Articles de revues : « Exonic DNA methylation »

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Emon, Isaac M., Ruaa Al-Qazazi, Michael J. Rauh et Stephen L. Archer. « The Role of Clonal Hematopoiesis of Indeterminant Potential and DNA (Cytosine-5)-Methyltransferase Dysregulation in Pulmonary Arterial Hypertension and Other Cardiovascular Diseases ». Cells 12, n^o 21 (26 octobre 2023) : 2528. http://dx.doi.org/10.3390/cells12212528.

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DNA methylation is an epigenetic mechanism that regulates gene expression without altering gene sequences in health and disease. DNA methyltransferases (DNMTs) are enzymes responsible for DNA methylation, and their dysregulation is both a pathogenic mechanism of disease and a therapeutic target. DNMTs change gene expression by methylating CpG islands within exonic and intergenic DNA regions, which typically reduces gene transcription. Initially, mutations in the DNMT genes and pathologic DNMT protein expression were found to cause hematologic diseases, like myeloproliferative disease and acute myeloid leukemia, but recently they have been shown to promote cardiovascular diseases, including coronary artery disease and pulmonary hypertension. We reviewed the regulation and functions of DNMTs, with an emphasis on somatic mutations in DNMT3A, a common cause of clonal hematopoiesis of indeterminant potential (CHIP) that may also be involved in the development of pulmonary arterial hypertension (PAH). Accumulation of somatic mutations in DNMT3A and other CHIP genes in hematopoietic cells and cardiovascular tissues creates an inflammatory environment that promotes cardiopulmonary diseases, even in the absence of hematologic disease. This review summarized the current understanding of the roles of DNMTs in maintenance and de novo methylation that contribute to the pathogenesis of cardiovascular diseases, including PAH.

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Anastasiadou, Christina, Andigoni Malousi, Nicos Maglaveras et Sofia Kouidou. « Human Epigenome Data Reveal Increased CpG Methylation in Alternatively Spliced Sites and Putative Exonic Splicing Enhancers ». DNA and Cell Biology 30, n^o 5 (mai 2011) : 267–75. http://dx.doi.org/10.1089/dna.2010.1094.

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Chen, Xiaona, Xinyu Duan, Qingqing Chong, Chunqing Li, Heng Xiao et Shanyuan Chen. « Genome-Wide DNA Methylation Differences between Bos indicus and Bos taurus ». Animals 13, n^o 2 (5 janvier 2023) : 203. http://dx.doi.org/10.3390/ani13020203.

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Disease risk is a persistent problem in domestic cattle farming, while economic traits are the main concern. This study aimed to reveal the epigenetic basis for differences between zebu (Bos indicus) and taurine cattle (Bos taurus) in disease, disease resistance, and economic traits, and provide a theoretical basis for the genetic improvement of domestic cattle. In this study, whole genome bisulfite sequencing (WGBS) was used to analyze the whole-genome methylation of spleen and liver samples from Yunnan zebu and Holstein cattle. In the genome-wide methylation pattern analysis, it was found that the methylation pattern of all samples was dominated by the CG type, which accounted for >94.9%. The DNA methylation levels of different functional regions and transcriptional elements in the CG background varied widely. However, the methylation levels of different samples in the same functional regions or transcriptional elements did not differ significantly. In addition, we identified a large number of differentially methylation region (DMR) in both the spleen and liver groups, of which 4713 and 4663 were annotated to functional elements, and most of them were annotated to the intronic and exonic regions of genes. GO and KEGG functional analysis of the same differentially methylation region (DMG) in the spleen and liver groups revealed that significantly enriched pathways were involved in neurological, disease, and growth functions. As a result of the results of DMR localization, we screened six genes (DNM3, INPP4B, PLD, PCYT1B, KCNN2, and SLIT3) that were tissue-specific candidates for economic traits, disease, and disease resistance in Yunnan zebu. In this study, DNA methylation was used to construct links between genotypes and phenotypes in domestic cattle, providing useful information for further screening of epigenetic molecular markers in zebu and taurine cattle.

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Park, Jee-Soo, Yun-Hee Shin et Young-Doo Park. « DNA Methylation Level Changes in Transgenic Chinese Cabbage (Brassica rapa ssp. pekinensis) Plants and Their Effects on Corresponding Gene Expression Patterns ». Genes 12, n^o 10 (30 septembre 2021) : 1563. http://dx.doi.org/10.3390/genes12101563.

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Plant tissue culture is an in vitro technique used to manipulate cells, tissues, or organs, and plays an important role in genetic transformation. However, plants cultured in vitro often exhibit unintended genetic and epigenetic variations. Since it is important to secure the stability of endogenous and exogenous gene expressions in transgenic plants, it is preferable to avoid the occurrence of such variations. In this study, we focused on epigenetic variations, exclusively on methylation level changes of DNA, in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) plants. To detect these methylation level changes of DNA, bisulfite sequencing was performed and the obtained sequences were compared with the ‘CT001’ reference genome. Differentially methylated regions (DMRs) of DNA between the non-transgenic and transgenic lines were detected by bisulfite sequencing, and ten DMRs located in exonic regions were identified. The regions with methylation variations that were inherited and consistently maintained in the next generation lines were selected and validated. We also analyzed the relationship between methylation status and expression levels of transformant-conserved DMR (TCD) genes by quantitative reverse transcription-PCR. These results suggested that the changes in methylation levels of these DMRs might have been related to the plant transformation process, affecting subsequent gene expression. Our findings can be used in fundamental research on methylation variations in transgenic plants and suggest that these variations affect the expression of the associated genes.

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Yu, Xiying, Ying Teng, Xingran Jiang, Hui Yuan et Wei Jiang. « Genome-Wide DNA Methylation Pattern of Cancer Stem Cells in Esophageal Cancer ». Technology in Cancer Research & ; Treatment 19 (1 janvier 2020) : 153303382098379. http://dx.doi.org/10.1177/1533033820983793.

