Novel imprints in mouse blastocysts are predominantly DNA methylation independent

ABSTRACTIn mammals, chromatin marks at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. This control is thought predominantly to involve parent-specific differentially methylated regions (DMR) in genomic DNA. However, neither parent-of-origin-specific transcription nor DMRs have been comprehensively mapped. We here address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos (blastocysts). Transcriptome-analysis identified 71 genes expressed with previously unknown parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expression). Uniparental expression of nBiX genes disappeared soon after implantation. Micro-whole-genome bisulfite sequencing (μWGBS) of individual uniparental blastocysts detected 859 DMRs. Only 18% of nBiXs were associated with a DMR, whereas 60% were associated with parentally-biased H3K27me3. This suggests a major role for Polycomb-mediated imprinting in blastocysts. Five nBiX-clusters contained at least one known imprinted gene, and five novel clusters contained exclusively nBiX-genes. These data suggest a complex program of stage-specific imprinting involving different tiers of regulation.

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Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3

Nature Communications ◽

10.1038/s41467-021-23510-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Laura Santini ◽

Florian Halbritter ◽

Fabian Titz-Teixeira ◽

Toru Suzuki ◽

Maki Asami ◽

...

Keyword(s):

Bisulfite Sequencing ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Imprinted Genes ◽

Specific Expression ◽

Histone 3 ◽

Complex Program ◽

Genome Bisulfite Sequencing ◽

Mammalian Genomes ◽

Parent Of Origin

AbstractIn mammalian genomes, differentially methylated regions (DMRs) and histone marks including trimethylation of histone 3 lysine 27 (H3K27me3) at imprinted genes are asymmetrically inherited to control parentally-biased gene expression. However, neither parent-of-origin-specific transcription nor imprints have been comprehensively mapped at the blastocyst stage of preimplantation development. Here, we address this by integrating transcriptomic and epigenomic approaches in mouse preimplantation embryos. We find that seventy-one genes exhibit previously unreported parent-of-origin-specific expression in blastocysts (nBiX: novel blastocyst-imprinted expressed). Uniparental expression of nBiX genes disappears soon after implantation. Micro-whole-genome bisulfite sequencing (µWGBS) of individual uniparental blastocysts detects 859 DMRs. We further find that 16% of nBiX genes are associated with a DMR, whereas most are associated with parentally-biased H3K27me3, suggesting a role for Polycomb-mediated imprinting in blastocysts. nBiX genes are clustered: five clusters contained at least one published imprinted gene, and five clusters exclusively contained nBiX genes. These data suggest that early development undergoes a complex program of stage-specific imprinting involving different tiers of regulation.

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Gamete imprinting: setting epigenetic patterns for the next generation

Reproduction Fertility and Development ◽

10.1071/rd05118 ◽

2006 ◽

Vol 18 (2) ◽

pp. 63 ◽

Cited By ~ 90

Author(s):

Jacquetta M. Trasler

Keyword(s):

Dna Methylation ◽

Genomic Dna ◽

Germ Line ◽

Imprinted Genes ◽

Specific Expression ◽

Placental Function ◽

Early Embryos ◽

Parent Of Origin ◽

Methylation Patterns ◽

Epigenetic Patterns

The acquisition of genomic DNA methylation patterns, including those important for development, begins in the germ line. In particular, imprinted genes are differentially marked in the developing male and female germ cells to ensure parent-of-origin-specific expression in the offspring. Abnormalities in imprints are associated with perturbations in growth, placental function, neurobehavioural processes and carcinogenesis. Based, for the most part, on data from the well-characterised mouse model, the present review will describe recent studies on the timing and mechanisms underlying the acquisition and maintenance of DNA methylation patterns in gametes and early embryos, as well as the consequences of altering these patterns.

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Ultra-deep whole genome bisulfite sequencing reveals a single methylation hotspot in human brain mitochondrial DNA

10.1101/2021.03.30.437685 ◽

2021 ◽

Author(s):

Romain Guitton ◽

Christian Dölle ◽

Guido Alves ◽

Ole-Bjørn Tysnes ◽

Gonzalo S. Nido ◽

...

