Gene Body Regions Research Articles

BIOM-13. COMBINED MGMT PROMOTER METHYLATION AND LOW GENE BODY METHYLATION IS A SUPERIOR PREDICTOR OF ALKYLATING AGENT RESPONSE THAN PROMOTER METHYLATION ALONE IN GLIOBLASTOMA

Abstract BACKGROUND The DNA methylation status of the promoter region of O-6-methylguanine-DNA methyltransferase (MGMT) is a critical predictor of temozolomide response in newly diagnosed glioblastoma. However, MGMT-STP27, the dominant method for MGMT promoter methylation determination from methylation array data, considers only methylation probes located in the promoter region. In this study, we propose a robust MGMT classifier that considers MGMT methylation probes in both the promoter and gene body regions. METHODS We leveraged glioblastoma DNA methylation and/or gene expression data published by the TCGA (N=312), GLASS (N=276), and Capper et al. (N=535). We correlated MGMT gene expression and MGMT probe methylation in datasets with paired expression and methylation data. We formulated an MGMT methylation score that measured the difference between MGMT promoter probe methylation and gene body probe methylation. We then compared the prognostic utility of this score with the MGMT-STP27 model on patients treated with upfront alkylating agents in multiple datasets in both univariate and multivariate analyses. RESULTS We found that MGMT expression was prognostic in TCGA (OS HR=1.5, p=0.027, PFS HR=1.9, p=0.001) and GLASS (OS HR=1.4, p=0.088, PFS HR=1.6, p=0.03) glioblastomas among patients treated with alkylating agents. MGMT expression was negatively correlated with MGMT promoter methylation and positively correlated with MGMT gene body methylation. We defined an MGMT score as the difference between the average MGMT DNA methylation in negatively correlated promoter probes and the average methylation in positively correlated gene body probes. This score was prognostic in the TCGA (OS HR=1.6, p=0.003, PFS HR=1.6, p=0.001) and GLASS (OS HR=3.1, p=0.01, PFS HR=3.9, p=0.004) and was also superior to the MGMT-STP27 classifiers in multivariate analyses (TCGA p=0.03). CONCLUSION Overall, we defined an MGMT methylation score that considers promoter and gene body methylation and may be a superior predictor of glioblastoma response to alkylating agents compared to MGMT-STP27.

Induction of FAM46C expression mediated by DNMT3A downregulation is involved in early-onset preeclampsia through gene body methylation

BackgroundAberrant methylation of genomic DNA has been found in preeclamptic placentas, which is characterized by elevated DNA methylation and hypermethylation of gene body regions, but the underlying mechanism is not yet fully understood. MethodsGlobal DNA methylation was assessed through ELISA and HPLC. The methylation sites were detected using the Illumina Human Methylation 450 K Microarray. The methylation level of FAM46C promoter and gene body was detected through the bisulfite sequencing. RNA-seq was utilized to investigate the mechanism by which DNMT3A and FAM46C mediate the migration and invasion of trophoblast cells. ResultsWe discovered that DNMT3A knockdown led to elevated levels of gene body methylation and FAM46C transcription. FAM46C downregulation completely rescued the suppressive effects caused by DNMT3A knockdown on the migration and invasion of trophoblast cells. Mechanistically, DNMT3A reduction led to an increase in the enrichment of DNMT3B and DNMT1 in the gene body region of FAM46C. The results of transcriptome sequencing showed that DNMT3A and FAM46C regulate the adhesion of trophoblast cells. Elevated expression of FAM46C and increased methylation levels within its gene body region were observed in extravillous trophoblast cells of early-onset preeclamptic placentas. ConclusionsDNMT3A-mediated aberrant FAM46C gene body methylation is relevant to the development of early-onset preeclampsia.

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock “Pd1” (Prunus tomentosa) on sweet cherry ‘Shuguang2’ scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock–scion interactions.

