DNA Methylation Silences Exogenous Gene Expression in Transgenic Birch Progeny

The genetic stability of exogenous genes in the progeny of transgenic trees is extremely important in forest breeding; however, it remains largely unclear. We selected transgenic birch (Betula platyphylla) and its hybrid F1 progeny to investigate the expression stability and silencing mechanism of exogenous genes. We found that the exogenous genes of transgenic birch could be transmitted to their offspring through sexual reproduction. The exogenous genes were segregated during genetic transmission. The hybrid progeny of transgenic birch WT1×TP22 (184) and WT1×TP23 (212) showed higher Bgt expression and greater insect resistance than their parents. However, the hybrid progeny of transgenic birch TP23×TP49 (196) showed much lower Bgt expression, which was only 13.5% of the expression in its parents. To elucidate the mechanism underlying the variation in gene expression between the parents and progeny, we analyzed the methylation rates of Bgt in its promoter and coding regions. The hybrid progeny with normally expressed exogenous genes showed much lower methylation rates (0–29%) than the hybrid progeny with silenced exogenous genes (32.35–45.95%). These results suggest that transgene silencing in the progeny is mainly due to DNA methylation at cytosine residues. We further demonstrated that methylation in the promoter region, rather than in the coding region, leads to gene silencing. We also investigated the relative expression levels of three methyltransferase genes: BpCMT, BpDRM, and BpMET. The transgenic birch line 196 with a silenced Gus gene showed, respectively, 2.54, 9.92, and 4.54 times higher expression levels of BpCMT, BpDRM, and BpMET than its parents. These trends are consistent with and corroborate the high methylation levels of exogenous genes in the transgenic birch line 196. Therefore, our study suggests that DNA methylation in the promoter region leads to silencing of exogenous genes in transgenic progeny of birch.

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A Sparse and Low-Rank Regression Model for Identifying the Relationships Between DNA Methylation and Gene Expression Levels in Gastric Cancer and the Prediction of Prognosis

Genes ◽

10.3390/genes12060854 ◽

2021 ◽

Vol 12 (6) ◽

pp. 854

Author(s):

Yishu Wang ◽

Lingyun Xu ◽

Dongmei Ai

Keyword(s):

Gene Expression ◽

Gastric Cancer ◽

Dna Methylation ◽

Regression Model ◽

The Cancer Genome Atlas ◽

Low Rank ◽

Expression Levels ◽

Rank Regression ◽

Prediction Of Prognosis ◽

Gene Expression Levels

DNA methylation is an important regulator of gene expression that can influence tumor heterogeneity and shows weak and varying expression levels among different genes. Gastric cancer (GC) is a highly heterogeneous cancer of the digestive system with a high mortality rate worldwide. The heterogeneous subtypes of GC lead to different prognoses. In this study, we explored the relationships between DNA methylation and gene expression levels by introducing a sparse low-rank regression model based on a GC dataset with 375 tumor samples and 32 normal samples from The Cancer Genome Atlas database. Differences in the DNA methylation levels and sites were found to be associated with differences in the expressed genes related to GC development. Overall, 29 methylation-driven genes were found to be related to the GC subtypes, and in the prognostic model, we explored five prognoses related to the methylation sites. Finally, based on a low-rank matrix, seven subgroups were identified with different methylation statuses. These specific classifications based on DNA methylation levels may help to account for heterogeneity and aid in personalized treatments.

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Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

10.1101/2020.03.28.012518 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel M. Sapozhnikov ◽

Moshe Szyf

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Region ◽

Dna Methyltransferase ◽

Dna Demethylation ◽

New Method ◽

Inducible Promoters ◽

Specific Targeting ◽

Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

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Targeted modification of promoters

Genome editing for precision crop breeding - Burleigh Dodds Series in Agricultural Science ◽

10.19103/as.2020.0082.29 ◽

2021 ◽

pp. 247-278

Author(s):

Andika Gunadi ◽

◽

Ning Zhang ◽

John J. Finer ◽

◽

...

