Dnmt3a and Dnmt3b-Decommissioned Fetal Enhancers are Linked to Kidney Disease

BackgroundCytosine methylation is an epigenetic mark that dictates cell fate and response to stimuli. The timing and establishment of methylation logic during kidney development remains unknown. DNA methyltransferase 3a and 3b are the enzymes capable of establishing de novo methylation.MethodsWe generated mice with genetic deletion of Dnmt3a and Dnmt3b in nephron progenitor cells (Six2CreDnmt3a/3b) and kidney tubule cells (KspCreDnmt3a/3b). We characterized KspCreDnmt3a/3b mice at baseline and after injury. Unbiased omics profiling, such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing and RNA sequencing were performed on whole-kidney samples and isolated renal tubule cells.ResultsKspCreDnmt3a/3b mice showed no obvious morphologic and functional alterations at baseline. Knockout animals exhibited increased resistance to cisplatin-induced kidney injury, but not to folic acid–induced fibrosis. Whole-genome bisulfite sequencing indicated that Dnmt3a and Dnmt3b play an important role in methylation of gene regulatory regions that act as fetal-specific enhancers in the developing kidney but are decommissioned in the mature kidney. Loss of Dnmt3a and Dnmt3b resulted in failure to silence developmental genes. We also found that fetal-enhancer regions methylated by Dnmt3a and Dnmt3b were enriched for kidney disease genetic risk loci. Methylation patterns of kidneys from patients with CKD showed defects similar to those in mice with Dnmt3a and Dnmt3b deletion.ConclusionsOur results indicate a potential locus-specific convergence of genetic, epigenetic, and developmental elements in kidney disease development.

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Efficient and accurate determination of genome-wide DNA methylation patterns in Arabidopsis thaliana with enzymatic methyl sequencing

Epigenetics & Chromatin ◽

10.1186/s13072-020-00361-9 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Suhua Feng ◽

Zhenhui Zhong ◽

Ming Wang ◽

Steven E. Jacobsen

Keyword(s):

Dna Methylation ◽

Bisulfite Sequencing ◽

Accurate Determination ◽

Gc Content ◽

Epigenetic Mark ◽

Whole Genome ◽

Whole Genome Bisulfite Sequencing ◽

Genome Wide ◽

A Genome ◽

Genome Bisulfite Sequencing

Abstract Background 5′ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing. Results Using an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions. Conclusion We suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

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Whole-genome bisulfite sequencing in systemic sclerosis provides novel targets to understand disease pathogenesis

BMC Medical Genomics ◽

10.1186/s12920-019-0602-8 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Tianyuan Lu ◽

Kathleen Oros Klein ◽

Inés Colmegna ◽

Maximilien Lora ◽

Celia M. T. Greenwood ◽

...

Keyword(s):

Dna Methylation ◽

Genetic Variation ◽

Systemic Sclerosis ◽

Cytosine Methylation ◽

Bisulfite Sequencing ◽

Whole Genome ◽

Disease Pathogenesis ◽

Whole Genome Bisulfite Sequencing ◽

Genome Wide ◽

Genome Bisulfite Sequencing

Abstract Background Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease whose pathogenesis remains incompletely understood. Increasing evidence suggests that both genetic susceptibilities and changes in DNA methylation influence pivotal biological pathways and thereby contribute to the disease. The role of DNA methylation in SSc has not been fully elucidated, because existing investigations of DNA methylation predominantly focused on nucleotide CpGs within restricted genic regions, and were performed on samples containing mixed cell types. Methods We performed whole-genome bisulfite sequencing on purified CD4+ T lymphocytes from nine SSc patients and nine controls in a pilot study, and then profiled genome-wide cytosine methylation as well as genetic variations. We adopted robust statistical methods to identify differentially methylated genomic regions (DMRs). We then examined pathway enrichment associated with genes located in these DMRs. We also tested whether changes in CpG methylation were associated with adjacent genetic variation. Results We profiled DNA methylation at more than three million CpG dinucleotides genome-wide. We identified 599 DMRs associated with 340 genes, among which 54 genes exhibited further associations with adjacent genetic variation. We also found these genes were associated with pathways and functions that are known to be abnormal in SSc, including Wnt/β-catenin signaling pathway, skin lesion formation and progression, and angiogenesis. Conclusion The CD4+ T cell DNA cytosine methylation landscape in SSc involves crucial genes in disease pathogenesis. Some of the methylation patterns are also associated with genetic variation. These findings provide essential foundations for future studies of epigenetic regulation and genome-epigenome interaction in SSc.

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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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Efficient and Accurate Determination of Genome-Wide DNA Methylation Patterns in Arabidopsis with Enzymatic Methyl Sequencing.