Keyword(s):

Gene Expression ◽

Parkinson’S Disease ◽

Mitochondrial Dna ◽

Prefrontal Cortex ◽

Cytosine Methylation ◽

Bisulfite Sequencing ◽

Whole Genome ◽

Whole Genome Bisulfite Sequencing ◽

Genome Bisulfite Sequencing ◽

Low Levels

ABSTRACTWhile DNA methylation is established as a major regulator of gene expression in the nucleus, the existence of mitochondrial DNA (mtDNA) methylation remains controversial. Here, we characterised the mtDNA methylation landscape in the prefrontal cortex of neurological healthy individuals (n=26) and patients with Parkinson’s disease (n=27), using a combination of whole genome bisulfite sequencing (WGBS) and bisulfite-independent methods. Accurate mtDNA mapping from WGBS data required alignment to an mtDNA reference only, to avoid misalignment to nuclear mitochondrial pseudogenes. Once correctly aligned, WGBS data provided ultra-deep mtDNA coverage (16,723±7,711), and revealed overall very low levels of cytosine methylation. The highest methylation levels (5.49±0.97%) were found on CpG position m.545, located in the heavy-strand promoter 1 region. The m.545 methylation was validated using a combination of methylation-sensitive DNA digestion and quantitative PCR analysis. We detected no association between mtDNA methylation profile and Parkinson’s disease. Interestingly, m.545 methylation correlated with the levels of mtDNA transcripts, suggesting a putative role in regulating mtDNA gene expression. In addition, we propose a robust framework for methylation analysis of mtDNA from WGBS data, which is less prone to false-positive findings due to misalignment of nuclear mitochondrial pseudogene sequences.Graphical abstract of the analyses and main findingsFresh-frozen brain tissue was obtained from the prefrontal cortex (Brodmann area 9) of 53 individuals, comprising 27 patients with idiopathic PD and 26 healthy controls. Tissue from the same samples was used in three different downstream analyses. WGBS was conducted on all 53 samples and the data were analysed using three different alignment strategies. Alignment against an mtDNA reference only was clearly superior as it gave the highest and most even depth of coverage. WGBS analysis revealed that mtDNA harbours very low levels of cytosine methylation, with the exception of the CpG position m.545 within the HSP1 region (lower right inset). The m.545 methylation was confirmed by bisulfite- and sequencing-independent methods, employing methylation-specific MspJI DNA digestion, followed by quantification with qPCR or fluorescent PCR and capillary electrophoresis. Finally, mtDNA transcript levels were determined by RT-qPCR and correlated to m.545 methylation levels, showing a positive association.

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DNA methylation affects pre-mRNA transcriptional initiation and processing in Arabidopsis

10.1101/2021.04.29.441938 ◽

2021 ◽

Author(s):

Qiuhui Li ◽

Shenjie Chen ◽

Amy Wing-Sze Leung ◽

Yaqin Liu ◽

Yan Xin ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Bisulfite Sequencing ◽

Regulation Of Gene Expression ◽

Mrna Processing ◽

Intergenic Sequence ◽

Whole Genome ◽

Transcriptional Initiation ◽

Whole Genome Bisulfite Sequencing ◽

Genome Bisulfite Sequencing

Background: DNA methylation may regulate pre-mRNA transcriptional initiation and processing, thus affecting gene expression. Unlike animal cells, plants, especially Arabidopsis thaliana, have relatively low DNA methylation levels, limiting our ability to observe any correlation between DNA methylation and pre-mRNA processing using typical short-read sequencing. However, with newly developed long-read sequencing technologies, such as Oxford Nanopore Technology Direct RNA sequencing (ONT DRS), combined with whole-genome bisulfite sequencing, we were able to precisely analyze the relationship between DNA methylation and pre-mRNA transcriptional initiation and processing using DNA methylation-related mutants. Results: Using ONT DRS, we generated more than 2 million high-quality full-length long reads of native mRNA for each of the wild type Col-0 and mutants defective in DNA methylation, identifying a total of 117,474 isoforms. We found that low DNA methylation levels around splicing sites tended to prevent splicing events from occurring. The lengths of the poly(A) tail of mRNAs were positively correlated with DNA methylation. DNA methylation before transcription start sites or around transcription termination sites tended to result in gene-silencing or read-through events. Furthermore, using ONT DRS, we identified novel transcripts that we could not have otherwise, since transcripts with intron retention and fusion transcripts containing the uncut intergenic sequence tend not to be exported to the cytoplasm. Using the met1-3 mutant with activated constitutive heterochromatin regions, we confirmed the effects of DNA methylation on pre-mRNA processing. Conclusion: The combination of ONT DRS with whole-genome bisulfite sequencing was a powerful tool for studying the effects of DNA methylation on splicing site selection and pre-mRNA processing, and therefore regulation of gene expression.

