scholarly journals ARPEGGIO: Automated Reproducible Polyploid EpiGenetic GuIdance workflOw

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Stefan Milosavljevic ◽  
Tony Kuo ◽  
Samuele Decarli ◽  
Lucas Mohn ◽  
Jun Sese ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
Sequence Similarity ◽  
Ground Truth ◽  
Whole Genome ◽  
Initial Dataset ◽  
Quality Checks ◽  
Living Organisms ◽  
Set Up

Abstract Background Whole genome duplication (WGD) events are common in the evolutionary history of many living organisms. For decades, researchers have been trying to understand the genetic and epigenetic impact of WGD and its underlying molecular mechanisms. Particular attention was given to allopolyploid study systems, species resulting from an hybridization event accompanied by WGD. Investigating the mechanisms behind the survival of a newly formed allopolyploid highlighted the key role of DNA methylation. With the improvement of high-throughput methods, such as whole genome bisulfite sequencing (WGBS), an opportunity opened to further understand the role of DNA methylation at a larger scale and higher resolution. However, only a few studies have applied WGBS to allopolyploids, which might be due to lack of genomic resources combined with a burdensome data analysis process. To overcome these problems, we developed the Automated Reproducible Polyploid EpiGenetic GuIdance workflOw (ARPEGGIO): the first workflow for the analysis of epigenetic data in polyploids. This workflow analyzes WGBS data from allopolyploid species via the genome assemblies of the allopolyploid’s parent species. ARPEGGIO utilizes an updated read classification algorithm (EAGLE-RC), to tackle the challenge of sequence similarity amongst parental genomes. ARPEGGIO offers automation, but more importantly, a complete set of analyses including spot checks starting from raw WGBS data: quality checks, trimming, alignment, methylation extraction, statistical analyses and downstream analyses. A full run of ARPEGGIO outputs a list of genes showing differential methylation. ARPEGGIO was made simple to set up, run and interpret, and its implementation ensures reproducibility by including both package management and containerization. Results We evaluated ARPEGGIO in two ways. First, we tested EAGLE-RC’s performance with publicly available datasets given a ground truth, and we show that EAGLE-RC decreases the error rate by 3 to 4 times compared to standard approaches. Second, using the same initial dataset, we show agreement between ARPEGGIO’s output and published results. Compared to other similar workflows, ARPEGGIO is the only one supporting polyploid data. Conclusions The goal of ARPEGGIO is to promote, support and improve polyploid research with a reproducible and automated set of analyses in a convenient implementation. ARPEGGIO is available at https://github.com/supermaxiste/ARPEGGIO.

scholarly journals ARPEGGIO: Automated Reproducible Polyploid EpiGenetic GuIdance workflOw

2020 ◽  
Author(s):  
Stefan Milosavljevic ◽  
Tony Kuo ◽  
Samuele Decarli ◽  
Lucas Mohn ◽  
Jun Sese ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
Sequence Similarity ◽  
Implementation Strategies ◽  
Ground Truth ◽  
Ease Of Use ◽  
Whole Genome ◽  
Quality Checks ◽  
Living Organisms

AbstractWhole genome duplication (WGD) events are common in the evolutionary history of many living organisms. For decades, researchers have been trying to understand the genetic and epigenetic impact of WGD and its underlying molecular mechanisms. Particular attention was given to allopolyploid study systems, species resulting from an hybridization event accompanied by WGD. Investigating the mechanisms behind the survival of a newly formed allopolyploid highlighted the key role of DNA methylation. With the improvement of high-throughput methods, such as whole genome bisulfite sequencing (WGBS), an opportunity opened to further understand the role of DNA methylation at a larger scale and higher resolution. However, only a few studies have applied WGBS to allopolyploids, which might be due to lack of genomic resources combined with a burdensome data analysis process. To overcome these problems, we developed the Automated Reproducible Polyploid EpiGenetic GuIdance workflOw (ARPEGGIO): the first workflow for the analysis of epigenetic data in polyploids. This workflow analyzes WGBS data from allopolyploid species via the genome assemblies of the allopolyploid’s parent species. ARPEGGIO utilizes an updated read classification algorithm (EAGLE-RC), to tackle the challenge of sequence similarity amongst parental genomes. ARPEGGIO offers automation, but more importantly, a complete set of analyses including spot checks starting from raw WGBS data: quality checks, trimming, alignment, methylation extraction, statistical analyses and downstream analyses. A full run of ARPEGGIO outputs a list of genes showing differential methylation. ARPEGGIO’s design focuses on ease of use and reproducibility. ARPEGGIO was made simple to set up, run and interpret, and its implementation includes both package management and containerization. Here we discuss all the steps, challenges and implementation strategies; example datasets are provided to show how to use ARPEGGIO. In addition, we also test EAGLE-RC with publicly available datasets given a ground truth, and we show that EAGLE-RC decreases the error rate by 3 to 4 times compared to standard approaches. The goal of ARPEGGIO is to promote, support and improve polyploid research with a reproducible and automated set of analyses in a convenient implementation.