Keyword(s):

Gene Expression ◽

Genome Editing ◽

Promoter Region ◽

Regulatory Region ◽

Regulatory Elements ◽

Upstream Regulatory Region ◽

Precise Control ◽

Coding Regions ◽

Gene Coding ◽

Altered Regulation

Although most genome editing efforts focus on modifications to gene coding regions, this chapter emphasizes genome editing of the upstream regulatory regions. Thoughtful editing of the promoter region will ultimately lead to improved plants, modified for more precise control of the intensity and specificity of native gene expression. In this chapter, we present an overview of the promoter or upstream regulatory region of a gene, and describe how this sequence is defined and studied. We then describe how the composition and arrangements of cis-regulatory elements within the promoter and the leading intron associated with the promoter region have been studied using classical transgenic approaches to reveal what regulatory components might be suitable for genome editing approaches. Finally, we offer some suggestions for pursuit of promoter editing and gene expression modulation, which will eventually lead to modified plants with an altered regulation of native gene expression.

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Occupational exposure to gases/fumes and mineral dust affect DNA methylation levels of genes regulating expression

Human Molecular Genetics ◽

10.1093/hmg/ddz067 ◽

2019 ◽

Vol 28 (15) ◽

pp. 2477-2485 ◽

Cited By ~ 1

Author(s):

Diana A van der Plaat ◽

Judith M Vonk ◽

Natalie Terzikhan ◽

Kim de Jong ◽

Maaike de Vries ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Occupational Exposure ◽

Health Effects ◽

Mineral Dust ◽

Occupational Exposures ◽

Expression Levels ◽

Adverse Health Effects ◽

Never Smokers ◽

Gene Expression Levels

Abstract Many workers are daily exposed to occupational agents like gases/fumes, mineral dust or biological dust, which could induce adverse health effects. Epigenetic mechanisms, such as DNA methylation, have been suggested to play a role. We therefore aimed to identify differentially methylated regions (DMRs) upon occupational exposures in never-smokers and investigated if these DMRs associated with gene expression levels. To determine the effects of occupational exposures independent of smoking, 903 never-smokers of the LifeLines cohort study were included. We performed three genome-wide methylation analyses (Illumina 450 K), one per occupational exposure being gases/fumes, mineral dust and biological dust, using robust linear regression adjusted for appropriate confounders. DMRs were identified using comb-p in Python. Results were validated in the Rotterdam Study (233 never-smokers) and methylation-expression associations were assessed using Biobank-based Integrative Omics Study data (n = 2802). Of the total 21 significant DMRs, 14 DMRs were associated with gases/fumes and 7 with mineral dust. Three of these DMRs were associated with both exposures (RPLP1 and LINC02169 (2×)) and 11 DMRs were located within transcript start sites of gene expression regulating genes. We replicated two DMRs with gases/fumes (VTRNA2-1 and GNAS) and one with mineral dust (CCDC144NL). In addition, nine gases/fumes DMRs and six mineral dust DMRs significantly associated with gene expression levels. Our data suggest that occupational exposures may induce differential methylation of gene expression regulating genes and thereby may induce adverse health effects. Given the millions of workers that are exposed daily to occupational exposures, further studies on this epigenetic mechanism and health outcomes are warranted.

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The Expression Level of mRNA, Protein, and DNA Methylation Status of FOSL2 of Uyghur in XinJiang in Type 2 Diabetes

Journal of Diabetes Research ◽

10.1155/2016/5957404 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Jun Li ◽

Siyuan Li ◽

Ying Hu ◽

Guolei Cao ◽

Siyao Wang ◽

...

