituted in vitro. The data support a model in which H1 plays an active role in helping direct two processes that lead to the formation of epigenetic silencing marks. The data also provide evidence for functional differences among the H1 subtypes expressed in somatic mammalian cells.mouse embryonic stem cell | H1 histone triple knockout T wo major types of epigenetic marks occur in mammalian genomes, DNA methylation and posttranslational modifications of histones (1, 2). The methylation of cytosines in mammalian DNA occurs primarily at CpG dinucleotides and is essential for normal mammalian development (1, 3). Perturbations of DNA methylation patterns are thought to play a role in cancer development (4). There are two classes of mammalian DNA methyltransferases (DNMTs), one that functions primarily to establish DNA methylation de novo (DNMT3A and DNMT3B) and the other to maintain it (DNMT1) (3). DNA methylation can have profound effects on gene expression and is most often associated with transcriptional silencing (1, 2). Accumulating evidence indicates the existence of crosstalk between the DNA methylation and core histone modification systems (1, 2). For example, mouse ES cells deficient for the histone H3 lysine 9 (H3K9) methyltransferases Suv39h1/h2 exhibit hypomethylation of CpGs at a subset of repetitive DNA elements (5). Additionally, several lines of evidence indicate that the presence of unmethylated lysine 4 of histone H3 (H3K4) in chromatin favors de novo methylation of DNA (6-9). Despite these advances, the factors in chromatin that coordinate DNA methylation and histone H3 methylation have not been identified.In this study, we investigated the role of the linker histone H1 in regulating DNA methylation and histone H3 methylation at the H19 and Gtl2 loci in mouse ES cells. H1 is the most distinct of the five histone proteins (H1, H2A, H2B, H3, and H4) in chromatin (10,11). Mammals contain at least eight histone H1 subtypes or variants that differ in amino acid sequences and expression during development (12-14). Functional differences among these subtypes have been difficult to identif...

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Section: Resultsmentioning

confidence: 81%

H1 linker histone promotes epigenetic silencing by regulating both DNA methylation and histone H3 methylation

Yang

Kim

Toro

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

103

104

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“…As methylation at gDMR was not completely restored to WT levels in the 3abKO + 3a2, we compared whole-genome methylation levels using LUMA [49], a bisulfite-independent quantitative assay that uses methylation-sensitive and methylation-insensitive enzymes to estimate total genomic methylation levels. LUMA analysis of 1KO and 3abKO cells shows clear loss of methylation compared to WT (set to 100%).…”

Section: Resultsmentioning

confidence: 99%

“…The Luminometric Methylation Assay (LUMA) using 300 ng/μl of genomic DNA from the respective cell lines was carried out exactly as described previously [13, 49]. HCT116 WT DNA (hypermethylated) and DKO DNA (hypomethylated) samples were used as a control (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

Thakur

Mackin

Irwin

et al. 2016

Epigenetics & Chromatin

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BackgroundImprinted loci are paradigms of epigenetic regulation and are associated with a number of genetic disorders in human. A key characteristic of imprints is the presence of a gametic differentially methylated region (gDMR). Previous studies have indicated that DNA methylation lost from gDMRs could not be restored by DNMT1, or the de novo enzymes DNMT3A or 3B in stem cells, indicating that imprinted regions must instead undergo passage through the germline for reprogramming. However, previous studies were non-quantitative, were unclear on the requirement for DNMT3A/B and showed some inconsistencies. In addition, new putative gDMR has recently been described, along with an improved delineation of the existing gDMR locations. We therefore aimed to re-examine the dependence of methylation at gDMRs on the activities of the methyltransferases in mouse embryonic stem cells (ESCs).ResultsWe examined the most complete current set of imprinted gDMRs that could be assessed using quantitative pyrosequencing assays in two types of ESCs: those lacking DNMT1 (1KO) and cells lacking a combination of DNMT3A and DNMT3B (3abKO). We further verified results using clonal analysis and combined bisulfite and restriction analysis. Our results showed that loss of methylation was approximately equivalent in both cell types. 1KO cells rescued with a cDNA-expressing DNMT1 could not restore methylation at the imprinted gDMRs, confirming some previous observations. However, nearly all gDMRs were remethylated in 3abKO cells rescued with a DNMT3A2 expression construct (3abKO + 3a2). Transcriptional activity at the H19/Igf2 locus also tracked with the methylation pattern, confirming functional reprogramming in the latter.ConclusionsThese results suggested (1) a vital role for DNMT3A/B in methylation maintenance at imprints, (2) that loss of DNMT1 and DNMT3A/B had equivalent effects, (3) that rescue with DNMT3A2 can restore imprints in these cells. This may provide a useful system in which to explore factors influencing imprint reprogramming.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0104-2) contains supplementary material, which is available to authorized users.

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“…As methylation was not restored to WT levels, we quantitated the global increase in methylation in rescued cells using the LUMA assay (Karimi et al, 2011a), which is a bisulfite-independent quantitative assay that uses methylation-sensitive and -insensitive restriction enzyme cleavage to estimate total genomic methylation levels, and showed that 1KO+1 cells had an overall gain of 28% versus 1KO cells (Fig. 6C).…”

Section: Resultsmentioning

confidence: 99%

“…Assays were conducted using the PyroMark PCR Kit (Qiagen); conditions were: 95°C, 15 minutes; followed by 45 cycles of 94°C for 30 seconds, 56°C for 30 seconds and 72°C for 30 seconds; with final elongation at 72°C, 10 minutes. LUMA analysis was carried out as previously described (Karimi et al, 2011a). …”

Section: Methodsmentioning

confidence: 99%

Ontogeny, conservation and functional significance of maternally inherited DNA methylation at two classes of non-imprinted genes

et al. 2014

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A functional role for DNA methylation has been well-established at imprinted loci, which inherit methylation uniparentally, most commonly from the mother via the oocyte. Many CpG islands not associated with imprinting also inherit methylation from the oocyte, although the functional significance of this, and the common features of the genes affected, are unclear. We identify two major subclasses of genes associated with these gametic differentially methylated regions (gDMRs), namely those important for brain and for testis function. The gDMRs at these genes retain the methylation acquired in the oocyte through preimplantation development, but become fully methylated postimplantation by de novo methylation of the paternal allele. Each gene class displays unique features, with the gDMR located at the promoter of the testis genes but intragenically for the brain genes. Significantly, demethylation using knockout, knockdown or pharmacological approaches in mouse stem cells and fibroblasts resulted in transcriptional derepression of the testis genes, indicating that they may be affected by environmental exposures, in either mother or offspring, that cause demethylation. Features of the brain gene group suggest that they might represent a pool from which many imprinted genes have evolved. The locations of the gDMRs, as well as methylation levels and repression effects, were also conserved in human cells.

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Global DNA Methylation Analysis Using the Luminometric Methylation Assay

Cited by 22 publications

References 23 publications

H1 linker histone promotes epigenetic silencing by regulating both DNA methylation and histone H3 methylation

H1 linker histone promotes epigenetic silencing by regulating both DNA methylation and histone H3 methylation

Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2

Ontogeny, conservation and functional significance of maternally inherited DNA methylation at two classes of non-imprinted genes

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