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PubMed2005

MSRE-PCR for analysis of gene-specific DNA methylation.

Abnormal DNA methylation is observed in certain promoters of neoplastic cells, although the likelihood of methylation for each individual promoter varies. Simultaneous analysis of many promoters in the same sample can allow use of statistical methods for identification of neoplasia. Here we describe an assay for such analysis, based on digestion of genomic DNA with methylation-sensitive restriction enzyme and multiplexed PCR with gene-specific primers (MSRE-PCR). MSRE-PCR includes extensive digestion of genomic DNA (uncut fragments cannot be identified by PCR), can be applied to dilute samples (<1 pg/microl), requires limited amount of starting material (42 pg or genomic equivalent of seven cells) and can identify methylation in a heterogeneous mix containing <2% of cells with methylated fragments. When applied to 53 promoters of breast cancer cell lines MCF-7, MDA-MB-231 and T47D, MSRE-PCR correctly identified the methylation status of genes analyzed by other techniques. For selected genes results of MSRE-PCR were confirmed by methylation-specific PCR and bisulfite sequencing. The assay can be configured for any number of desired targets in any user-defined set of genes.

Breast Neoplasms↗
PubMed2005

Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level.

Promoters are molecular 'modules', which are controlled as individual entities yet are often analyzed by nuclease digestion methodologies which, a priori, destroy this modularity. About 40% of mammalian genes contain CpG islands in their promoters and exonic regions, which are normally unmethylated. We developed a footprinting strategy to map the chromatin structure at unmethylated CpG islands by treatment of isolated nuclei with the CpG-specific DNA methyltransferase SssI (M.SssI), followed by genomic bisulfite sequencing of individual progeny DNA molecules. This gave single molecule resolution over the promoter region and allowed for the physical linkage between binding sites on individual promoter molecules to be maintained. Comparison of the p16 promoters in two human cell lines, J82 and LD419, expressing the p16 gene at 25-fold different levels showed that the two cell lines contain remarkably different, heterogeneously positioned nucleosomes over the promoter region, which were not distinguishable by standard methods using nucleases. Our high resolution approach gives a 'digitized' visualization of each promoter providing information regarding nucleosome occupancy and may be utilized to define transcription factor binding and chromatin remodeling.

Base Sequence↗
PubMed2026

Methylation histology reveals the molecular mechanism by which red light-mediated DNA methylation delays leaf senescence in pak choi (Brassica rapa subsp. chinensis).

Leaf senescence is a key factor affecting the postharvest quality and shelf life of vegetables. The specific mechanisms by which light environment and DNA methylation mediate leaf senescence remain unclear. This study explored the molecular mechanism by which red light (RL) LED delays leaf senescence through DNA methylation in pak choi (Brassica rapa subsp. chinensis). In this study, RL treatment significantly suppressed leaf senescence in pak choi during postharvest storage and downregulated the expression of senescence-associated genes (SAGs). Experiments with methylation inhibitors confirmed its association with DNA methylation. Furthermore, whole-genome bisulfite sequencing revealed that during storage-induced senescence, pak choi exhibited significantly reduced methylation levels across its genome, particularly in promoter regions, and RL treatment reversed this effect. Furthermore, virus-induced gene silencing and overexpression experiments confirmed the central role of the demethylase BrDML3 (BraA01g004250.3.5C) in this process. Subsequently, a transcription factor under its regulation, BrNAC55 (BraA05g032630.3.5C), was identified and shown to promote leaf senescence by activating downstream SAGs (BrSGR1, BrPPH, BrSAUR36) to promote leaf senescence. In addition, this study found that BrNAC55 can also form a feedback loop with BrDML3, continuously amplifying leaf senescence. This study elucidates the mechanism by which RL-mediated DNA methylation delays leaf senescence, providing a foundation for postharvest preservation technologies.

DNA Methylation↗
PubMed2006

Phenobarbital induces progressive patterns of GC-rich and gene-specific altered DNA methylation in the liver of tumor-prone B6C3F1 mice.

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.

Animals↗
PubMed2003

Transcriptional silencing of geminiviral promoter-driven transgenes following homologous virus infection.