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Background: Cancer stem cells (CSCs) are considered the main cause of cancer recurrence and metastasis, and DNA methylation is involved in the maintenance of CSCs. However, the methylation profile of esophageal CSCs remains unknown. Methods: Side population (SP) cells were isolated from esophageal squamous cell carcinoma (ESCC) cell lines KYSE150 and EC109. Sphere-forming cells were collected from human primary esophageal cancer cells. SP cells and sphere-forming cells were used as substitutes for cancer stem-like cells. We investigated the genome-wide DNA methylation profile in esophageal cancer stem-like cells using reduced representation bisulfite sequencing (RRBS). Results: Methylated cytosine (mC) was found mostly in CpG dinucleotides, located mostly in the intronic, intergenic, and exonic regions. Forty intersected differentially methylated regions (DMRs) were identified in these 3 groups of samples. Thirteen differentially methylated genes with the same alteration trend were detected; these included OTX1, SPACA1, CD163L1, ST8SIA2, TECR, CADM3, GRM1, LRRK1, CHSY1, PROKR2, LINC00658, LOC100506688, and NKD2. DMRs covering ST8SIA2 and GRM1 were located in exons. These differentially methylated genes were involved in 10 categories of biological processes and 3 cell signaling pathways. Conclusions: When compared to non-CSCs, cancer stem-like cells have a differential methylation status, which provides an important biological base for understanding esophageal CSCs and developing therapeutic targets for esophageal cancer.

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Xiao, Chunlin, et Valerie Schneider. « Abstract 3743 : Genome-wide profiling of DNA N6-methylation from a breast cancer and a matched normal cell lines ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 3743. http://dx.doi.org/10.1158/1538-7445.am2022-3743.

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Abstract DNA N6-methyladenine (m6A) modification has been found widely presented in the human genome, and genome-wide DNA methylation profiling in cancer may reveal epigenetic signatures with significant clinical outcomes. Whole genome sequencing data from PacBio single-molecule real-time (SMRT) system provides signals for identifying the presence of m6A in human genomic DNA. We identified 343,199 m6A modification sites with an average density of 122 per Mb in the HCC1395 breast cancer cell line, whereas 722,303 m6A modification sites with average density of 257 per Mb were observed in a matched normal HCC1395BL cell line, meaning that the total number and the average density of m6A methylation sites in the cancer cell line were reduced more than 50% than that in normal cell line. Only small fraction of the methylation sites was found to be shared between the two cell lines, indicating that significant de-methylation (loss) and new methylation (gain) events occurred in HCC1395 cancer cells. A broad distribution of m6A methylation sites across autosomal chromosomes was observed, but the density of m6A methylation sites on chromosome X was extremely low for both HCC1395 and HCC1395BL cell lines. In contrast, the m6A densities on chromosome 7 and chromosome 22, particularly on their q-arms, from HCC1395 were substantially higher (hypermethylation) than other autosomal chromosomes, suggesting that copy number variations may be associated with these chromosomal regions. Most of the m6A methylation sites were located in intergenic and intronic regions, whereas only about 2~3% of the m6A methylation sites were situated in exonic regions, and 12% on ncRNAs. Understanding the genome-wide distinction of DNA methylation between cancer and matched normal cell lines makes it possible to ask more targeted questions and further investigate the role of methylation in cancer. Citation Format: Chunlin Xiao, Valerie Schneider. Genome-wide profiling of DNA N6-methylation from a breast cancer and a matched normal cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3743.

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Verma, Pratima, Amrita Singh, Supriya Purru, Kangila Venkataramana Bhat et Suman Lakhanpaul. « Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.) ». Biology 11, n^o 7 (23 juin 2022) : 954. http://dx.doi.org/10.3390/biology11070954.

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Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.

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Nishida, Hiromi. « Nucleosome Positioning ». ISRN Molecular Biology 2012 (15 octobre 2012) : 1–5. http://dx.doi.org/10.5402/2012/245706.

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Nucleosome positioning is not only related to genomic DNA compaction but also to other biological functions. After the chromatin is digested by micrococcal nuclease, nucleosomal (nucleosome-bound) DNA fragments can be sequenced and mapped on the genomic DNA sequence. Due to the development of modern DNA sequencing technology, genome-wide nucleosome mapping has been performed in a wide range of eukaryotic species. Comparative analyses of the nucleosome positions have revealed that the nucleosome is more frequently formed in exonic than intronic regions, and that most of transcription start and translation (or transcription) end sites are located in nucleosome linker DNA regions, indicating that nucleosome positioning influences transcription initiation, transcription termination, and gene splicing. In addition, nucleosomal DNA contains guanine and cytosine (G + C)-rich sequences and a high level of cytosine methylation. Thus, the nucleosome positioning system has been conserved during eukaryotic evolution.

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Claus, Rainer, Manfred Fliegauf, Michael Stock, Jesus Duque, Mateusz Kolanczyk et Michael Lübbert. « AML1/ETO-Mediated Lysozyme Repression Is Independently Relieved by Inhibitors of DNA Methylation and Histone Deacetylation. » Blood 108, n^o 11 (16 novembre 2006) : 4310. http://dx.doi.org/10.1182/blood.v108.11.4310.4310.

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Abstract The human lysozyme (LZM) gene, a marker gene for myeloid-specific development, is highly methylated in immature myeloid and in non-myeloid cells (all LZM-negative), and unmethylated in LZM-expressing mature phagocyte cells. Thus this gene provides an excellent model for investigating differentation-associated DNA methylation changes during myelopoiesis. There is now increasing evidence that LZM (containing five perfect consensus binding sites for AML1/RUNX1 in its 5′ region) is repressed by the AML1/ETO chimeric transcription factor (Fliegauf et al, Oncogene 23:9070–81, 2004), and this repression can be relieved by siRNA-mediated AML1/ETO depletion in AML1/ETO-positive Kasumi-1 cells (Dunne et al., Oncogene, 2006). Recently, AML1/ETO has also been implicated in gene-specific epigenetic repression of interleukin-3 (Liu et al, Cancer Res 65, 1277–84, 2005). By extensive methylation analyses of the LZM gene including bisulfite sequencing, we now demonstrate marked demethylation in both the CpG-poor 5′ region and the exonic CpG island after treatment of Kasumi-1 cells with non-cytotoxic concentrations of the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-azaCdR), which was not associated with cellular differentiation. By Northern blot analysis, LZM mRNA levels in Kasumi-1 cells but not in AML1/ETO-negative HL-60 and U-937 cell lines were specifically and independently upregulated upon treatment with 5-azaCdR and, to a lesser extent, with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). Combined treatment with subliminal concentrations of 5-azaCdR and TSA applied in different schedules did not reveal synergistic effects on LZM transcription. Relative chromatin accessibility of the LZM 5′ region, as detected by “MspI protection” assay, and associated with partial demethylation in several myeloid cell lines, was increased in Kasumi-1 with 5-azaCdR-induced further DNA demethylation, but not by TSA. As shown by chromatin immunoprecitation, TSA increased the acetylation of histones H3 and H4 both in the 5′ flanking region and exonic CpG island. In a U-937 inducible model, antagonization of AML1/ETO-mediated repression of LZM was achieved by TSA, implying that the histone deacetylation in this region of the human LZM gene is mediated by AML1/ETO protein. In conclusion, we demonstrate functional interactions between DNA methylation and histone modifications in mediating LZM gene repression which implicate AML1/ETO as one component involved in local chromatin remodelling. Interestingly, inhibitors of DNA methylation and histone deacetylation independently relieve repression of this CpG-poor gene in AML1/ETO-positive cells.