scholarly journals DNA methylation regulates transcriptional homeostasis of algal endosymbiosis in the coral modelAiptasia

2017 ◽  
Author(s):  
Yong Li ◽  
Yi Jin Liew ◽  
Guoxin Cui ◽  
Maha J Cziesielski ◽  
Noura Zahran ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
Model System ◽  
Epigenetic Mechanism ◽  
Symbiotic Relationship ◽  
Epigenetic Mechanisms ◽  
Genome Wide ◽  
Spurious Transcription ◽  
Coral Reef Ecosystems

The symbiotic relationship between cnidarians and dinoflagellates is the cornerstone of coral reef ecosystems. Although research is focusing on the molecular mechanisms underlying this symbiosis, the role of epigenetic mechanisms, which have been implicated in transcriptional regulation and acclimation to environmental change, is unknown. To assess the role of DNA methylation in the cnidarian-dinoflagellate symbiosis, we analyzed genome-wide CpG methylation, histone associations, and transcriptomic states of symbiotic and aposymbiotic anemones in the model systemAiptasia. We find methylated genes are marked by histone H3K36me3 and show significant reduction of spurious transcription and transcriptional noise, revealing a role of DNA methylation in the maintenance of transcriptional homeostasis. Changes in DNA methylation and expression show enrichment for symbiosis-related processes such as immunity, apoptosis, phagocytosis recognition and phagosome formation, and unveil intricate interactions between the underlying pathways. Our results demonstrate that DNA methylation provides an epigenetic mechanism of transcriptional homeostasis during symbiosis.

scholarly journals Evolutionary and functional genomics of DNA methylation in maize domestication and improvement

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Gen Xu ◽  
Jing Lyu ◽  
Qing Li ◽  
Han Liu ◽  
Dafang Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Direct Selection ◽  
Whole Genome ◽  
Evolutionary Forces ◽  
Weak Evidence ◽  
Modern Maize ◽  
Genome Bisulfite Sequencing ◽  
Maize Domestication ◽  
Chromatin Feature

Abstract DNA methylation is a ubiquitous chromatin feature, present in 25% of cytosines in the maize genome, but variation and evolution of the methylation landscape during maize domestication remain largely unknown. Here, we leverage whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (WGBS) data on populations of modern maize, landrace, and teosinte (Zea mays ssp. parviglumis) to estimate epimutation rates and selection coefficients. We find weak evidence for direct selection on DNA methylation in any context, but thousands of differentially methylated regions (DMRs) are identified population-wide that are correlated with recent selection. For two trait-associated DMRs, vgt1-DMR and tb1-DMR, HiChIP data indicate that the interactive loops between DMRs and respective downstream genes are present in B73, a modern maize line, but absent in teosinte. Our results enable a better understanding of the evolutionary forces acting on patterns of DNA methylation and suggest a role of methylation variation in adaptive evolution.

scholarly journals Mosaic DNMT3A Germline Mutation As a Model for Mutant DNMT3A Competitive Advantage in the Blood Lineage

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 173-173
Author(s):  
Ayala Tovy ◽  
Hyun Jung Park ◽  
Jaime M. Reyes ◽  
Anna Guzman ◽  
Rachel E. Rau ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cell ◽  
Competitive Advantage ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Clonal Expansion ◽  
Whole Genome ◽  
Genomic Regions ◽  
The Impact ◽  
Mutant Cells