Promoters isolated from the Tomato leaf curl virus (TLCV) drive both constitutive and tissue-specific expression in transgenic tobacco. Following systemic TLCV infection of plants stably expressing TLCV promoter:GUS transgenes, transgene expression driven by all six TLCV promoters was silenced. Silencing in the TLCV coat protein promoter:GUS plants (V2:GUSdeltaC) was characterized in more detail. Transgene silencing observed in leaf, stem, and pre-anthesis floral tissue occurred with the continued replication of TLCV in host tissues. Infection of the V2:GUSdeltaC plants with heterologous geminiviruses did not result in transgene silencing, indicating that silencing was specifically associated with TLCV infection. Nuclear run-on assays indicated that silencing was due to the abolition of transcription from the V2:GUSdeltaC transgene. Bisulfite sequencing showed that silencing was associated with cytosine hypermethylation of the TLCV-derived promoter sequences of the V2:GUSdeltaC transgene. Progeny derived from V2:GUSdeltaC plants silenced by TLCV infection were analyzed. Transgene expression was silenced in progeny seedlings but was partially reactivated in the majority of plants by 75 days postgermination. Progeny seedlings treated with the nonmethylatable cytosine analog 5-azacytidine or the histone deacetylase inhibitor sodium butyrate exhibited partial reactivation of expression. This is the first report of the hypermethylation of a virus-derived transgene associated with a DNA virus infection.

Base Sequence↗
PubMed2004

Epigenetic and genomic imprinting analysis in nuclear transfer derived Bos gaurus/Bos taurus hybrid fetuses.

Somatic cell nuclear transfer (NT) in cattle is an inefficient process, whereby the production of calves is hindered by low pregnancy rates as well as fetal and placental abnormalities. Interspecies models have been previously used to facilitate the identification of single nucleotide polymorphisms (SNPs) within coding regions of genes to discriminate between parental alleles in the offspring. Here we report the use of a bovine interspecies model (Bos gaurus x Bos taurus) for the assessment and characterization of epigenetic modifications and genomic imprinting in Day 40-old female NT-derived fetuses and placenta. Analysis of NT and control pregnancies indicated disruption of genomic imprinting at the X inactivation-specific transcript (XIST) locus in the chorion, but not the fetus of clones, whereas proper allelic expression of the insulin-like growth factor II (IGF2) and gene trap locus 2 (GTL2) loci was maintained in both the fetus and placenta. Analysis of the XIST differentially methylated region (DMR) in clones indicated normal patterns of methylation; however, bisulfite sequencing of the satellite I repeat element and epidermal cytokeratin promoter indicated hypermethylation in the chorion of clones when compared with controls. No differences were detected in methylation levels in the fetus proper. These results indicate that the nuclear transfer process affects gene expression patterns in the trophectoderm- and inner cell mass-derived tissues to different extents.

Animals↗
PubMed2006

Bovine SNRPN methylation imprint in oocytes and day 17 in vitro-produced and somatic cell nuclear transfer embryos.

Findings from recent studies have suggested that the low survival rate of animals derived via somatic cell nuclear transfer (SCNT) may be in part due to epigenetic abnormalities brought about by this procedure. DNA methylation is an epigenetic modification of DNA that is implicated in the regulation of imprinted genes. Genes subject to genomic imprinting are expressed monoallelically in a parent of origin-dependent manner and are important for embryo growth, placental function, and neurobehavioral processes. The vast majority of imprinted genes have been studied in mice and humans. Herein, our objectives were to characterize the bovine SNRPN gene in gametes and to compare its methylation profile in in vivo-produced, in vitro-produced, and SCNT-derived Day 17 elongating embryos. A CpG island within the 5' region of SNRPN was identified and examined using bisulfite sequencing. SNRPN alleles were unmethylated in sperm, methylated in oocytes, and approximately 50% methylated in somatic samples. The examined SNRPN region appeared for the most part to be normally methylated in three in vivo-produced Day 17 embryos and in eight in vitro-produced Day 17 embryos examined, while alleles from Day 17 SCNT embryos were severely hypomethylated in seven of eight embryos. In this study, we showed that the SNRPN methylation profiles previously observed in mouse and human studies are also conserved in cattle. Moreover, SCNT-derived Day 17 elongating embryos were abnormally hypomethylated compared with in vivo-produced and in vitro-produced embryos, which in turn suggests that SCNT may lead to faulty reprogramming or maintenance of methylation imprints at this locus.