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Ackah, Michael, Liangliang Guo, Shaocong Li, Xin Jin, Charles Asakiya, Evans Tawiah Aboagye, Feng Yuan et al. « DNA Methylation Changes and Its Associated Genes in Mulberry (Morus alba L.) Yu-711 Response to Drought Stress Using MethylRAD Sequencing ». Plants 11, n^o 2 (12 janvier 2022) : 190. http://dx.doi.org/10.3390/plants11020190.

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Drought stress remains one of the most detrimental environmental cues affecting plant growth and survival. In this work, the DNA methylome changes in mulberry leaves under drought stress (EG) and control (CK) and their impact on gene regulation were investigated by MethylRAD sequencing. The results show 138,464 (37.37%) and 56,241 (28.81%) methylation at the CG and CWG sites (W = A or T), respectively, in the mulberry genome between drought stress and control. The distribution of the methylome was prevalent in the intergenic, exonic, intronic and downstream regions of the mulberry plant genome. In addition, we discovered 170 DMGs (129 in CG sites and 41 in CWG sites) and 581 DMS (413 in CG sites and 168 in CWG sites). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicates that phenylpropanoid biosynthesis, spliceosome, amino acid biosynthesis, carbon metabolism, RNA transport, plant hormone, signal transduction pathways, and quorum sensing play a crucial role in mulberry response to drought stress. Furthermore, the qRT-PCR analysis indicates that the selected 23 genes enriched in the KEGG pathways are differentially expressed, and 86.96% of the genes share downregulated methylation and 13.04% share upregulation methylation status, indicating the complex link between DNA methylation and gene regulation. This study serves as fundamentals in discovering the epigenomic status and the pathways that will significantly enhance mulberry breeding for adaptation to a wide range of environments.

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Epstein, Richard J., Frank P. Y. Lin, Robert A. Brink et James Blackburn. « Synonymous alterations of cancer-associated Trp53 CpG mutational hotspots cause fatal developmental jaw malocclusions but no tumors in knock-in mice ». PLOS ONE 18, n^o 4 (13 avril 2023) : e0284327. http://dx.doi.org/10.1371/journal.pone.0284327.

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Intragenic CpG dinucleotides are tightly conserved in evolution yet are also vulnerable to methylation-dependent mutation, raising the question as to why these functionally critical sites have not been deselected by more stable coding sequences. We previously showed in cell lines that altered exonic CpG methylation can modify promoter start sites, and hence protein isoform expression, for the human TP53 tumor suppressor gene. Here we extend this work to the in vivo setting by testing whether synonymous germline modifications of exonic CpG sites affect murine development, fertility, longevity, or cancer incidence. We substituted the DNA-binding exons 5–8 of Trp53, the mouse ortholog of human TP53, with variant-CpG (either CpG-depleted or -enriched) sequences predicted to encode the normal p53 amino acid sequence; a control construct was also created in which all non-CpG sites were synonymously substituted. Homozygous Trp53-null mice were the only genotype to develop tumors. Mice with variant-CpG Trp53 sequences remained tumor-free, but were uniquely prone to dental anomalies causing jaw malocclusion (p < .0001). Since the latter phenotype also characterises murine Rett syndrome due to dysfunction of the trans-repressive MeCP2 methyl-CpG-binding protein, we hypothesise that CpG sites may exert non-coding phenotypic effects via pre-translational cis-interactions of 5-methylcytosine with methyl-binding proteins which regulate mRNA transcript initiation, expression or splicing, although direct effects on mRNA structure or translation are also possible.

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Kolb, Kathleen Liedtke, Ana Luiza Sprotte Mira, Eduardo Delabio Auer, Isabela Dall’Oglio Bucco, Carla Eduarda de Lima e Silva, Priscila Ianzen dos Santos, Valéria Bumiller-Bini Hoch et al. « Glucocorticoid Receptor Gene (NR3C1) Polymorphisms and Metabolic Syndrome : Insights from the Mennonite Population ». Genes 14, n^o 9 (15 septembre 2023) : 1805. http://dx.doi.org/10.3390/genes14091805.

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The regulation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with polymorphisms and the methylation degree of the glucocorticoid receptor gene (NR3C1) and is potentially involved in the development of metabolic syndrome (MetS). In order to evaluate the association between MetS with the polymorphisms, methylation, and gene expression of the NR3C1 in the genetically isolated Brazilian Mennonite population, we genotyped 20 NR3C1 polymorphisms in 74 affected (MetS) and 138 unaffected individuals without affected first-degree relatives (Co), using exome sequencing, as well as five variants from non-exonic regions, in 70 MetS and 166 Co, using mass spectrometry. The methylation levels of 11 1F CpG sites were quantified using pyrosequencing (66 MetS and 141 Co), and the NR3C1 expression was evaluated via RT-qPCR (14 MetS and 25 Co). Age, physical activity, and family environment during childhood were associated with MetS. Susceptibility to MetS, independent of these factors, was associated with homozygosity for rs10482605*C (OR = 4.74, pcorr = 0.024) and the haplotype containing TTCGTTGATT (rs3806855*T_ rs3806854*T_rs10482605*C_rs10482614*G_rs6188*T_rs258813*T_rs33944801*G_rs34176759*A_rs17209258*T_rs6196*T, OR = 4.74, pcorr = 0.048), as well as for the CCT haplotype (rs41423247*C_ rs6877893*C_rs258763*T), OR = 6.02, pcorr = 0.030), but not to the differences in methylation or gene expression. Thus, NR3C1 polymorphisms seem to modulate the susceptibility to MetS in Mennonites, independently of lifestyle and early childhood events, and their role seems to be unrelated to DNA methylation and gene expression.

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Geng, Huimin, Mignon L. Loh, Richard C. Harvey, I.-Ming Chen, Meenakshi Devidas, Tanja M. Davidsen, Jaime M. Guidry Auvil et al. « Genome-Wide DNA Methylation Analysis Reveals Biological and Clinical Insights In Relapsed Childhood Acute Lymphoblastic Leukemia : A Report From The COG ALL Target Project ». Blood 122, n^o 21 (15 novembre 2013) : 3736. http://dx.doi.org/10.1182/blood.v122.21.3736.3736.