Abstract The DNA Methyltransferase 3A (DNMT3A) gene is recurrently mutated in a large spectrum of hematologic malignancies, including acute myeloid leukemia (AML). About 25% of adult AML patients carry mutations in DNMT3A and these mutations are generally associated with poor prognosis. DNMT3A mutations have been also associated with aged-related clonal hematopoiesis of indeterminate potential (CHIP). The high prevalence of DNMT3A somatic mutations in AML and CHIP implies that cells with mutated DNMT3A have a competitive advantage over wild-type (WT) cells, resulting in clonal expansion. However, the downstream molecular mechanisms that underlie this phenotype are not clear. Tatton-Brown-Raman syndrome (TBRS) is a rare genetic disorder caused by heterozygous germline mutations in DNMT3A, characterized by overgrowth and developmental delay. In one particular family, a group of 4 children out of 12 were diagnosed with TBRS and were found to be heterozygous carriers of DNMT3A-R771Q mutation (DNMT3AR771Q) inherited from their mosaic father. Thus, this individual provides a unique opportunity to study the long-term consequences of DNMT3A mutations, as he harbors both WT and mutant cells. From this mosaic individual, we generated lymphoblastoid cell lines (LCLs) from the peripheral blood (PB) and measured DNMT3AR771Q variant allele frequency (VAF) in the LCL pool as well as in PB, saliva and urine, all collected at the same time. Strikingly, DNMT3AR771Q VAF in the LCL pool and in PB was substantially higher than in saliva and urine (respectively 30%, 45%, 10%, 4%), implying that levels of DNMT3A mosaicism are tissue-specific and that cells with mutated DNMT3A tend to expand in the blood but not in epithelia (figure 1A and figure1B). One hypothesis for the prevalence of DNMT3A mutations in AML is that its loss reduces the effectiveness of DNA repair leading to increased mutational rates. In order to test this, we compared the mutational loads in individual LCL clones that were WT or DNMT3A mutant using whole genome sequencing. Surprisingly, no clear differences were observed between WT and DNMT3AR771Q mutant cells, indicating that clonal expansion is unlikely to be secondary to a general increase in mutational burden. To explore the impact of DNMT3AR771Q mutation on DNA methylation, we performed whole-genome bisulfite sequencing (WGBS) on two WT and two DNMT3AR771Q LCL clones. We identified ~31,500 differentially methylated regions (DMRs) between WT and mutant clones, with the majority of DMRs being hypomethylated. Hypomethylated DMRs were associated with gene regulatory regions, mainly promoters and enhancer regions. These data suggest that the DNMT3AR771Q mutation affects DNA methylation setting at genomic regions that can directly affect transcription. Canyons are large genomic regions of low methylation that often occur around master regulators such as homeobox-containing genes. We previously showed in mice that DNMT3A regulates DNA methylation at canyon edges, with loss of DNMT3A resulting in canyon expansion. In agreement, DNMT3AR771Q mutant clones displayed larger canyons, particularly at loci marked by H3K27Ac and H3K4me3 (figure 1C). Gene Ontology analysis of genes falling into expanded canyons showed a significant enrichment for leukemia and stem cell-related genes, including members of the HOX family. RNAseq analysis of DNMT3AR771Q mutant LCL clones confirmed the upregulation of key cancer-associated genes. These data suggest that DNMT3A mutations may promote clonal expansion through hypomethylation and overexpression of stem cell and cancer-related genes In conclusion, by comparing WT and DNMT3Amutant LCL clones generated from the same individual, we show that DNMT3A mutations lead to significant hypomethylation and overexpression of key cancer-associated genes. Further studies on specific target genes will reveal critical pathways responsible for the clonal expansion of cells with mutated DNMT3A, paving the way for the development of new therapeutic strategies for malignancies with mutated DNMT3A. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals BoostMe accurately predicts DNA methylation values in whole-genome bisulfite sequencing of multiple human tissues

2017 ◽  
Author(s):  
Luli S. Zou ◽  
Michael R. Erdos ◽  
D. Leland Taylor ◽  
Peter S. Chines ◽  
Arushi Varshney ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bisulfite Sequencing ◽  
Gradient Boosting ◽  
Whole Genome ◽  
Human Tissues ◽  
Whole Genome Bisulfite Sequencing ◽  
Genome Bisulfite Sequencing ◽  
Boosting Algorithm ◽  
Multiple Samples

AbstractBisulfite sequencing is widely employed to study the role of DNA methylation in disease; however, the data suffer from biases due to coverage depth variability. Here we describe BoostMe, a method for imputing low quality DNA methylation estimates within whole-genome bisulfite sequencing (WGBS) data. BoostMe uses a gradient boosting algorithm, XGBoost, and leverages information from multiple samples for prediction. We find that BoostMe outperforms existing algorithms in speed and accuracy when applied to WGBS of human tissues. We also show that imputation improves concordance between WGBS and the MethylationEPIC array at low WGBS depth, suggesting improved WGBS accuracy after imputation.