Keyword(s):

Gene Expression ◽

Type 2 Diabetes ◽

Dna Methylation ◽

Methylation Status ◽

Methylation Data ◽

Biochemical Indicators ◽

Expression Levels ◽

Protein Levels ◽

Cpg Sites

Objective. We investigated the expression levels of both FOSL2 mRNA and protein as well as evaluating DNA methylation in the blood of type 2 diabetes mellitus (T2DM) Uyghur patients from Xinjiang. This study also evaluated whether FOSL2 gene expression had demonstrated any associations with clinical and biochemical indicators of T2DM. Methods. One hundred Uyghur subjects where divided into two groups, T2DM and nonimpaired glucose tolerance (NGT) groups. DNA methylation of FOSL2 was also analyzed by MassARRAY Spectrometry and methylation data of individual units were generated by the EpiTyper v1.0.5 software. The expression levels of FOS-like antigen 2 (FOSL2) and the protein expression levels were analyzed. Results. Significant differences were observed in mRNA and protein levels when compared with the NGT group, while methylation rates of eight CpG units within the FOSL2 gene were higher in the T2DM group. Methylation of CpG sites was found to inversely correlate with expression of other markers. Conclusions. Results show that a correlation between mRNA, protein, and DNA methylation of FOSL2 gene exists among T2DM patients from Uyghur. FOSL2 protein and mRNA were downregulated and the DNA became hypermethylated, all of which may be involved in T2DM pathogenesis in this population.

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Enhancer variants associated with Alzheimer’s disease affect gene expression via chromatin looping

BMC Medical Genomics ◽

10.1186/s12920-019-0574-8 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 7

Author(s):

Masataka Kikuchi ◽

Norikazu Hara ◽

Mai Hasegawa ◽

Akinori Miyashita ◽

Ryozo Kuwano ◽

...

Keyword(s):

Gene Expression ◽

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Regulatory Elements ◽

Genome Wide Association Studies ◽

Expression Levels ◽

Chromatin Loops ◽

Coding Regions ◽

Affect Gene Expression ◽

Gene Expression Levels

Abstract Background Genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) that may be genetic factors underlying Alzheimer’s disease (AD). However, how these AD-associated SNPs (AD SNPs) contribute to the pathogenesis of this disease is poorly understood because most of them are located in non-coding regions, such as introns and intergenic regions. Previous studies reported that some disease-associated SNPs affect regulatory elements including enhancers. We hypothesized that non-coding AD SNPs are located in enhancers and affect gene expression levels via chromatin loops. Methods To characterize AD SNPs within non-coding regions, we extracted 406 AD SNPs with GWAS p-values of less than 1.00 × 10− 6 from the GWAS catalog database. Of these, we selected 392 SNPs within non-coding regions. Next, we checked whether those non-coding AD SNPs were located in enhancers that typically regulate gene expression levels using publicly available data for enhancers that were predicted in 127 human tissues or cell types. We sought expression quantitative trait locus (eQTL) genes affected by non-coding AD SNPs within enhancers because enhancers are regulatory elements that influence the gene expression levels. To elucidate how the non-coding AD SNPs within enhancers affect the gene expression levels, we identified chromatin-chromatin interactions by Hi-C experiments. Results We report the following findings: (1) nearly 30% of non-coding AD SNPs are located in enhancers; (2) eQTL genes affected by non-coding AD SNPs within enhancers are associated with amyloid beta clearance, synaptic transmission, and immune responses; (3) 95% of the AD SNPs located in enhancers co-localize with their eQTL genes in topologically associating domains suggesting that regulation may occur through chromatin higher-order structures; (4) rs1476679 spatially contacts the promoters of eQTL genes via CTCF-CTCF interactions; (5) the effect of other AD SNPs such as rs7364180 is likely to be, at least in part, indirect through regulation of transcription factors that in turn regulate AD associated genes. Conclusion Our results suggest that non-coding AD SNPs may affect the function of enhancers thereby influencing the expression levels of surrounding or distant genes via chromatin loops. This result may explain how some non-coding AD SNPs contribute to AD pathogenesis.