Animals↗
PubMed2006

Promoter hypomethylation of Toll-like receptor-2 gene is associated with increased proinflammatory response toward bacterial peptidoglycan in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) is the most common lethal inherited disorder caused by mutation in the gene encoding CF transmembrane regulator (CFTR). The clinical course of CF is characterized by recurrent pulmonary infections and chronic inflammation. Here, we show that toll-like receptor-2 (TLR2) expression and response were strongly enhanced in the human CF bronchial epithelial cell line, CFBE41o-. Treatment of the cells with 5-azacytidine decreased the promoter methylation within TLR2 proximal promoter and increased endogenous expression of TLR2 in non-CF 16HBE14o- cells, suggesting that TLR2 expression is epigenetically regulated by CpG methylation. Moreover, bisulfite sequence analysis revealed that TLR2 promoters were highly demethylated in CFBE41o- cells, implying that decreased methylation of the TLR2 promoter is responsible for CF-related up-regulation of TLR2. Finally, stable expression of WT-CFTR in CFBE41o- cells (CFBE41o-/WT-CFTR cells) reduced TLR2 expression and the response to its ligand peptidoglycan (PGN), implying a causal relationship between CFTR dysfunction and TLR2 up-regulation. Consistent with reduced expression of TLR2 in CFBE41o-/WT-CFTR cells, CpG methylation was increased in CFBE41o-/WT-CFTR cells. Taken together, our results demonstrate that TLR2 expression is epigenetically up-regulated in CF bronchial epithelial cells and suggest that TLR2 overexpression or prolonged activation of TLR2 signaling might be critical in CF pathogenesis.

Azacitidine↗
PubMed2000

Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.

Azacitidine↗
PubMed2003

Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene silencing by aberrant CpG methylation.

Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.

Base Sequence↗
PubMed2019

Genome-wide analysis of polymerase III-transcribed Alu elements suggests cell-type-specific enhancer function.

Alu elements are one of the most successful families of transposons in the human genome. A portion of Alu elements is transcribed by RNA Pol III, whereas the remaining ones are part of Pol II transcripts. Because Alu elements are highly repetitive, it has been difficult to identify the Pol III-transcribed elements and quantify their expression levels. In this study, we generated high-resolution, long-genomic-span RAMPAGE data in 155 biosamples all with matching RNA-seq data and built an atlas of 17,249 Pol III-transcribed Alu elements. We further performed an integrative analysis on the ChIP-seq data of 10 histone marks and hundreds of transcription factors, whole-genome bisulfite sequencing data, ChIA-PET data, and functional data in several biosamples, and our results revealed that although the human-specific Alu elements are transcriptionally repressed, the older, expressed Alu elements may be exapted by the human host to function as cell-type-specific enhancers for their nearby protein-coding genes.

Alu Elements↗
PubMed2006

Oocyte growth-dependent progression of maternal imprinting in mice.

In mammals, some genes categorized as imprinted genes are exclusively expressed either from maternal or paternal allele. This parental-origin-specific gene expression is regulated by epigenetic modification of DNA methylation in differentially methylated region (DMR), which is independently imposed during oogenesis and spermatogenesis. It is known that methylation of DMR in the female germ line is established during oocyte growth phase. However, the cause of the progression of methylation on DMR, due to either aging of mice or growth-size of oocyte was unclear up to now. Here, we analyzed the methylation of DMR for each eight imprinted genes (Igf2r, Lit1, Zac1, Snrpn, Peg1/Mest, Impact, Meg1/Grb10, and H19) by bisulfite sequencing methylation assay, using oocytes from 10 dpp (days post partum), 15 dpp, 20 dpp, and adult mice. To find whether the size of oocytes is the cause of methylation, above oocytes were classified into seven groups (each oocyte diameter ranging from 40 to 75 microm with intervals of 5 microm). The results from juvenile mice oocytes showed that DMR methylation progressed according to oocyte growth each imprinted gene. More than 85% of DMR methylation was achieved for both Igf2r, Zac1 & Lit1 with oocyte size of reaching 55 microm and Snrpn, Peg1/Mest, Impact, and Meg1/Grb10 with oocyte size of reaching 60 microm. Preferential methylation of maternal allele was observed in Zac1 and Peg1/Mest of juvenile oocytes and in Snrpn of juvenile and adult oocytes. The oocyte size-dependent-methylation progressed equally for all three different-age juvenile mice. The size-dependent-methylation was also recognized in the growing oocytes collected from adult mice, although the progress is slightly slower than that of juvenile mice. From these results, we concluded that DNA methylation is established with oocyte size dependent manner, not with aging of mice.

Alleles↗
PubMed2006

A differentially methylated region of the DAZ1 gene in spermatic and somatic cells.

AIM: To investigate the methylation status of the deleted in azoospermia 1 (DAZ1) gene promoter region in different cell types. METHODS: Using CpG island Searcher software, a CpG island was found in the promoter region of the DAZ1 gene. The methylation status of this region was analyzed in sperm and leukocytes by bisulfited sequencing. RESULTS: The methylation status of the CpG island in the DAZ1 gene promoter region differed in leukocytes and sperm: it was methylated in leukocytes, but unmethylated in sperm. CONCLUSION: A differentially methylated region of the DAZ1 gene exists in spermatic and somatic cells, suggesting that methylation of this region may regulate DAZ1 gene expression in different tissues.

