MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation

Lee, Dong‐Hoon; Tran, Diana A.; Singh, Purnima; Oates, Nathan; Rivas, Guillermo; Larson, Garrett P.; Pfeifer, Gerd P.; Szabó, Piroska E.

doi:10.4161/epi.6.2.13699

Cited by 8 publications

(7 citation statements)

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“…We isolated DNA and collected the CpG-methylated fraction using MIRA [ 40 ]. We quantified the percentage of parental alleles in the total methylated fraction at 14 DMRs (18 SNPs) using multiplex SNuPE assays [ 41 ]. The average percentage of paternal or maternal allele methylation is displayed in Figure 3 C and Additional file 1 .…”

Section: Resultsmentioning

confidence: 99%

“…Allele-specific DNA methylation and gene expression was measured by multiplex SNuPE assays [ 29 , 61 ] on the Sequenom platform, as we have done previously [ 32 , 41 , 62 , 63 ]. These assays are based on single nucleotide polymorphisms (SNPs) that distinguish between the inbred JF1/Ms (JF1) and 129S1 (129) mouse strains, or between the JF1 and the TgOG2 (OG2) transgenic mouse strains.…”

Section: Methodsmentioning

confidence: 99%

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Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

et al. 2015

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BackgroundExposure to environmental endocrine-disrupting chemicals during pregnancy reportedly causes transgenerationally inherited reproductive defects. We hypothesized that to affect the grandchild, endocrine-disrupting chemicals must alter the epigenome of the germ cells of the in utero-exposed G1 male fetus. Additionally, to affect the great-grandchild, the aberration must persist in the germ cells of the unexposed G2 grandchild.ResultsHere, we treat gestating female mice with vinclozolin, bisphenol A, or di-(2-ethylhexyl)phthalate during the time when global de novo DNA methylation and imprint establishment occurs in the germ cells of the G1 male fetus. We map genome-wide features in purified G1 and G2 prospermatogonia, in order to detect immediate and persistent epigenetic aberrations, respectively. We detect changes in transcription and methylation in the G1 germline immediately after endocrine-disrupting chemicals exposure, but changes do not persist into the G2 germline. Additional analysis of genomic imprints shows no persistent aberrations in DNA methylation at the differentially methylated regions of imprinted genes between the G1 and G2 prospermatogonia, or in the allele-specific transcription of imprinted genes between the G2 and G3 soma.ConclusionsOur results suggest that endocrine-disrupting chemicals exert direct epigenetic effects in exposed fetal germ cells, which are corrected by reprogramming events in the next generation. Avoiding transgenerational inheritance of environmentally-caused epigenetic aberrations may have played an evolutionary role in the development of dual waves of global epigenome reprogramming in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0619-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

et al. 2015

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“…Parental allele-specific CpG methylation was measured using multiplex single nucleotide extension (SNuPE) by sequenom allelotyping assays (20). The primer sequences are listed in Supplementary Table S1.…”

Section: Methodsmentioning

confidence: 99%

“…The methylated fraction of sonicated genomic DNA from MEFs was captured using recombinant MBD2b and MBD3L1 proteins as described earlier ( 19 ). Parental allele-specific CpG methylation was measured using multiplex single nucleotide extension (SNuPE) by sequenom allelotyping assays ( 20 ). The primer sequences are listed in Supplementary Table S1 .…”

Section: Methodsmentioning

confidence: 99%

Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing

Tran

Bai

Singh

et al. 2013

Nucleic Acids Research

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Mouse embryo fibroblasts (MEFs) are convenient sources for biochemical studies when cell number in mouse embryos is limiting. To derive the imprinting signature of MEFs and potentially detect novel imprinted genes we performed strand- and allele-specific RNA deep sequencing. We used sequenom allelotyping in embryo and adult organs to verify parental allele-specific expression. Thirty-two known ubiquitously imprinted genes displayed correct parental allele-specific transcripts in MEFs. Our analysis did not reveal any novel imprinted genes, but detected extended parental allele-specific transcripts in several known imprinted domains: maternal allele-specific transcripts downstream of Grb10 and downstream of Meg3, Rtl1as and Rian in the Dlk1-Dio3 cluster, an imprinted domain implicated in development and pluripotency. We detected paternal allele-specific transcripts downstream of Nespas, Peg3, Peg12 and Snurf/Snrpn. These imprinted transcript extensions were not unique to MEFs, but were also present in other somatic cells. The 5′ end points of the imprinted transcript extensions did not carry opposing chromatin marks or parental allele-specific DNA methylation, suggesting that their parental allele-specific transcription is under the control of the extended imprinted genes. Based on the imprinting signature of MEFs, these cells provide valid models for understanding the biochemical aspects of genomic imprinting.

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“…It is well known that the expression of IGF-II is imprinted and regulated by methylation and, normally, only the paternal allele is transcribed [ 34 – 36 ]. Nevertheless, our results demonstrated that biallelic expression of IGF-II was present in 100% of the normal paired samples, thus demonstrating that “loss of imprinting” (LOI) is not the result of carcinogenesis in these samples.…”

Section: Discussionmentioning

confidence: 99%

Expression of Intratumoral IGF-II Is Regulated by the Gene Imprinting Status in Triple Negative Breast Cancer from Vietnamese Patients

Radhakrishnan

Hernandez

Anderson

et al. 2015

International Journal of Endocrinology

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African American women suffer higher incidence and mortality of triple negative breast cancer (TNBC) than Caucasian women. TNBC is very aggressive, causing the worst clinical outcome. We previously demonstrated that tumors from these patients express high IGF-II and exhibit high activation of the IGF signaling pathways. IGF-II gene expression is imprinted (monoallelic), promotes tumor progression, and metastasis and regulates Survivin, a TNBC prognostic marker. Since BC mortality has increased among young Vietnamese women, we analyzed 48 (paired) TNBC samples from Vietnamese patients to assess IGF-II expression. We analyzed all samples by qrtPCR for identification of IGF-II heterozygosity and to determine allelic expression of the IGF-II gene. We also analyzed the tissues for proIGF-II and Survivin by RT-PCR and Western blotting. A total of 28 samples displayed IGF-II heterozygosity of which 78% were biallelic. Tumors with biallelic IGF-II gene expression exhibited the highest levels of proIGF-II and Survivin. Although 100% of these tissues corresponding normal samples were biallelic, they expressed significantly lower levels of or no proIGF-II and Survivin. Thus, IGF-II biallelic gene expression is differentially regulated in normal versus tumor tissues. We propose that intratumoral proIGF-II is dependent on the IGF-II gene imprinting status and it will promote a more aggressive TNBC.

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MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation

Abstract: (2011) MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation , Epigenetics, 6:2, 212-223,

Cited by 8 publications

References 74 publications

Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

Deleterious effects of endocrine disruptors are corrected in the mammalian germline by epigenome reprogramming

Characterization of the imprinting signature of mouse embryo fibroblasts by RNA deep sequencing

Expression of Intratumoral IGF-II Is Regulated by the Gene Imprinting Status in Triple Negative Breast Cancer from Vietnamese Patients

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