P–529 Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S D Persio ◽  
E Leitão ◽  
M Wöste ◽  
T Tekath ◽  
J F Cremers ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Small Sample ◽  
Global Dna Methylation ◽  
Obstructive Azoospermia ◽  
Whole Genome ◽  
Ploidy Analysis ◽  
Normal Spermatogenesis

Abstract Study question Do DNA methylation changes occur in testicular germ cells (TGCs) from patients with impaired spermatogenesis? Summary answer TGCs from men with cryptozoospermia exhibit altered DNA methylation levels at several genomic regions, many of which are associated with genes involved in spermatogenesis. What is known already In the last 15 years, several studies have described DNA methylation changes in sperm of infertile men. More recently, using whole genome bisulfite sequencing (WGBS) we were able to refute these findings by demonstrating that somatic DNA contamination and genetic variation confound methylation studies in swim-up purified sperm of severely oligozoospermic men. However, it remains unknown whether altered DNA methylation plays a role during the development of the germ cells in the testes of these patients. Study design, size, duration For identifying DNA methylation differences associated with impaired spermatogenesis, we compared the TGC methylomes of men with cryptozoospermia (CZ) and men with obstructive azoospermia (n = 4 each), who had normal spermatogenesis and served as controls (CTR). Study participants were selected among an age-matched cohort of 24 CTR and 10 CZ. The selection was based on similar composition of the TGC suspension evaluated by ploidy analysis and absence of somatic DNA. Participants/materials, setting, methods TGCs were isolated from biopsies after short-term cell culture. Presence of somatic DNA was evaluated by analyzing the DNA methylation levels of H19, MEST, DDX4 and XIST. WGBS was performed at ∼14× coverage. Bioinformatic tools were used to compare global DNA methylation levels, identify differentially methylated regions (DMRs) and functionally annotate the DMRs. Single-cell RNA sequencing (scRNA-seq) was used to associate the DNA methylation changes to gene expression. Main results and the role of chance We could not identify any difference in the global DNA methylation level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 DMRs, 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10–16). The DMRs are associated with 132 genes, 61 of which are known to be differentially expressed at various stages of spermatogenesis according to scRNA-seq studies. Almost all of the DMRs associated with the 61 genes are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As assessed by scRNA-seq, 13 DMR-associated genes, which were mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter was hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, most of which downregulated in CZ, germ cell-specific enhancers may be affected. Limitations, reasons for caution The small sample size constitutes a limitation of this study. Furthermore, even though the cellular composition of samples was similar by ploidy analysis, we cannot rule out that the observed DNA methylation changes might be due to differences in the relative proportion of different germ cell types. Wider implications of the findings: Impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome, which points to an important role of DNA methylation in normal spermatogenesis. The DNA methylation changes may contribute to premature abortion of spermatogenesis and therefore not appear in mature sperm. Trial registration number N/A

Semenogelin 1 Expression in Myeloma Cells: Interaction between DNA Methylation, MeCP2 Protein and Specific Cytokines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2242-2242
Author(s):  
Yana Zhang ◽  
Zhiqing Wang ◽  
Jian Zhang ◽  
Benjamin Farmer ◽  
Seah H. Lim
Keyword(s):  
Dna Methylation ◽  
Protein Binding ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Hematologic Malignancies ◽  
Cytosine Residue ◽  
Normal Tissues ◽  
Mecp2 Protein ◽  
Cpg Dinucleotide