Base Sequence↗
PubMed2026

Integrative Multi-Omics Analysis of Stem Growth Habit Divergence in Wild Soybean (Glycine soja).

Stem architecture is a major determinant of lodging resistance, biomass accumulation, and harvest efficiency in soybean. However, the molecular features associated with contrasting stem growth habits in wild soybean remain incompletely characterised. Here, we performed an integrated transcriptomic, metabolomic, and epigenomic analysis of stem growth-habit divergence in wild soybean, comparing the wild-type accession ZYD7068 with contrasting vining and erect mutant lines derived from carbon-ion beam mutagenesis. Pairwise transcriptomic comparisons identified between 20&#x2009;311 and 28&#x2009;705 differentially expressed genes per contrast, with a core set of 2672 genes consistently altered across the comparisons. Functional enrichment, gene set variation analysis, and gene set enrichment analysis converged on xylem and phloem pattern formation as a prominent molecular pathway associated with growth-habit divergence. Random forest analysis identified BBR-BPC and ARF transcription factor families as major molecular discriminators, while metabolomic profiling revealed distinct metabolic profiles involving amino-acid-derived and lipid-associated metabolites. Whole-genome bisulfite sequencing revealed context-specific DNA methylation differences, including substantial variation in CHG methylation among erect mutant lines. Integrated network and in silico perturbation analyses prioritised four candidate genes associated with vascular development for future functional validation. Together, these results provide a multi-layer molecular resource for investigating stem growth-habit divergence in G. soja and establish testable candidate pathways and genes for subsequent functional studies and soybean improvement.

glycine soja↗
PubMed2025

GhDMT7-mediated DNA methylation dynamics enhance starch and sucrose metabolism pathways to confer salt tolerance in cotton.

This study provides a comprehensive analysis of the impact of DNA methylation in cotton under salt stress conditions, elucidating its effects on gene expression and biological processes. Here, we determined the structures of the DNA methylation landscape across the cotton genome subjected to salt stress using whole-genome bisulfite sequencing (WGBS) and RNA-seq methodologies. We identified 4938 differentially methylated regions (DMRs) correlated with alterations in gene expression. Salt stress induced significant shifts in DNA methylation patterns, particularly in CHH contexts, suggesting context-dependent epigenetic regulation. DMRs were found to be implicated in diverse biological processes and pathways, encompassing protein metabolism, cellular homeostasis, starch and sucrose metabolism, and plant hormone signaling, all pivotal for cotton's adaptation to salt stress. Furthermore, RNA-seq analysis confirmed the impact of DNA methylation on gene expression, uncovering 9642 salt stress-responsive differentially expressed genes (DEGs). These DEGs exhibited enrichment in pathways such as carbohydrate metabolism, cell wall synthesis, and defense response, underscoring the intricate interplay between methylation and gene regulation in stress response. Moreover, the study investigated the role of the key DNA methyltransferase gene GhDMT7 in modulating cotton's response to salt stress, revealing that its downregulation enhanced cotton's salt tolerance, potentially attributed to decreased DNA methylation levels, reduced membrane damage, and enhanced antioxidant capacity. These findings elucidate the role of DNA methylation in abiotic stress resilience and provide insights for crop improvement.

Gossypium↗
PubMed2025

Beyond parental lines: multi-omics analyses reveal epigenetic and transcriptional mechanisms underlying heterosis in Oryza sativa &#xd7; Oryza rufipogon hybrids.

Heterosis, or hybrid vigor, refers to the superior phenotypes of a hybrid compared with their parents and is widely exploited in agriculture. Interspecific hybrids within the Oryza genus demonstrate significant potential for the systematic improvement of rice varieties. Nevertheless, the mechanistic basis underlying heterosis in interspecific Oryza hybrids remains poorly understood. Here, we systematically performed phenotypic characterization, whole-genome bisulfite sequencing, RNA sequencing, and small RNA profiling using Oryza sativa L. ssp. japonica cv. Nipponbare (NIP), Oryza rufipogon Griff. acc. CWR, and their resulting F1 hybrid (named as NC). NIP and CWR showed distinct phenotypic and molecular differences. The interspecific hybrid, NC, exhibited significant yield heterosis. In the hybrid, most epigenetic and transcriptional features displayed additive inheritance patterns relative to parental lines. Analysis revealed that domestication-selected genes maintained relatively low DNA methylation coupled with high expression levels in both hybrid and parental lines. Additionally, we identified that non-additive miRNAs were potentially involved in regulating fertility, cell growth, and cell division processes in the hybrid. A significant negative correlation was observed between DNA methylation level and gene expression. Functional enrichment analysis revealed that hybrid-MPV DEGs were significantly associated with flowering time regulation, carbohydrate metabolism, photosynthesis, protein phosphorylation, seed development, and defense responses. Through weighted gene co-expression network analysis, we identified 102 functional gene modules, six of which were significantly associated with yield-related heterosis. Collectively, our results provide a multi-omics framework for understanding interspecific hybridization between elite cultivars and wild rice relatives, highlighting CWR as an untapped genetic reservoir for rice improvement.