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Abstract Although survival of children with B-cell acute lymphoblastic leukemia (B-ALL) has improved substantially over time, 15% to 20% of patients will relapse, and most of those who experience a bone marrow relapse will die. A better understanding of genetic and epigenetic aberrations in relapsed ALL will facilitate new strategies for risk stratification and targeted therapy. In this collaborative study with the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project, we performed high resolution genome-wide DNA methylation profiling using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) array on a total of 178 (110 diagnosis, 68 relapse) leukemia samples from 111 patients with childhood B-ALL enrolled on the Children’s Oncology Group (COG) clinical trials who experienced relapsed, and 12 normal preB samples isolated from the bone marrows of 12 healthy individuals. The HELP array covers 117,521 CpG sites, annotated to ∼22,000 gene promoters. For eight diagnosis/relapse pairs, base-pair resolution DNA methylation using the eRRBS (enhanced Reduced Representation Bisulfite Sequencing) method was also performed on Illumina HiSeq2000. The median relapse time for the 111 patients was 21.8 months (range 2.1 to 56.2). Unsupervised clustering analysis using the HELP data revealed seven clusters: one cluster contained only the 12 normal preB samples; four clusters were enriched with MLLr, ETV6/RUNX1, Trisomy 4+10, and TCF3/PBX1 samples, respectively. The sixth cluster was not enriched for specific cytogenetic cases, but interestingly, all cases in this cluster were NCI High Risk (age>10 years or WBC>=50,000; p<0.0001, Fisher’s Exact test) while the seventh cluster has a mixture of other cases. Supervised analysis of HELP profiles between paired relapse/diagnosis samples (n=67) revealed a markedly aberrant DNA methylation signature (1011 probesets, 888 genes, FDR<0.01 and methylation difference dx >25%, paired t-test), with 70% of the genes hyper- and 30% hypo-methylated in relapse samples. Using a Bayesian predictor and leave-one-out cross validation, this methylation signature could predict a sample as diagnosis or relapse with 95.3% accuracy. When comparing early (<36 months; n=50) versus late relapses (>=36 months; n=18), we detected a profound hypermethylation signature in early relapse (96.6% of the 610 probesets, 544 genes, FDR<0.01, dx >25%). Finally, we identified 1800 probesets (1658 genes) as differentially methylated within all cytogenetic subtypes described above compared to the normal preB samples (Dunnett’s test with normal preB as reference, FDR<0.01, dx>25%). Again the majority (70%) of those genes were hypermethylated in relapse as compared to diagnostic and normal preB. The base-pair resolution and more comprehensive eRRBS methylation analysis for the eight pairs of samples identified 39,679 CpG sites as differentially methylated (dx >25%, FDR<0.01), with 78.2% CpG sites hyper- and 21.2% hypo-methylated in relapse samples. Remarkably, the hypermethylated CpGs are primarily in promoter regions (50%, defined as +/-1kb to TSS), followed by intergenic (26%), then intragenic (14%), and exonic (10%) regions. In contrast, the hypomethylated CpGs are mainly in intragenic (48%), followed by intergenic (31%), exonic (14%) and promoter (7%) regions. The hypermethylated CpGs were mainly in CpG islands (86%) or CpG shores (10%), while hypomethylated CpGs were not (CpG islands: 8%, CpG shores: 27%). We further identified 3040 differentially methylated regions (DMRs) with a median size 426 bp. 78.4% of those DMRs were hyper- (1362 gene promoters) and 21.6% hypo-methylated (98 promoters) in relapse compared to diagnostic samples. Gene set enrichment and Ingenuity pathway analysis showed epigenetically disrupted pathways that are highly involved in cell signaling, and embryonic and organismal development. Taken together, our genome-wide high resolution DNA methylation analysis on a large cohort of relapsed childhood B-ALL from the COG trial identified unique methylation signatures that correlated with relapse and with specific genetic subsets. Those methylation signatures featured prevailing promoter hypermethylation and to a lesser extent, intrageneic hypomethylation. Epigenetically dysregulated gene networks in those relapse samples involved cell signaling, and embryonic and organismal development. Disclosures: No relevant conflicts of interest to declare.

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Swierczek#, Sabina, Neeraj Agarwal#, Gerald Rothstein, Roberto Nussenzveig et Josef Prchal. « Is Hematopoiesis Clonal in Healthy Elderly Females ? » Blood 110, n^o 11 (16 novembre 2007) : 2223. http://dx.doi.org/10.1182/blood.v110.11.2223.2223.

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Abstract Clonality studies can establish the single cell origin of tumors and differentiate nonmalignant from malignant states. Detection of clonal cells may be genotype-based relying on somatic mutations to mark the clonal population (e.g. 9q+:22q– translocation in CML), or phenotype-based, where the clonal population is identified by expression of surrogate genes which facilitate tracking the clonal process. Methods for determining phenotypic clonality rely on the principle of X chromosome inactivation (XCIP), unique to women, and are based on differentiating transcriptionally active from inactive X-chromosomal genes. Detection of the polymorphic state of genes subjected to inactivation may be done by either: discrimination of DNA methylation status, detection of mRNA transcripts, or polymorphic isozyme protein products. Extreme skewing of X-chromosome allelic usage by methylation-based clonality assay has been reported in ∼30% of healthy elderly females precluding clonality studies in this population. In contrast, by X-chromosome quantitative transcriptional clonality assay (TCA), we previously reported a normal skewing range based on our determination of random X-chromosome inactivation in 8 progenitors of pluripotent hematopoietic stem cells at the time of inactivation during the blastocyst stage of development (J Exp Med, 183:748, 1996). Moreover, we have not observed clonal XCIP in TCA studies involving over 200 healthy heterozygous females, indicating the rarity of this phenomenon. However, we did not systematically study females >65 years old. Furthermore, our TCA protocol was laborious, technically demanding and required significant amounts of highly radioactive isotopes. In addition, due to susceptibility of DNA methylation to environmental factors we decided to re-investigate the issue of clonality in older females using a novel quantitative real time PCR assay based on a unique primer design, we previously reported for the JAK2V617F mutation (Exp Hematol. 2007;35(1):32–8). Females >65 years of age with no history of malignant disorders, unexplained anemia, or autoimmune disorders were recruited for our IRB approved study. Genomic DNA and total RNA was isolated from peripheral blood granulocytes, reticulocytes, and platelets where applicable. Clonality studies were performed using BTK, FHL1, IDS, G6PD, and MPP1 exonic polymorphisms (∼95% females are informative for at least one marker; Blood101:3294–301, 2003). Genomic DNA was used for genotyping exonic polymorphisms by TaqMan based allelic-discrimination assays. Thirteen elderly females (age range 65–92, mean 75.5, median 75) and 5 younger females (age range 30–40, median 36, mean 35), heterozygous for one or more markers, were identified. TCA was performed using total RNA on markers found to be informative. Neither clonal XCIP, nor extreme skewing XCIP was noted in any of the study subjects. Based on reported data, ∼30% elderly women were found to have extremely skewed XCIP; hence, we expected to find 4/13 elderly women with clonal XCIP in our study group. Statistical analysis, using an exact binomial test, indicates a low probability of false positive results by our assay (p=0.014, exact 95% CI [0,0.22]). In conclusion, hematopoiesis is not clonal in healthy elderly females. #equal contribution.