Intra species dissection of phytophthora capsici resistance in black pepper

IntroductionBlack pepper, a financially significant tropical crop, assumes a pivotal role in global agriculture for the major source of specie flavor. Nonetheless, the growth and productivity of black pepper face severe impediments due to the destructive pathogen Phytophthora capsici, ultimately resulting in black pepper blight. The dissecting for the genetic source of pathogen resistance for black pepper is beneficial for its global production. The genetic sources include the variations on gene coding sequences, transcription capabilities and epigenetic modifications, which exerts hierarchy of influences on plant defense against pathogen. However, the understanding of genetic source of disease resistance in black pepper remains limited. MethodsThe wild species Piper flaviflorum (P. flaviflorum, Pf) is known for blight resistance, while the cultivated species P. nigrum is susceptible. To dissecting the genetic sources of pathogen resistance for black pepper, the chromatin modification on H3K4me3 and transcriptome of black pepper species were profiled for genome wide comparative studies, applied with CUT&Tag and RNA sequencing technologies. ResultsThe intraspecies difference between P. flaviflorum and P. nigrum on gene body region led to coding variations on 5137 genes, including 359 gene with biotic stress responses and regulation. P. flaviflorum exhibited a more comprehensive resistance response to Phytophthora capsica in terms of transcriptome features. The pathogen responsive transcribing was significant associated with histone modification mark of H3K4me3 in black pepper. The collective data on variations of sequence, transcription activity and chromatin structure lead to an exclusive jasmonic acid-responsive pathway for disease resistance in P. flaviflorum was revealed. This research provides a comprehensive frame work to identify the fine genetic source for pathogen resistance from wild species of black pepper.

Decoding the Root Lignification Mechanism of Angelica sinensis through Genome-wide Methylation Analysis.

Angelica sinensis is a traditional Chinese herbal medicine with significant economic and medicinal value. However, early bolting and flowering can occur during the second year of the vegetative growth period, rendering the roots unviable for medicinal use and resulting in substantial economic losses. Consequently, the growing interest in studying the molecular mechanisms underlying early bolting or increased root lignification in A. sinensis. Here, we conducted whole-genome bisulfite sequencing and observed an increase in whole-genome DNA methylation levels on chromosomes after bolting in A. sinensis. Comparative analysis revealed methylation patterns in the upstream, gene body, and downstream regions in the context of CG, CHG, and CHH, suggesting a possible association between CHH-type methylation of promoters and phenylpropanoid biosynthesis. Furthermore, joint analysis of transcriptomic and methylomics data revealed a positive correlation between DNA methylation and gene expression. We identified the hyperDMR gene in the CHH background within the promoter region; this gene is also a key gene (AsCOMT1), exhibiting dual catalytic activity and facilitating the synthesis of both ferulic acid and lignin. Enzyme kinetic analysis demonstrated that AsCOMT1 preferentially catalyzes the synthesis of lignin monomer precursors. These findings highlight the important regulatory role of DNA methylation in bolting and the synthesis of secondary metabolites in A. sinensis, providing valuable insights into the underlying molecular mechanisms. Therefore, as DNA methylation plays an important regulatory role in A. sinensis bolting and secondary metabolite synthesis, it has potential significance in the analysis of the underlying molecular mechanism.

Epigenome-850K-wide profiling reveals peripheral blood differential methylation in term low birth weight.

Aim: We investigate the genome-wide DNA methylation (DNAm) patterns of term low birth weight (TLBW) neonates.Methods: In the discovery phase, we assayed 32 samples (TLBW/control:16/16) using the EPIC 850k BeadChip Array. Targeted pyrosequencing of in 60 samples (TLBW/control:28/32) using targeted pyrosequencing during the replication phase.Results: The 850K array identified TLBW-associated 144differentially methylated positions (DMPs) and 149DMRs. Nearly 77% DMPs exhibited hypomethylation, located in the opensea and gene body regions. The most significantly enriched pathway in KEGG is sphingolipid metabolism (hsa00600), and the genes GALC and SGMS1 related to this pathway both show hypomethylation.Conclusion: Our analysis provides evidence of genome-wide DNAm alterations in TLBW. Further investigations are needed to elucidate the functional significance of these DNAm changes.

Whole-genome DNA methylomes of Tree shrew brains reveal conserved and divergent roles of DNA methylation on sex chromosome regulation.