Oryza↗
PubMed2025

DNA methylation controls the expression of tanshinone synthesis genes and the tanshinone accumulation in Salvia&#xa0;miltiorrhiza and Salvia bowleyana.

DNA methylation plays pivotal roles in regulating gene expression and the secondary metabolism in plants. Salvia miltiorrhiza and Salvia bowleyana are traditional Chinese medicinal plants with roots enriched with tanshinone components. However, the regulatory mechanism of DNA methylation on tanshinone production remains elusive. Here, we analyzed 30-day-old hairy roots of S. miltiorrhiza and S. bowleyana using targeted high-performance liquid chromatography analysis and found significantly higher tanshinone content in S. miltiorrhiza. Whole-genome bisulfite sequencing revealed elevated DNA methylation levels in S. miltiorrhiza, potentially due to the upregulation of methylation-related genes, including DOMAINS REARRANGED METHYLTRANSFERASE 1 (DRM1), DECREASE IN DNA METHYLATION 1 (DDM1), CHROMOMETHYLASE 2 (CMT1), and CHROMOMETHYLASE 3 (CMT3), alongside the low expression of the demethylase gene REPRESSOR OF SILENCING 1 (ROS1) in S. miltiorrhiza. Additionally, four genes that are involved in tanshinone biosynthesis, including 1-DEOXY-D-XYLULOSE-5-PHOSPHATE REDUCTASE (DXS1), GERANYLGERANYL DIPHOSPHATE SYNTHASE (GGPPS2), 4-HYDROXY-3-METHYLBUT-2-ENYL PYROPHOSPHATE REDUCTASE (HDR2), and COPALYL PYROPHOSPHATE SYNTHASE (CPS3), showed lower methylation levels in the promoters of DXS1, GGPPS2, and CPS3 and a higher DNA methylation level in the gene body of HDR2 in S. miltiorrhiza, which may lead to their high expression and the accumulation of tanshinones. Consistently, overexpression of the SmCMT3 in S. miltiorrhiza significantly reduced the contents of cryptotanshinone, tanshinone I, and tanshinone IIA. Transcriptomic and methylome analyses confirmed that the expression levels of the tanshinone biosynthesis-related genes, including SmMK, SmCPS1, SmDXS2, and SmAACT1, were correlated with their promoter or gene body DNA methylation levels. Our findings reveal that DNA methylation critically regulates tanshinone biosynthesis in S. miltiorrhiza and S. bowleyana, offering valuable insights for breeding.

Abietanes↗
PubMed2004

Specific histone tail modification and not DNA methylation is a determinant of herpes simplex virus type 1 latent gene expression.

During herpes simplex virus type 1 (HSV-1) latency, gene expression is tightly repressed except for the latency-associated transcript (LAT). The mechanistic basis for this repression is unknown, but its global nature suggests regulation by an epigenetic mechanism such as DNA methylation. Previous work demonstrated that latent HSV-1 genomes are not extensively methylated, but these studies lacked the resolution to examine methylation of individual CpGs that could repress transcription from individual promoters during latency. To address this point, we employed established models to predict genomic regions with the highest probability of being methylated and, using bisulfite sequencing, analyzed the methylation profiles of these regions. We found no significant methylation of latent DNA isolated from mouse dorsal root ganglia in any of the regions examined, including the ICP4 and LAT promoters. This analysis indicates that methylation is unlikely to play a major role in regulating HSV-1 latent gene expression. Subsequently we focused on differential histone modification as another epigenetic mechanism that could regulate latent transcription. Chromatin immunoprecipitation analysis of the latent HSV-1 DNA repeat regions demonstrated that a portion of the LAT region is associated with histone H3 acetylated at lysines 9 and 14, consistent with a euchromatic and nonrepressed structure. In contrast, the chromatin associated with the HSV-1 DNA polymerase gene located in the unique long segment was not enriched in H3 acetylated at lysines 9 and 14, suggesting a transcriptionally inactive structure. These data suggest that histone composition may be a major regulatory determinant of HSV latency.

Acetylation↗