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Natarajan, Umamaheswari, Sultan E. Ahmed, Steve Weinstein et Appu Rathinavelu. « Abstract 2117 : Changes of the DNA methylation status in multiple myeloma patients experiencing chemotherapy induced peripheral neuropathy ». Cancer Research 83, n^o 7_Supplement (4 avril 2023) : 2117. http://dx.doi.org/10.1158/1538-7445.am2023-2117.

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Abstract Chemotherapy-Induced Peripheral Neuropathy (CIPN) is considered to be one of the most common side effects caused by anti-neoplastic agents (1) such as Bortezomib (BTZ), Cisplatin (CSP), Taxol (TAX), and Vincristine (VINC). Clinical manifestations of CIPN include deficits of varying intensities of sensory, motor, and autonomic functions. Growing evidence suggests that epigenetic alterations could strongly underlie the induction and maintenance of PN (Peripheral Neuropathy) caused by many factors, including chemotherapy. Recently, a few groups have reported epigenetic alterations as well as changes in the Gene Expression Profiles (GEP) using the DNA and RNA isolated from patients experiencing CIPN. Therefore, our goal was to determine epigenetic alterations that may serve as predictive biomarkers and/or diagnostic biomarkers in the DNA isolated from the Peripheral Blood Mononuclear Cells (PBMCs) of human patients who were exhibiting varying levels of CIPN. To obtain preliminary data, we analyzed the methylation status of the DNA isolated from 3 Multiple Myeloma (MM) patients who experienced different levels (grades 1-7) of CIPN. When the methylation status of the MM patients was analyzed, the DNA was differentially methylated in 12,230 regions, in comparison to a normal control. Out of the above-mentioned total, about 4,563 regions were hypermethylated and 7,667 regions were hypomethylated. These differentially methylated regions included CpG islands in the promoter, intronic, and exonic regions of various genes within the 23 pairs of chromosomes including X and Y. When the most enriched Gene Ontology (GO) terms were identified, about 20 of them exhibited DNA hyper-methylation with fold changes ranging from 1-4 with the highest significance of 2.0e-12. The ranking of the genes involved in the top 3 GO terms related to neuronal function revealed that a total of 112 were hypermethylated in all three patients compared to the control. Similarly, ranking of the genes in the top GO terms with the highest fold change of hypo-methylation revealed 176 genes. Though few other GO terms showed significant changes, they were excluded in our CIPN-related ranking analysis, because they appeared to be linked to cancer or cancer growth-related pathways. It appears that these 288 hyper or hypomethylated genes may be directly involved in the onset, progression, or maintenance of CIPN. Analysis of the functional significance of these genes would provide greater insight into the relevance of the methylation status and its impact on the functions of the neurons that might be involved in pre-disposing or causing CIPN. Our research outcomes are expected to facilitate the design of an assessment method to detect the onset and progression of CIPN in cancer patients. (This research was supported by the Community Foundation of Broward and the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida). Citation Format: Umamaheswari Natarajan, Sultan E. Ahmed, Steve Weinstein, Appu Rathinavelu. Changes of the DNA methylation status in multiple myeloma patients experiencing chemotherapy induced peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2117.

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Yeung, Kit San, Matthew Sai Pong Ho, So Lun Lee, Anita Sik Yau Kan, Kelvin Yuen Kwong Chan, Mary Hoi Yin Tang, Christopher Chun Yu Mak et al. « Paternal uniparental disomy of chromosome 19 in a pair of monochorionic diamniotic twins with dysmorphic features and developmental delay ». Journal of Medical Genetics 55, n^o 12 (14 juillet 2018) : 847–52. http://dx.doi.org/10.1136/jmedgenet-2018-105328.

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BackgroundWe report here clinical, cytogenetic and molecular data for a pair of monochorionic diamniotic twins with paternal isodisomy for chromosome 19. Both twins presented with dysmorphic features and global developmental delay. This represents, to our knowledge, the first individual human case of paternal uniparental disomy for chromosome 19 (UPD19).MethodsWhole-exome sequencing, together with conventional karyotype and SNP array analysis were performed along with genome-wide DNA methylation array for delineation of the underlying molecular defects.ResultsConventional karyotyping on amniocytes and lymphocytes showed normal karyotypes for both twins. Whole-exome sequencing did not identify any pathogenic sequence variants but >5000 homozygous exonic variants on chromosome 19, suggestive of UPD19. SNP arrays on blood and buccal DNA both showed paternal isodisomy for chromosome 19. Losses of imprinting for known imprinted genes on chromosome 19 were identified, including ZNF331, PEG3, ZIM2 and MIMT1. In addition, imprinting defects were also identified in genes located on other chromosomes, including GPR1-AS, JAKMP1 and NHP2L1.ConclusionImprinting defects are the most likely cause for the dysmorphism and developmental delay in this first report of monozygotic twins with UPD19. However, epigenotype-phenotype correlation will require identification of additional individuals with UPD19 and further molecular analysis.

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Moll, Matthew, Victoria E. Jackson, Bing Yu, Megan L. Grove, Stephanie J. London, Sina A. Gharib, Traci M. Bartz et al. « A systematic analysis of protein-altering exonic variants in chronic obstructive pulmonary disease ». American Journal of Physiology-Lung Cellular and Molecular Physiology 321, n^o 1 (1 juillet 2021) : L130—L143. http://dx.doi.org/10.1152/ajplung.00009.2021.