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A Genome-Wide Study of DNA Methylation Patterns and Gene Expression Levels in Multiple Human and Chimpanzee Tissues

PLoS Genetics ◽

10.1371/journal.pgen.1001316 ◽

2011 ◽

Vol 7 (2) ◽

pp. e1001316 ◽

Cited By ~ 142

Author(s):

Athma A. Pai ◽

Jordana T. Bell ◽

John C. Marioni ◽

Jonathan K. Pritchard ◽

Yoav Gilad

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Expression Levels ◽

Genome Wide ◽

A Genome ◽

Methylation Patterns ◽

Genome Wide Study ◽

Gene Expression Levels

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Study on DNA Methylation Status of WT1 Gene in Its Promoter Region in Hematologic Neoplasm Cell Lines.

Blood ◽

10.1182/blood.v106.11.4386.4386 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4386-4386

Author(s):

Ye Zhao ◽

Zi-xing Chen ◽

Shao-yan Hu ◽

Jian-nong Cen

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Cell Lines ◽

Promoter Region ◽

U937 Cell ◽

Methylation Status ◽

Gene Promoter ◽

Epigenetic Mechanism ◽

Wt1 Gene ◽

Gene Promoter Region

Abstract The methylation at CpG island in the promoter region of a gene is one of the important epigenetic mechanism which regulates the gene activity. To study the DNA methylation pattern of WT1 gene promoter region within hematologic neoplastic cell lines and its correlation with WT1 gene expression by using the PCR-based methods. RT-PCR and Methylation-specific PCR were performed to study the WT1 gene expression in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines and the DNA methylation status in promoter region of WT1 gene. After treatment of U937 cell line by 5-aza-CdR, a demethylation inducing agent, the changes of WT1 gene expression level and the methylation status in its promter region in U937 cells was determined. Our Results showed that HL-60, K562, KG-1, NB4, SHI-1 cell lines demonstrated higher level of WT1 expression, while extremely low level was found in 8226, Jurkat, Raji, U266 and U937. The DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cell lines. The WT1 gene expression in U937 was markedly enhanced after treatment with 5-aza-CdR in company with the decrease of methylated level and the increase of unmethylated level in its promoter region. These results indicate that modulation of the DNA methylation in WT1 promoter region is one of the epigenetic mechanisms to regulate its expression.

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Epigenetic Changes of the Cyp11a1 Promoter Region in Granulosa Cells Undergoing Luteinization During Ovulation in Female Rats

Endocrinology ◽

10.1210/en.2016-1264 ◽

2016 ◽

Vol 157 (9) ◽

pp. 3344-3354 ◽

Cited By ~ 14

Author(s):

Maki Okada ◽

Lifa Lee ◽

Ryo Maekawa ◽

Shun Sato ◽

Takuya Kajimura ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Granulosa Cells ◽

Chromatin Immunoprecipitation ◽

Promoter Region ◽

Binding Protein ◽

Female Rats ◽

Proximal Promoter ◽

Lh Surge ◽

Enhancer Binding Protein

The ovulatory LH surge induces rapid up-regulation of Cyp11a1 in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic controls including histone modifications and DNA methylation in the promoter region are associated with the rapid increase of Cyp11a1 gene expression after LH surge. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and 4 h and 12 h after human (h)CG injection. Cyp11a1 mRNA levels rapidly increased after hCG injection, reached a peak at 4 hours, and then remained elevated until 12 hours. DNA methylation status in the Cyp11a1 proximal promoter region was hypomethylated and did not change at any of the observed times after hCG injection. Chromatin immunoprecipitation assays revealed that the levels of trimethylation of lysine 4 on histone H3 (H3K4me3), an active mark for transcription, increased, whereas the levels of H3K9me3 and H3K27me3, which are marks associated with repression of transcription, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin immunoprecipitation assays also showed that the binding activity of CAATT/enhancer-binding protein-β to the Cyp11a1 proximal promoter increased after hCG injection. Luciferase assays revealed that the CAATT/enhancer-binding protein-β-binding site had transcriptional activity and contributed to basal and cAMP-induced Cyp11a1 expression. These results suggest that changes in histone modification and chromatin structure in the Cyp11a1 proximal promoter are involved in the rapid increase of Cyp11a1 gene expression in GCs undergoing luteinization during ovulation.

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