The tree shrew (Tupaia belangeri) is a promising emerging model organism in biomedical studies, notably due to their evolutionary proximity to primates. To enhance our understanding of how DNA methylation is implicated in regulation of gene expression and the X chromosome inactivation (XCI) in tree shrew brains, here we present their first genome-wide, single-base-resolution methylomes integrated with transcriptomes from prefrontal cortices. We discovered both divergent and conserved features of tree shrew DNA methylation compared to that of other mammals. DNA methylation levels of promoter and gene body regions are negatively correlated with gene expression, consistent with patterns in other mammalian brains studied. Comparing DNA methylation patterns of the female and male X chromosomes, we observed a clear and significant global reduction (hypomethylation) of DNA methylation across the entire X chromosome in females. Our data suggests that the female X hypomethylation does not directly contribute to the gene silencing of the inactivated X chromosome nor does it significantly drive sex-specific gene expression of tree shrews. However, we identified a putative regulatory region in the 5' end of the X inactive specific transcript (Xist) gene, a key gene for XCI, whose pattern of differential DNA methylation strongly relate to its differential expression between male and female tree shrews. We show that differential methylation of this region is conserved across different species. Moreover, we provide evidence suggesting that the observed difference between human and tree shrew X-linked promoter methylation is associated with the difference in genomic CpG contents. Our study offers novel information on genomic DNA methylation of tree shrews, as well as insights into the evolution of X chromosome regulation in mammals.

Integrated analysis of the DNA methylome and RNA transcriptome during the development of skeletal muscle in Duroc pigs.

Skeletal muscle development plays a crucial role in yield and quality of pork; however, this process is influenced by various factors. In this study, we employed whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing to comprehensively investigate the longissimus dorsi muscle (LDM), aiming to identify key genes that impact the growth and development of Duroc pigs with different average daily gains (ADGs). Eight pigs were selected and divided into two groups based on ADGs: H (774.89 g) group and L (658.77 g) group. Each pair of the H and L groups were half-siblings. The results of methylation sequencing revealed 2631 differentially methylated genes (DMGs) involved in metabolic processes, signalling, insulin secretion, and other biological activities. Furthermore, a joint analysis was conducted on these DMGs and the differentially expressed genes (DEGs) obtained from transcriptome sequencing of the same individual. This analysis identified 316 differentially methylated and differentially expressed genes (DMEGs), including 18 DMEGs in promoter regions and 294 DMEGs in gene body regions. Finally, LPAR1 and MEF2C were selected as candidate genes associated with muscle development. Bisulfite sequencing PCR (BSP) and quantitative real-time PCR (qRT-PCR) revealed that the promoter region of LPAR1 exhibited significantly lower methylation levels (P < 0.05) and greater expression levels (P < 0.05) in the H group than in the L group. Additionally, hypermethylation was observed in the gene body region of MEF2C, as was a low expression level, in the H group (P < 0.05). These results suggest that the differences in the ADGs of Duroc pigs fed the same diet may be influenced by the methylation levels and expression levels of genes related to skeletal muscle development.

ISWI chromatin remodeling complexes recruit NSD2 and H3K36me2 in pericentromeric heterochromatin.

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.

Ficus carica ERF12 improves fruit firmness at ripening

Fig fruit firmness decreases rapidly during ripening and after harvest, resulting in poor storability and transportation loss, which severely restricts development of the fresh fig industry. APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors are downstream components of the ethylene-signaling pathway that play crucial roles in quality formation during fruit ripening. In this study, Ficus carica (Fc) ERF12 was clustered in repressor subfamily VIII of ERFs through phylogenetic analysis, and further recruited by its two EAR motifs and expression pattern during fig ripening. DNA affinity purification sequencing analysis indicated that FcERF12 binds to the promoter or gene body regions of multiple ripening-related genes, including cell wall-modification genes FcPG, FcXTH and FcPME, and ethylene-biosynthesis genes FcACS and FcACO. Yeast two-hybrid assay demonstrated that FcERF12 interacts with TOPLESS (TPL) co-repressors FcTPL1, FcTPL4 and FcTPL5, and histone deacetylases FcHDA6 and FcHDA19; interaction with FcTPL4 and FcTPL5 relied on the C-terminal EAR motif. Overexpressing FcERF12 in tomato did not change fruit size or yield, but resulted in an 18.37% increment in fruit firmness and a 49.62% reduction in ethylene-release rate at fruit ripening, accompanied by a significant decrease in seed number per fruit. Transcriptomic analysis revealed downregulation of tomato cell wall-modification genes SlPL, SlEXP and SlPG, and ethylene-synthesis genes SlACO and SlACS. Metabolomic profiling identified 82 differentially accumulated flavonoid metabolites, 61 of them showing significantly decreased contents. Taken together, our results exhibit the negative regulatory role of FcERF12 in fig ethylene-signal transduction, providing new information on precise control of fruit firmness and other quality traits at ripening.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight

BackgroundThe Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes.ResultsTo provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate.ConclusionsFENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

Abstract 7019: Identifying cell free DNA methylation signatures in cerebrospinal fluids for the early detection of brain metastasis in non-small cell lung cancer

Abstract Brain metastasis (BM) is major complication for adult cancer patients. Up to 2% of all cancer patients develop BM. Non-small cell lung cancer (NSCLC) contributes up to 55% of all BM. Approximately 15-20% of NSCLC patients show metastases at diagnosis, and approximately half will develop BM during their clinical course. Even after treatments like surgery, radiation and immunotherapy, the prognosis is dismal with median overall survival less than 13 months. No standard screening or early diagnostic testing exists for BM now. There is an urgent need to identify genomic features that predispose towards brain metastasis - these biomarkers could be used for early detection and disease monitoring. The cfDNA methylation and fragmentation patterns provide potential epigenetic biomarkers for detecting cancer. These patterns are specific to cell/tissue origin and cancer types. DNA methylation refers to a chemical modification of a specific carbon with an added methyl group in the cytosine. Hypomethylation in the gene body and hypermethylation in the promoter region lead to cancer development. We hypothesize that there are specific cfDNA methylation and fragmentation signatures in CSF that reflect the epigenetic states of BMs, and cancer predisposition for brain metastasis. We applied a novel single molecule sequencing approach to study CSF cfDNA methylation of BM from NSCLC. Our cohort consisted of ten patients with NSCLC-BM (samples N=14) and eleven healthy controls (N=11). Briefly, extracted cfDNA was obtained from CSF samples. Multiplexed sequencing libraries were prepared following an optimized protocol, and sequenced on PromethION flow cell for 72hr by Oxford nanopore technologies. Data were processed with the onboard base and methylation calling, followed by our bioinformatic pipelines for cfDNA size and methylation analysis. Distinct methylation patterns were found on significant CpG sites (fdr-corrected p&amp;lt;0.01) between cancer patients and controls, in both gene-body and promoter region. Biological pathway analysis showed enrichment in essential functions involving PI3K, ERBB2, TNF-α signaling (p&amp;lt;0.03), all of which play a role in NSCLC. Enriched mono-nucleosome levels (p = 1.07e-03) and differentiated mono-/tri- nucleosome ratios (p = 1.34e-03) were found in cancer vs. controls. Overall, our results suggest that a combination of cfDNA methylation and fragmentation can detect NSCLC-BM. This study will benefit BM patients with early detection and better prognosis, and extend our understanding in brain metastases in NSCLC. Citation Format: Tianqi (Kiki) Chen, Billy T. Lau, Xiangqi Bai, Melanie Hayden Gephart, Hanlee P. Ji. Identifying cell free DNA methylation signatures in cerebrospinal fluids for the early detection of brain metastasis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7019.

The super-pangenome of Populus unveils genomic facets for its adaptation and diversification in widespread forest trees

Understanding the underlying mechanisms and links between genome evolution and adaptive innovations stands as a key goal in evolutionary studies. Poplars, among the world’s most widely distributed and cultivated trees, exhibit extensive phenotypic diversity and environmental adaptability. In this study, we present a genus-level super-pangenome comprising 19 Populus genomes, revealing the likely pivotal role of private genes in facilitating local environmental and climate adaptation. Through the integration of pangenomes with transcriptomes, methylomes, and chromatin accessibility mapping, we unveil that the evolutionary trajectories of pangenes and duplicated genes are closely linked to local genomic landscapes of regulatory and epigenetic architectures, notably CG methylation in gene-body regions. Further comparative genomic analyses have enabled the identification of 142 202 structural variants across species that intersect with a significant number of genes and contribute substantially to both phenotypic and adaptive divergence. We have experimentally validated a ∼180-bp presence/absence variant affecting the expression of the CUC2 gene, crucial for leaf serration formation. Finally, we developed a user-friendly web-based tool encompassing the multi-omics resources associated with the Populus super-pangenome (http://www.populus-superpangenome.com). Together, the present pioneering super-pangenome resource in forest trees not only aids in the advancement of breeding efforts of this globally important tree genus but also offers valuable insights into potential avenues for comprehending tree biology.

Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

The chromosome-scale genome and population genomics reveal the adaptative evolution of Populus pruinosa to desertification environment.

The Populus pruinosa is a relic plant that has managed to survive in extremely harsh desert environments. Owing to intensifying global warming and desertification, research into ecological adaptation and speciation of P. pruinosa has attracted considerable interest, but the lack of a chromosome-scale genome has limited adaptive evolution research. Here, a 521.09Mb chromosome-level reference genome of P. pruinosa was reported. Genome evolution and comparative genomic analysis revealed that tandemly duplicated genes and expanded gene families in P. pruinosa contributed to adaptability to extreme desert environments (especially high salinity and drought). The long terminal repeat retrotransposons (LTR-RTs) inserted genes in the gene body region might drive the adaptive evolution of P. pruinosa and species differentiation in saline-alkali desert environments. We recovered genetic differentiation in the populations of the northern Tianshan Mountain and southern Tianshan Mountain through whole-genome resequencing of 156 P. pruinosa individuals from 25 populations in China. Further analyses revealed that precipitation drove the local adaptation of P. pruinosa populations via some genetic sites, such as MAG2-interacting protein 2 (MIP2) and SET domain protein 25 (SDG25). This study will provide broad implications for adaptative evolution and population studies by integrating internal genetic and external environmental factors in P. pruinosa.

Protocol for Analyzing Epigenetic Regulation Mechanisms in Breast Cancer.

DNA methylation and gene expression are two critical aspects of the epigenetic landscape that contribute significantly to cancer pathogenesis. Analysis of aberrant genome-wide methylation patterns can provide insights into how these affect the cancer transcriptome and possible clinical implications for cancer diagnosis and treatment. The role of tumor suppressors and oncogenes is well known in tumorigenesis. Epigenetic alterations can significantly impact the expression and function of these critical genes, contributing to the initiation and progression of cancer. This protocol chapter presents a unified workflow to explore the role of DNA methylation in gene expression regulation in breast cancer by identifying differentially expressed genes whose promoter or gene body regions are differentially methylated using various Bioconductor packages in R environment. Functional enrichment analysis of these genes can help in understanding the mechanisms leading to tumorigenesis due to epigenetic alterations.

Prophylactic vitamin C supplementation regulates DNA demethylation to protect against cisplatin-induced acute kidney injury in mice

Cisplatin-induced acute kidney injury (AKI) restricts the use of cisplatin as a first-line chemotherapeutic agent. Our previous study showed that prophylactic vitamin C supplementation may act as an epigenetic modulator in alleviating cisplatin-induced AKI in mice. However, the targets of vitamin C and the mechanisms underlying the epigenetics changes remain largely unknown. Herein, whole-genome bisulfite sequencing and bulk RNA sequencing were performed on the kidney tissues of mice treated with cisplatin with prophylactic vitamin C supplementation (treatment mice) or phosphate-buffered saline (control mice) at 24 h after cisplatin treatment. Ascorbyl phosphate magnesium (APM), an oxidation-resistant vitamin C derivative, was found that led to global hypomethylation in the kidney tissue and regulated different functional genes in the promoter region and gene body region. Integrated evidence suggested that APM enhanced renal ion transport and metabolism, and reduced apoptosis and inflammation in the kidney tissues. Strikingly, Mapk15, Slc22a6, Cxcl5, and Cd44 were the potential targets of APM that conferred protection against cisplatin-induced AKI. Moreover, APM was found to be difficult to rescue cell proliferation and apoptosis caused by cisplatin in the Slc22a6 knockdown cell line. These results elucidate the mechanism by which vitamin C as an epigenetic regulator to protects against cisplatin-induced AKI and provides a new perspective and evidence support for controlling the disease process through regulating DNA methylation.