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Genome-wide association studies (GWASs) have identified regions associated with chronic obstructive pulmonary disease (COPD). GWASs of other diseases have shown an approximately 10-fold overrepresentation of nonsynonymous variants, despite limited exonic coverage on genotyping arrays. We hypothesized that a large-scale analysis of coding variants could discover novel genetic associations with COPD, including rare variants with large effect sizes. We performed a meta-analysis of exome arrays from 218,399 controls and 33,851 moderate-to-severe COPD cases. All exome-wide significant associations were present in regions previously identified by GWAS. We did not identify any novel rare coding variants with large effect sizes. Within GWAS regions on chromosomes 5q, 6p, and 15q, four coding variants were conditionally significant ( P < 0.00015) when adjusting for lead GWAS single-nucleotide polymorphisms A common gasdermin B ( GSDMB) splice variant (rs11078928) previously associated with a decreased risk for asthma was nominally associated with a decreased risk for COPD [minor allele frequency (MAF) = 0.46, P = 1.8e-4]. Two stop variants in coiled-coil α-helical rod protein 1 ( CCHCR1), a gene involved in regulating cell proliferation, were associated with COPD (both P < 0.0001). The SERPINA1 Z allele was associated with a random-effects odds ratio of 1.43 for COPD (95% confidence interval = 1.17–1.74), though with marked heterogeneity across studies. Overall, COPD-associated exonic variants were identified in genes involved in DNA methylation, cell-matrix interactions, cell proliferation, and cell death. In conclusion, we performed the largest exome array meta-analysis of COPD to date and identified potential functional coding variants. Future studies are needed to identify rarer variants and further define the role of coding variants in COPD pathogenesis.

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Alfimova, Margarita, Nikolay Kondratyev, Galina Korovaitseva, Tatyana Lezheiko, Victoria Plakunova, Marina Gabaeva et Vera Golimbet. « A Role of DNA Methylation within the CYP17A1 Gene in the Association of Genetic and Environmental Risk Factors with Stress-Related Manifestations of Schizophrenia ». International Journal of Molecular Sciences 23, n^o 20 (20 octobre 2022) : 12629. http://dx.doi.org/10.3390/ijms232012629.

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As genetic and environmental influences on schizophrenia might converge on DNA methylation (DNAm) within loci which are both associated with the disease and implicated in response to environmental stress, we examined whether DNAm within CYP17A1, a hypothalamus–pituitary–adrenal axis gene which is situated within the schizophrenia risk locus 10q24.32, would mediate genetic and environmental effects on stress-related schizophrenia symptoms. DNAm within an exonic–intronic fragment of CYP17A1 was assessed in the blood of 66 schizophrenia patients and 63 controls using single-molecule real-time bisulfite sequencing. Additionally, the VNTR polymorphism of the AS3MT gene, a plausible causal variant within the 10q24.32 locus, was genotyped in extended patient and control samples (n = 700). The effects of local haplotype, VNTR and a polyenviromic risk score (PERS) on DNAm, episodic verbal memory, executive functions, depression, and suicidality of patients were assessed. Haplotype and PERS differentially influenced DNAm at four variably methylated sites identified within the fragment, with stochastic, additive, and allele-specific effects being found. An allele-specific DNAm at CpG-SNP rs3781286 mediated the relationship between the local haplotype and verbal fluency. Our findings do not confirm that the interrogated DNA fragment is a place where genetic and environmental risk factors converge to influence schizophrenia symptoms through DNAm.

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Anas, Muhammad, Alison K. Ward, Kacie L. McCarthy, Pawel P. P. Borowicz, Lawrence P. P. Reynold, Joel S. Caton, Carl R. Dahlen et Wellison J. j. S. Diniz. « 131 DNA methylation profile in bovine fetal liver is affected by maternal vitamin and mineral supplementation during early gestation ». Journal of Animal Science 102, Supplement_2 (1 mai 2024) : 54–55. http://dx.doi.org/10.1093/jas/skae102.063.

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Abstract Early gestation is the critical period for successful pregnancy establishment. During this period, maternal nutrition affects fetal development with potentially long-lasting consequences on offspring performance. Despite the known effects of vitamins and minerals on embryonic development, supplementation is still not a widely adopted practice. Here we focused on the effects of maternal vitamin and mineral supplementation from pre-breeding to d 83 of gestation. We hypothesized that the DNA methylation pattern of genes involved with fetal hepatic metabolism and function would be altered in response to vitamin and mineral supplementation during early gestation. Sixteen Angus crossbred heifers (~16 mo of age) were randomly assigned to either a treatment (vitamin and mineral supplementation, VTM, n = 8) or control (corn-based carrier without supplementation, NoVTM, n = 8) group. Supplemented heifers received 0.45 kg •heifer-1•d-1 of to provide 113 g of vitamin-mineral premix (Purina Wind & Rain Storm All-Season 7.5 Complete, Land O’Lakes, Inc., Arden Hills, MN) from d 71 to 148 before breeding to d 83 of gestation. All heifers received 100% of NRC recommended nutritional requirements targeting 0.28 kg •heifer-1•d-1 of gain. The heifers were bred through artificial insemination following a 7-d Co-Synch + CIDR estrus synchronization protocol. All pregnant heifers were surgically ovariohysterectomized on d 83, and liver samples were collected from the fetuses. DNA methylation profiles from 16 fetuses (8 per treatment) were determined using Reduced Representation Bisulfite Sequencing (RRBS). The analysis was conducted using methylKit pipeline where data was normalized using principal components. We identified 794 differentially methylated cytosines (DMCs) for the control (NoVTM) vs VTM comparison. Most of the DMCs were found in introns followed by intergenic, promoter, and exonic regions. Among these DMCs, 60% (479) were hypermethylated and 40% (315) were hypomethylated, potentially regulating 739 nearby genes (FDR < 0.1). Biological processes over-represented by these genes included transport of ions through voltage gated channels (KCNK13, KCNT1, CACNA1B, KCNK9, LRRC55, KCNAB2, CACNA1S, KCNJ2, CACNG5, CLIC5, TPCN2, KCNQ1), which may be associated to pregnancy establishment by inducing pregnancy-associated relaxation and uterine artery dilatation. Some genes associated with structural development were also regulated by VTM supplementation (i.e., TIAM1, COL18A1, DYSF, BMP10, RXRA, EHMT1, COL4A2, PLEC, ZFAT, MTMR2, NAV1, ANHX, ACACB, and LIMK2). Collectively, these results suggest that periconceptual maternal vitamin and mineral supplementation affects the methylation pattern and potentially regulates the expression of genes involved in fetal liver development and ion transport.

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Salilew-Wondim, D., M. Hoelker, U. Besenfelder, V. Havlicek, F. Rings, D. Gagné, E. Fournier et al. « 199 IN VITRO-DEVELOPED BOVINE BLASTOCYSTS ARE MARKED WITH ABERRANT HYPER- AND HYPO-METHYLATED GENOMIC REGIONS ». Reproduction, Fertility and Development 27, n^o 1 (2015) : 190. http://dx.doi.org/10.1071/rdv27n1ab199.