10.21203/rs.3.rs-41816/v1 ◽

2020 ◽

Author(s):

Suhua Feng ◽

Zhenhui Zhong ◽

Ming Wang ◽

Steven E. Jacobsen

Keyword(s):

Dna Methylation ◽

Bisulfite Sequencing ◽

Accurate Determination ◽

Gc Content ◽

Epigenetic Mark ◽

Whole Genome ◽

Whole Genome Bisulfite Sequencing ◽

Genome Wide ◽

A Genome ◽

Genome Bisulfite Sequencing

Abstract Background: 5’ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may thus provide advantages over bisulfite sequencing.Results: Using an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions. Conclusion: We suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

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DNA Methylation Analysis of the Citrullus lanatus Response to Cucumber Green Mottle Mosaic Virus Infection by Whole-Genome Bisulfite Sequencing

Genes ◽

10.3390/genes10050344 ◽

2019 ◽

Vol 10 (5) ◽

pp. 344 ◽

Cited By ~ 8

Author(s):

Yuyan Sun ◽

Min Fan ◽

Yanjun He

Keyword(s):

Dna Methylation ◽

Mosaic Virus ◽

Calcium Binding ◽

Bisulfite Sequencing ◽

Citrullus Lanatus ◽

Epigenetic Mark ◽

Post Inoculation ◽

Whole Genome ◽

Whole Genome Bisulfite Sequencing ◽

Genome Bisulfite Sequencing

DNA methylation is an important epigenetic mark associated with plant immunity, but little is known about its roles in viral infection of watermelon. We carried out whole-genome bisulfite sequencing of watermelon leaves at 0 h (ck), 48 h, and 25 days post-inoculation with Cucumber green mottle mosaic virus (CGMMV). The number of differentially methylated regions (DMRs) increased during CGMMV infection and 2788 DMR-associated genes (DMGs) were screened out among three libraries. Most DMRs and DMGs were obtained under the CHH context. These DMGs were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of secondary biosynthesis and metabolism, plant–pathogen interactions, Toll-like receptor signaling, and ABC transporters. Additionally, DMGs encoding PR1a, CaMs, calcium-binding protein, RIN4, BAK1, WRKYs, RBOHs, STKs, and RLPs/RLKs were involved in the watermelon–CGMMV interaction and signaling. The association between DNA methylation and gene expression was analyzed by RNA-seq and no clear relationship was detected. Moreover, downregulation of genes in the RdDM pathway suggested the reduced RdDM-directed CHH methylation plays an important role in antiviral defense in watermelon. Our findings provide genome-wide DNA methylation profiles of watermelon and will aid in revealing the molecular mechanism in response to CGMMV infection at the methylation level.

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Whole-genome bisulfite sequencing in systemic sclerosis provides novel targets to understand disease pathogenesis

10.21203/rs.2.12858/v3 ◽

2019 ◽

Author(s):

Tianyuan Lu ◽

Kathleen Oros Klein ◽

Inés Colmegna ◽

Maximilien Lora ◽

Celia M.T. Greenwood ◽

...

Keyword(s):

Dna Methylation ◽

Genetic Variation ◽

Systemic Sclerosis ◽

Cytosine Methylation ◽

Bisulfite Sequencing ◽

Whole Genome ◽

Disease Pathogenesis ◽

Whole Genome Bisulfite Sequencing ◽

Genome Wide ◽

Genome Bisulfite Sequencing

Abstract Background Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease whose pathogenesis remains incompletely understood. Increasing evidence suggests that both genetic susceptibilities and changes in DNA methylation influence pivotal biological pathways and thereby contribute to the disease. The role of DNA methylation in SSc has not been fully elucidated, because existing investigations of DNA methylation predominantly focused on nucleotide CpGs within restricted genic regions, and were performed on samples containing mixed cell types. Methods We performed whole-genome bisulfite sequencing on purified CD4+ T lymphocytes from nine SSc patients and nine controls in a pilot study, and then profiled genome-wide cytosine methylation as well as genetic variations. We adopted robust statistical methods to identify differentially methylated genomic regions (DMRs). We then examined pathway enrichment associated with genes located in these DMRs. We also tested whether changes in CpG methylation were associated with adjacent genetic variation. Results We profiled DNA methylation at more than three million CpG dinucleotides genome-wide. We identified 599 DMRs associated with 340 genes, among which 54 genes exhibited further associations with adjacent genetic variation. We also found these genes were associated with pathways and functions that are known to be abnormal in SSc, including Wnt/-catenin signaling pathway, skin lesion formation and progression, and angiogenesis. Conclusion The CD4+ T cell DNA cytosine methylation landscape in SSc involves crucial genes in disease pathogenesis. Some of the methylation patterns are also associated with genetic variation. These findings provide essential foundations for future studies of epigenetic regulation and genome-epigenome interaction in SSc.