Abstract Semenogelin (SEMG) 1 is a protein of semen coagulum with limited expression in normal tissues. It plays an important role in sperm clotting and is normally degraded into smaller fragments by prostate-specific antigen. The gene encoding SEMG 1 has been localized to the long arm of chromosome 20, a region of chromosome 20 that is frequently deleted in myeloproliferative diseases and myelodysplastic syndrome. We previously found SEMG 1 to be aberrantly expressed by tumor cells of hematologic malignancies, including multiple myeloma (MM). The aberrant expression of SEMG 1 in tumor cells of hematologic malignancies is associated in vivo with the generation of high titers IgG directed at SEMG 1 protein, suggesting the immunogenicity of the protein in the cancer-bearing patients. The combination of being immunogenic in cancer patients and limited expression in normal tissue expression makes SEMG 1 a potential candidate protein for tumor vaccines. In this study, we have set out to determine the molecular mechanisms associated with SEMG 1 expression in MM. Treatment of SEMG 1-positive MM cells with IL-4 and IL-6 resulted in the upregulation of SEMG 1 expression. In SEMG 1-negative MM cells, SEMG 1 expression could only be upregulated by IL-4 and IL-6 after pre-treatment with 5-azacytidine, suggesting that DNA methylation is likely the primary regulatory mechanism for SEMG 1 expression. Treatment of SEMG 1-negative MM cells induced SEMG 1 gene and protein expression. SEMG 1 promoter only has one CpG dinucleotide, located at position -11 of the gene. Bisulfite conversion and nucleotide sequencing was carried out on the genomic DNA from MM cells. MM cells that did not express SEMG 1 were 100% methylated. In contrast, 100% of the sequences obtained from SEMG 1-positive MM cells were unmethylated at the cytosine residue of the CpG dinucleotide. Induction of SEMG 1 expression by 5-azacytidine was associated with a decrease in the % of methylation of this cytosine residue, from 100% to 20%. These results, therefore, further implicate the role of DNA methylation in the primary regulation of SEMG 1 expression. Applying antibodies directed at MeCP2 in chromatin immunoprecipiation, MeCP2 protein binding to the SEMG 1 promoter sequence of MM cell lines and fresh MM cells was correlated to SEMG 1 gene silencing, suggesting the likely role of the MeCP2 protein in SEMG 1 gene repression. Further analysis by promoter truncation studies indicated the dependence of the promoter function on the sequence spanning the two putative GATA-1 binding sites within the gene. Using a reporter gene expression system, both IL-4 and IL-6 were found to upregulate SEMG 1 via their effect on the hypomethylated promoter gene. The effects of IL-4 and IL-6 on the function of the SEMG 1 promoter were dose dependent. In conclusion, the present study demonstrates that SEMG 1 expression in MM cells is regulated through the interaction between primary regulatory effect of promoter methylation, MeCP2 protein binding and the secondary effect of specific cytokines. Our findings provide insight into the molecular mechanisms affecting SEMG 1 expression and suggest the possible use of hypomethylating agents to upregulate SEMG 1 expression in tumor cells. Obviously, it remains to be determined whether or not there is a differential dose response among the different normal tissues in their sensitivity to the antigen-inducing effect of these agents.

scholarly journals Phosphatidic acid – a simple phospholipid with multiple faces

2018 ◽  
Vol 65 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Jolanta Zegarlinska ◽  
Magda Piaścik ◽  
Aleksander F Sikorski ◽  
Aleksander Czogalla
Keyword(s):  
Phosphatidic Acid ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Gene Expression Regulation ◽  
Large Degree ◽  
Cellular Lipids ◽  
Living Organisms ◽  
Protein Recruitment ◽  
Membrane Shape

Phosphatidic acid (PA) is the simplest glycerophospholipid naturally occurring in living organisms, and even though its content among other cellular lipids is minor, it is drawing more and more attention due to its multiple biological functions. PA is a precursor for other phospholipids, acts as a lipid second messenger and, due to its structural properties, is also a modulator of membrane shape. Although much is known about interaction of PA with its effectors, the molecular mechanisms remain unresolved to a large degree. Throughout many of the well-characterized PA cellular sensors, no conserved binding domain can be recognized. Moreover, not much is known about the cellular dynamics of PA and how it is distributed among subcellular compartments. Remarkably, PA can play distinct roles within each of these compartments. For example, in the nucleus it behaves as a mitogen, influencing gene expression regulation, and in the Golgi membrane it plays a role in membrane trafficking. Here we discuss how a biophysical experimental approach enabled PA behavior to be described in the context of a lipid bilayer and to what extent various physicochemical conditions may modulate the functional properties of the lipid. Understanding these aspects would help to unravel specific mechanisms of PA-driven membrane transformation and protein recruitment and thus would lead to a clearer picture of the biological role of PA.

scholarly journals The Role of fnx1, a Fission Yeast Multidrug Resistance Protein, in the Transition of Cells to a Quiescent G0 State

1998 ◽  
Vol 18 (9) ◽  
pp. 5239-5246 ◽  
Author(s):  
Krassen Dimitrov ◽  
Shelley Sazer
Keyword(s):  
Multidrug Resistance ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Sequence Similarity ◽  
Cell Communication ◽  
Major Facilitator Superfamily ◽  
Natural Habitats ◽  
Resistance Protein ◽  
Major Facilitator

ABSTRACT Most microorganisms live in conditions of nutrient limitation in their natural habitats. When exposed to these conditions they respond with physiological and morphological changes that enable them to survive. To obtain insights into the molecular mechanisms of this response a systematic genetic screen was performed to identify genes that when overexpressed can induce a starvation-like response in the yeast species Schizosaccharomyces pombe. One gene that meets these criteria, fnx1 +, induces, transcriptionally correlates with, and is required for the entry into the quiescent G0 state that is normally induced by nitrogen starvation. fnx1 + encodes a protein with sequence similarity to the proton-driven plasma membrane transporters from the multidrug resistance group of the major facilitator superfamily of proteins. We propose that fnx1 +plays a role in the entry into G0, possibly by facilitating the release of a signaling substance into the environment as a means of cell-to-cell communication.

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