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Most often, in vitro produced embryos display poor quality and altered gene expression patterns compared to their in vivo counterparts. Aberrant DNA methylation occurring during in vitro embryo development is believed to be one of the multifaceted factors which may cause altered gene expression and poor embryo quality. Here, we investigated the genome-wide DNA methylation patterns of in vitro derived embryos using the recently developed Bovine EmbryoGENE Methylation Platform (BEGMP) array (Shojaei Saadi et al. BMC Genomics 2014 15, 451. doi: 10.1186/1471-2164-15-451) to unravel the aberrantly methylated genomic region in in vitro developed embryos. For this, in vitro and in vivo produced blastocysts were produced and used for genome-wide DNA methylation analysis. In vitro blastocysts were produced from oocytes retrieved from ovaries collected from the local abattoir and matured, fertilized, and cultured in vitro using SOF media. The in vivo blastocysts were produced by superovulation and AI of Simmental heifers followed by uterine flushing. Genomic DNA (gDNA) was then isolated from four replicates (each 10 blastocysts) of in vivo and in vitro derived blastocysts using Allprep DNA/RNA micro kit (Qiagen, Valencia, CA, USA) and the gDNA was then fragmented using the MseI enzyme. Following this, MseLig21 and MseLig were ligated to the MseI-digested genomic fragments in the presence of Ligase enzyme. Methyl-sensitive enzymes, HpaII, AciI, and Hinp1I, were used to cleave unmethlayted genomic regions within the MseI-MseI region of the fragmented DNA. The gDNA was subjected to two rounds of ligation-mediated polymerase chain reaction (LM-PCR) amplification. After removal of the adapters, the amplified gDNA samples from in vivo or in vitro groups were labelled either Cy-3 or Cy-5 dyes in dye-swap design using ULS Fluorescent gDNA labelling kit (Kreatech Biotechnology BV, Amsterdam, The Netherlands). Hybridization was performed for 40 h at 65°C. Slides were scanned using Agilent's High-Resolution C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) and features were extracted with Agilent's Feature Extraction software (Agilent Technologies Inc.). The results have shown that from a total of 414 566 probes harboured by the BEGMP array, 248 453 and 253 147 probes were detected in in vitro and in vivo derived blastocysts, respectively. Data analysis using the linear modelling for microarray (LIMMA) package and R software (The R Project for Statistical Computing, Vienna, Austria) revealed a total of 3434 differentially methylated regions (DMRs; Fold change ≥1.5, P-value <0.05), of which 42 and 58% were hyper- and hypo-methylated, respectively, in in vitro derived blastocysts compared to their in vivo counterparts. The DMRs were found to be localised in the intronic, exonic, promoter, proximal promoter, and distal promoter, and some of the probes did not have nearby genes. In addition, 10.8% of the DMRs were found to be stretched in short, long, or intermediate CpG islands. Thus, this study demonstrated genome-wide dysregulation in the epigenome landscape of in vitro-derived embryos by the time they reach to the blastocysts stage.

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Franzini, Anca, Jamshid S. Khorashad, Hein Than, Anthony D. Pomicter, Dongqing Yan, Thomas O'Hare et Michael W. Deininger. « Molecular Alterations in Chronic Myelomonocytic Leukemia Monocytes : Transcriptional and Methylation Profiling ». Blood 132, Supplement 1 (29 novembre 2018) : 3889. http://dx.doi.org/10.1182/blood-2018-99-115077.

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Abstract Chronic myelomonocytic leukemia (CMML) is a genetically heterogeneous hematopoietic stem cell disorder that combines features of a myelodysplastic syndrome and a myeloproliferative neoplasm and exhibits a strong bias towards older age. The prognosis of CMML is poor, with overall survival of less than 3 years in most studies, however recurrent somatic mutations explain only 15-24% of the clinical heterogeneity of CMML (Elena C. et al. Blood 128:1408-17, 2016). The extreme skewing of the CMML age distribution suggests that CMML reflects the malignant conversion of the myelomonocytic-biased differentiation characteristic of an aged hematopoietic system. We hypothesized that separating the contribution of the normal aging process from bona fide CMML-specific alterations will improve the molecular characterization and biological understanding of CMML. We decided to focus on monocytes as the phenotypic minimal common denominator of genetically heterogeneous diseases. CD14+ monocytes were sorted from the blood of untreated CMML patients (N=12, median age 77 years, range 61-90), age-matched healthy controls (old controls: N=12, median age 68 years, range 62-74) and young healthy controls (young controls: N=16, median age 29 years, range 24-44) and subjected to RNA sequencing and DNA methylation profiling. Differentially expressed genes in CMML monocytes compared to healthy controls were identified with DESeq2 using a 1% false discovery rate (FDR) and a fold-change cutoff set at >│2│ (Figure 1A). We identified the 2480 CMML-specific genes by subtracting all genes with significant differences in the young controls vs. old controls comparison from the CMML vs. old controls comparison. The top-25 most significantly upregulated genes (Figure 1B) included transcription factors, TNFα signaling genes, genes that regulate genomic stability, and genes involved in apoptosis. The most significantly downregulated transcripts were genes involved in response to DNA damage, RNA binding, monocyte differentiation and mediators of inflammatory process. To link these observations to function, we imputed the 2480 CMML-specific differentially expressed genes into the ingenuity pathway analysis (IPA) application. This analysis uncovered significant enrichment of pathways involved in: mitotic roles of Polo-like kinase, G2/M DNA damage checkpoint regulation, lymphotoxin β receptor signaling, IL-6 signaling and ATM signaling (Figure 1C). DNA methylation profiling revealed 909 differentially methylated regions (DMRs) between CMML and age-matched controls, with most regions being hypermethylated in CMML monocytes. Of these, 37% of the DMRs were intronic, 22% were exonic, 14 % were in the promoter region (Figure 1D), 10% were downstream, 10% were upstream, the remainder were 3' and 5'-overlaps. We also performed integrated analysis using the promoter DMRs and the gene expression profile to identify CMML-associated genes that are likely to be regulated by specific changes in methylation. We observed concomitant changes in CMML-specific mRNA transcripts and DNA methylation promoter regions in the CMML vs. old controls contrast for 10 genes (Figure 1E). AOAH, SERINC5, TAF3 and AHCYL1 were downregulated and hypermethylated; MS4A3, TNF, VCAM1, and IFT80, were upregulated and hypermethylated; TUBA1B was upregulated and hypomethylated and PITPNA was downregulated and hypomethylated. Our study is the first to combine transcriptional and methylation profiling for molecular characterization of CMML monocytes. Conclusions: (i) age-related gene expression changes contribute significantly to the CMML transcriptome; (ii) the CMML-specific transcriptome is characterized by differential regulation of transcription factors, inflammatory response genes and anti-apoptotic pathway genes; (iii) differences in promoter methylation represent only a small proportion of overall differences in methylation, suggesting that intragenic or intronic methylation is a major contributor to the leukemic phenotype; (iv) age-related changes may be necessary, but are not sufficient to realize the CMML phenotype. Figure 1. Figure 1. Disclosures Deininger: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint: Consultancy.