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Whole-genome bisulfite sequencing in systemic sclerosis provides novel targets to understand disease pathogenesis

10.21203/rs.2.12858/v1 ◽

2019 ◽

Author(s):

Tianyuan Lu ◽

Kathleen Oros Klein ◽

Inés Colmegna ◽

Maximilien Lora ◽

Celia M.T. Greenwood ◽

...

Keyword(s):

Dna Methylation ◽

Genetic Variation ◽

Systemic Sclerosis ◽

Cytosine Methylation ◽

Bisulfite Sequencing ◽

Whole Genome ◽

Disease Pathogenesis ◽

Whole Genome Bisulfite Sequencing ◽

Genome Wide ◽

Genome Bisulfite Sequencing

Abstract Background Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease whose pathogenesis remains incompletely understood. Increasing evidence suggests that both genetic susceptibilities and changes in DNA methylation influence pivotal biological pathways and thereby contribute to the disease. The role of DNA methylation in SSc has not been fully elucidated, because existing investigations of DNA methylation predominantly focused on nucleotide CpGs within restricted genic regions, and were performed on samples containing mixed cell types. Methods We performed whole-genome bisulfite sequencing on purified CD4+ T lymphocytes from nine SSc patients and nine controls in a pilot study, and then profiled genome-wide cytosine methylation as well as genetic variations. We adopted robust statistical methods to identify differentially methylated genomic regions (DMRs). We then examined pathway enrichment associated with genes located in these DMRs. We also tested whether changes in CpG methylation were associated with adjacent genetic variation. Results We profiled DNA methylation at more than three million CpG dinucleotides genome-wide. We identified 599 DMRs associated with 340 genes, among which 54 genes exhibited further associations with adjacent genetic variation. We also found these genes were associated with pathways and functions that are known to be abnormal in SSc, including Wnt/-catenin signaling pathway, skin lesion formation and progression, and angiogenesis. Conclusion The CD4+ T cell DNA cytosine methylation landscape in SSc involves crucial genes in disease pathogenesis. Some of the methylation patterns are also associated with genetic variation. These findings provide essential foundations for future studies of epigenetic regulation and genome-epigenome interaction in SSc.

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Whole-genome bisulfite sequencing in systemic sclerosis provides novel targets to understand disease pathogenesis

10.21203/rs.2.12858/v2 ◽

2019 ◽

Author(s):

Tianyuan Lu ◽

Kathleen Oros Klein ◽

Inés Colmegna ◽

Maximilien Lora ◽

Celia M.T. Greenwood ◽

...

Keyword(s):

Dna Methylation ◽

Genetic Variation ◽

Systemic Sclerosis ◽

Cytosine Methylation ◽

Bisulfite Sequencing ◽

Whole Genome ◽

Disease Pathogenesis ◽

Whole Genome Bisulfite Sequencing ◽

Genome Wide ◽

Genome Bisulfite Sequencing

Abstract Background Systemic sclerosis (SSc) is a rare autoimmune connective tissue disease whose pathogenesis remains incompletely understood. Increasing evidence suggests that both genetic susceptibilities and changes in DNA methylation influence pivotal biological pathways and thereby contribute to the disease. The role of DNA methylation in SSc has not been fully elucidated, because existing investigations of DNA methylation predominantly focused on nucleotide CpGs within restricted genic regions, and were performed on samples containing mixed cell types. Methods We performed whole-genome bisulfite sequencing on purified CD4+ T lymphocytes from nine SSc patients and nine controls in a pilot study, and then profiled genome-wide cytosine methylation as well as genetic variations. We adopted robust statistical methods to identify differentially methylated genomic regions (DMRs). We then examined pathway enrichment associated with genes located in these DMRs. We also tested whether changes in CpG methylation were associated with adjacent genetic variation. Results We profiled DNA methylation at more than three million CpG dinucleotides genome-wide. We identified 599 DMRs associated with 340 genes, among which 54 genes exhibited further associations with adjacent genetic variation. We also found these genes were associated with pathways and functions that are known to be abnormal in SSc, including Wnt/-catenin signaling pathway, skin lesion formation and progression, and angiogenesis. Conclusion The CD4+ T cell DNA cytosine methylation landscape in SSc involves crucial genes in disease pathogenesis. Some of the methylation patterns are also associated with genetic variation. These findings provide essential foundations for future studies of epigenetic regulation and genome-epigenome interaction in SSc.

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