Analysis of Chromatin Accessibility and DNA Methylation to Reveal the Functions of Epigenetic Modifications in Cyprinus carpio Gonads.

Epigenetic modifications are critical in precisely regulating gene expression. The common carp (Cyprinus carpio) is an economically important fish species, and females exhibit faster growth rates than males. However, the studies related to epigenetic modifications in the common carp gonads are limited. In this study, we conducted the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Bisulfite sequencing (BS-seq) to explore the roles of epigenetic modifications in the common carp gonads. We identified 84,207 more accessible regions and 77,922 less accessible regions in ovaries compared to testes, and some sex-biased genes showed differential chromatin accessibility in their promoter regions, such as sox9a and zp3. Motif enrichment analysis showed that transcription factors (TFs) associated with embryonic development and cell proliferation were heavily enriched in ovaries, and the TFs Foxl2 and SF1 were only identified in ovaries. We also analyzed the possible regulations between chromatin accessibility and gene expression. By BS-seq, we identified 2087 promoter differentially methylated genes (promoter-DMGs) and 5264 gene body differentially methylated genes (genebody-DMGs) in CG contexts. These genebody-DMGs were significantly enriched in the Wnt signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway, indicating that methylation in gene body regions could play an essential role in sex maintenance, just like methylation in promoter regions. Combined with transcriptomes, we revealed that the expression of dmrtb1-like, spag6, and fels was negatively correlated with their methylation levels in promoter regions. Our study on the epigenetic modifications of gonads contributes to elucidating the molecular mechanism of sex differentiation and sex maintenance in the common carp.

Duck pan-genome reveals two transposon insertions caused bodyweight enlarging and white plumage phenotype formation during evolution.

Structural variations (SVs) are a major source of domestication and improvement traits. We present the first duck pan-genome constructed using five genome assemblies capturing ∼40.98 Mb new sequences. This pan-genome together with high-depth sequencing data (∼46.5×) identified 101,041 SVs, of which substantial proportions were derived from transposable element (TE) activity. Many TE-derived SVs anchoring in a gene body or regulatory region are linked to duck's domestication and improvement. By combining quantitative genetics with molecular experiments, we, for the first time, unraveled a 6945 bp Gypsy insertion as a functional mutation of the major gene IGF2BP1 associated with duck bodyweight. This Gypsy insertion, to our knowledge, explains the largest effect on bodyweight among avian species (27.61% of phenotypic variation). In addition, we also examined another 6634 bp Gypsy insertion in MITF intron, which triggers a novel transcript of MITF, thereby contributing to the development of white plumage. Our findings highlight the importance of using a pan-genome as a reference in genomics studies and illuminate the impact of transposons in trait formation and livestock breeding.

A telomere-to-telomere reference genome of ficus (Ficus hispida) provides new insights into sex determination.

A high-quality reference genome is indispensable for resolving biologically essential traits. Ficus hispida is a dioecious plant. A complete Ficus reference genome will be crucial for understanding their sex evolution and important biological characteristics, such as aerial roots, mutualistic symbiosis with ficus-wasps, and fruiting from old stems. Here, we generated a telomere-to-telomere (T2T) genome for F. hispida using PacBio HiFi and Oxford Nanopore Ultra-long sequencing technologies. The genome contiguity and completeness has shown improvement compared with the previously released genome, with the annotation of six centromeres and 28 telomeres. We have refined our previously reported 2-Mb male-specific region into a 7.2-Mb genomic region containing 51 newly predicted genes and candidate sex-determination genes AG2 and AG3. Many of these genes showed extremely low expression, likely attributed to hypermethylation in the gene body and promoter regions. Gene regulatory networks (GRNs) revealed that AG2 and AG3 are related to the regulation of stamen development in male flowers, while the AG1 gene is responsible for regulating female flowers' defense responses and secondary metabolite processes. Comparative analysis of GRNs showed that the NAC, WRKY, and MYB transcription factor families dominate the female GRN, whereas the MADS and MYB transcription factor families are prevalent in the male GRN.