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Investigation of Gene Expression and DNA Methylation From Seven Different Brain Regions of a Crab-Eating Monkey as Determined by RNA-Seq and Whole-Genome Bisulfite Sequencing

Frontiers in Genetics ◽

10.3389/fgene.2019.00694 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 3

Author(s):

Won-Jun Lim ◽

Kyoung Hyoun Kim ◽

Jae-Yoon Kim ◽

Hee-Jin Kim ◽

Mirang Kim ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Bisulfite Sequencing ◽

Brain Regions ◽

Whole Genome ◽

Rna Seq ◽

Whole Genome Bisulfite Sequencing ◽

Genome Bisulfite Sequencing

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Divergence among rice cultivars reveals roles for transposition and epimutation in ongoing evolution of genomic imprinting

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2104445118 ◽

2021 ◽

Vol 118 (29) ◽

pp. e2104445118

Author(s):

Jessica A. Rodrigues ◽

Ping-Hung Hsieh ◽

Deling Ruan ◽

Toshiro Nishimura ◽

Manoj K. Sharma ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Genomic Imprinting ◽

Small Rnas ◽

Imprinted Genes ◽

Dna Hypomethylation ◽

Transcription Start ◽

Specific Expression ◽

Parent Of Origin

Parent-of-origin–dependent gene expression in mammals and flowering plants results from differing chromatin imprints (genomic imprinting) between maternally and paternally inherited alleles. Imprinted gene expression in the endosperm of seeds is associated with localized hypomethylation of maternally but not paternally inherited DNA, with certain small RNAs also displaying parent-of-origin–specific expression. To understand the evolution of imprinting mechanisms in Oryza sativa (rice), we analyzed imprinting divergence among four cultivars that span both japonica and indica subspecies: Nipponbare, Kitaake, 93-11, and IR64. Most imprinted genes are imprinted across cultivars and enriched for functions in chromatin and transcriptional regulation, development, and signaling. However, 4 to 11% of imprinted genes display divergent imprinting. Analyses of DNA methylation and small RNAs revealed that endosperm-specific 24-nt small RNA–producing loci show weak RNA-directed DNA methylation, frequently overlap genes, and are imprinted four times more often than genes. However, imprinting divergence most often correlated with local DNA methylation epimutations (9 of 17 assessable loci), which were largely stable within subspecies. Small insertion/deletion events and transposable element insertions accompanied 4 of the 9 locally epimutated loci and associated with imprinting divergence at another 4 of the remaining 8 loci. Correlating epigenetic and genetic variation occurred at key regulatory regions—the promoter and transcription start site of maternally biased genes, and the promoter and gene body of paternally biased genes. Our results reinforce models for the role of maternal-specific DNA hypomethylation in imprinting of both maternally and paternally biased genes, and highlight the role of transposition and epimutation in rice imprinting evolution.

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Whole Genome Bisulfite Sequencing of Purified Mouse Promyelocytes Reveals Differentially Methylated Regions in Cells Expressing PML-Rara

Blood ◽

10.1182/blood.v124.21.3531.3531 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3531-3531

Author(s):

Shamika Ketkar-Kulkarni ◽

Christopher B Cole ◽

David H. Spencer ◽

Angela M. Verdoni ◽

Nichole Havey ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Latent Period ◽

Bisulfite Sequencing ◽

De Novo ◽

Chromosome 8 ◽

Whole Genome ◽

Chromosome 4 ◽

Differentially Methylated Regions ◽

Genome Bisulfite Sequencing

Abstract Acute promyelocytic leukemia (APL) is an AML subtype that is characterized by aberrant expansion of immature myeloid progenitors and precursors that are arrested at the promyelocyte stage. Almost all APL cases are characterized by the t(15;17)(q22;q11.2) translocation that creates the PML-RARA fusion oncogene. Human APL cells are known to have a canonical expression signature and a specific methylation phenotype that is unique to this form of AML. Our laboratory previously created a mouse model of APL by expressing a human PML-RARA cDNA from the mouse Cathepsin G (Ctsg) locus (Ctsg-PML-RARA), which activates human PML- RARA expression in early myeloid progenitor cells, with peak expression in promyelocytes. After a long latent period (6-12 months), ~60% of these mice develop a clonal, APL-like myeloid malignancy. The long latent period is probably due to the requirement for cooperating mutations that synergize with PML-RARA to accelerate the disease. Human APL samples have a unique gene expression signature that distinguishes them from all other subtypes of AML. We evaluated RNA-Seq data derived from Poly A+ enriched cDNAs obtained from purified promyelocytes derived from 3 young (6 week old) WT and 3 Ctsg-PML-RARA mice. We identified 779 annotated genes that are significantly dysregulated in murine promyelocytes expressing PML-RARA with a log2 fold change >= 2 and P<0.05. Some of these genes included Spib/Pu.1, Pou2af1, Jak2, Runx1, and many others. We also identified a set of 24,018 RNAs in promyelocytes that were defined as novel transcripts. This set contains 7,413 lncRNAs with an FPKM value of >= 2. Differential expression analysis yielded 56 dysregulated lncRNA regions in PML-RARA expressing promyelocytes. To explore the association between gene dysregulation and DNA methylation in promyelocytes, we carried out whole-genome bisulfite sequencing using DNA derived from the purified promyelocytes of a 6 week old Ctsg-PML-RARA mouse, and a WT littermate. We generated a total of approximately 800 million sequencing reads, of which 78% mapped uniquely to the reference genome (mm9); we were able to map ~19 million CpGs with at least 10x coverage. Differential methylation analysis performed on ~4.5 million 1 Kb windows spanning the entire genome identified 17,633 differentially methylated regions with a mean difference of >= 25% and a q-value of < 0.01, the vast majority of which (17,264, 98%) were hypomethylated in the Ctsg-PML-RARA promyelocytes. These windows overlap several known genes, including Runx1, Jak2, Dnmt3a, Gata2, and the Hoxa and Hoxb gene clusters. Using more strict criteria (> 50% mean methylation difference), we identified 87 differentially methylated regions of at least 2 Kb in size. Of these 87 distinct regions, 74 (85%) were hypomethylated in PML-RARA promyelocytes, and 13 were hypermethylated; examples of both as shown in Figure 1. These data strongly suggest that PML-RARA has at least two distinct mechanisms by which it can modify DNA methylation. In regions where CpGs are hypomethylated, PML-RARA may be blocking the normal methylation of CpGs by the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b. In contrast, PML-RARA may be directing de novo methyltransferases to act on the hypermethylated regions. Regardless, these data, when coupled with comprehensive chromatin accessibility mapping and complete RNA sequencing data, should provide new insights into the mechanisms used by PML-RARA to alter gene expression and initiate APL. Figure1. Examples of differentially methylated regions. Black=WT cells. Red=PML-RARA expressing cells. Each CpG in the region is represented as a dot. Scale is 0-100% methylated at each position. Top panel: a region on chromosome 8 that is hypomethylated in PML-RARA expressing promyelocytes. Bottom panel: a region on chromosome 4 that is hypermethylated in PML-RARA expressing promyelocytes. Figure1. Examples of differentially methylated regions. Black=WT cells. Red=PML-RARA expressing cells. Each CpG in the region is represented as a dot. Scale is 0-100% methylated at each position. Top panel: a region on chromosome 8 that is hypomethylated in PML-RARA expressing promyelocytes. Bottom panel: a region on chromosome 4 that is hypermethylated in PML-RARA expressing promyelocytes. Disclosures No relevant conflicts of interest to declare.

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Parent of origin gene expression in a founder population identifies two new imprinted genes at known imprinted regions

10.1101/344457 ◽

2018 ◽

Author(s):

Sahar V. Mozaffari ◽

Michelle M. Stein ◽

Kevin M. Magnaye ◽

Dan L. Nicolae ◽

Carole Ober

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Gene Expression Data ◽

Imprinted Genes ◽

Genotype Data ◽

Expression Data ◽

Rna Seq ◽

Specific Expression ◽

Allele Specific ◽

Parent Of Origin

AbstractGenomic imprinting is the phenomena that leads to silencing of one copy of a gene inherited from a specific parent. Mutations in imprinted regions have been involved in diseases showing parent of origin effects. Identifying genes with evidence of parent of origin expression patterns in family studies allows the detection of more subtle imprinting. Here, we use allele specific expression in lymphoblastoid cell lines from 306 Hutterites related in a single pedigree to provide formal evidence for parent of origin effects. We take advantage of phased genotype data to assign parent of origin to RNA-seq reads in individuals with gene expression data. Our approach identified known imprinted genes, two putative novel imprinted genes, and 14 genes with asymmetrical parent of origin gene expression. We used gene expression in peripheral blood leukocytes (PBL) to validate our findings, and then confirmed imprinting control regions (ICRs) using DNA methylation levels in the PBLs.Author SummaryLarge scale gene expression studies have identified known and novel imprinted genes through allele specific expression without knowing the parental origins of each allele. Here, we take advantage of phased genotype data to assign parent of origin to RNA-seq reads in 306 individuals with gene expression data. We identified known imprinted genes as well as two novel imprinted genes in lymphoblastoid cell line gene expression. We used gene expression in PBLs to validate our findings, and DNA methylation levels in PBLs to confirm previously characterized imprinting control regions that could regulate these imprinted genes.

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