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Walker, Brian A., Paola E. Leone, Nicholas J. Dickens, Kevin D. Boyd, David Gonzalez, Faith E. Davies et Gareth J. Morgan. « UTX, a Histone Demethylase, Is Inactivated through Homozygous Deletion, Mutation, and DNA Methylation in Multiple Myeloma. » Blood 114, n^o 22 (20 novembre 2009) : 1798. http://dx.doi.org/10.1182/blood.v114.22.1798.1798.

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Abstract Abstract 1798 Poster Board I-824 Histone modifications are known to mediate transcriptional regulation through changes in chromatin condensation and as such can lead to aberrant transcriptional patterns resulting in malignant transformation. Modulation of chromatin structure via histone modification is becoming recognised as an important pathogenic mechanism in myeloma and has been suggested by the over-expression of MMSET, a histone methyltransferase, by the t(4;14) chromosomal rearrangement. More recently inactivation of UTX, a histone demethylase, has also been suggested to have a role in myeloma pathogenesis and both UTX and MMSET are mediators of transcriptional repression. UTX is inactivated in a number of different cancer cell lines but importantly, mutations and deletions have been detected in myeloma cell lines and we wished to follow up on this observation in uniformly treated clinical cases. UTX is a large gene found on the X chromosome covering 240 kb of genomic DNA and consists of 29 exons encoding a protein with both JmjC-domains and tricopeptide repeats responsible for histone demethylation and polycomb protein interactions. Inactivation of UTX occurs through deletions of individual exons through to large whole gene deletions as well as by mutations scattered throughout the 29 exons. A further mechanism of UTX inactivation which has not been looked for to date is via DNA methylation of the CpG island upstream of the transcriptional start site. We set out to determine the status of UTX in our dataset which includes expression, mapping, and methylation array data from presenting myeloma samples entered into the MRC Myeloma IX clinical trial. The gene expression of UTX was measured on 272 samples using Affymetrix U133 Plus 2.0 arrays and showed that 80% of samples do not express UTX transcripts but using expression quartile analysis we could not detect an effect on overall survival. The mechanism underlying the abrogation of expression was investigated further using the Affymetrix 500K SNP mapping array on a subset of 114 samples to detect copy number alterations. UTX was hemizygously deleted in 21 (42%) female samples and was completely deleted in 1 male sample, at the resolution of the arrays. In order to determine if individual exons were deleted, at a resolution below that detectable by mapping arrays, we performed quantitative PCR coupled with high resolution melting (HRM) analysis using the Rotor-gene Q real-time cycler (Qiagen). Exons were amplified, over 40 cycles, to obtain products of ∼200 bp using LC Green Plus mastermix (Idaho Technologies) in a 10 μl reaction on the Rotor-gene Q with a final HRM step from 72-95 °C with increments of 0.1 °C. Amplification plots combined with the HRM step allows us to identify both homozygous deletions and mutations within the exons. We screened all 114 samples for micro-deletions and mutations and found homozygous deletions in ∼7% of samples and identified a significant proportion of mutations using the HRM method which accounted for a total of ∼10% of gene inactivation. In order to determine if methylation could be responsible for inactivation of the remaining allele we used the Illumina Infinium humanmethylation27 array to study the methylation status at the UTX locus. This array interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. The results of this analysis are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. In samples with a diploid copy number of UTX the methylation signals covered 2 ranges: hemi-methylated (0.35-0.55, n=7) and hyper-methylated (0.73-0.89, n=14). In samples with 1 copy of UTX, which includes all males, there were 3 ranges: hypomethylated (0.08-0.21, n=5), hemi-methylated (0.35-0.51, n=3), and hypermethylated (0.66-0.88, n=48). All of the hypomethylated samples with a single copy of UTX were male, and at least 1 of these samples contained an inactivating exonic deletion resulting in complete loss of function. These data indicate that methylation of the residual allele contributes significantly to the inactivation of UTX along with interstitial deletions and mutations. We will go on to present data on the interaction of UTX with variation at the UTY locus and how this modulates behaviour of the myeloma clone. Disclosures No relevant conflicts of interest to declare.

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Zampini, Matteo, Claudia Tregnago, Valeria Bisio, Benedetta Accordi, Valentina Serafin, Paolo Pierani, Nicola Santoro et al. « Dna Methylation Is Linked to a Specific Cell-Adhesion Program in Relapsed Pediatric t(8;21)(q22;q22)RUNX1-RUNX1T1 Patients ». Blood 128, n^o 22 (2 décembre 2016) : 1524. http://dx.doi.org/10.1182/blood.v128.22.1524.1524.

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Abstract t(8;21)(q22;q22)RUNX1-RUNX1T1 is a recurrent somatic lesion detected at diagnosis in approximately 12-15% of children with acute myeloid leukemia (AML). Children with this isolated translocation are usually considered at standard risk, but our last multicenter trial revealed a higher than expected cumulative incidence of relapse for these patients1. Genetic and epigenetic heterogeneity is emerging as a fundamental property of AML in the context of the clonal architecture dynamic evolution. In view of this observation, we hypothesized that within t(8;21) patients there may coexist a complex mosaic of cells containing combinations of the same genetic t(8,21) lesion together with different epigenetic variants, and that epigenetic complexity may play a crucial role in predisposing patients to relapse. The importance of the identification of molecular markers distinctive of t(8,21)-rearranged patients prone to develop relapse could be instrumental to improve their cure rate. We performed high throughput DNA methylation profiling (RRBS-seq) and integrated results with gene expression profiling (Affymetrix HTA 2.0) of 16 isolated t(8;21) AML samples collected at diagnosis, and analyzed data by comparing patients who did or did not experience relapse. We applied a logistic regression algorithm to identify differentially methylated regions (DMRs) considering a minimum change in methylation level of 25%. We validated results in a proteome context by reverse phase protein array (RPPA) in an independent cohort of 35 t(8;21) AML patients. DNA methylation profiling analysis identified 337 DMRs able to correctly predict t(8;21) patients who did relapse from those who did not. In particular, 23 DMRs (7%) were located at promoters, while most of them were equally distributed between intronic (48%) and exonic (45%) regions. Globally, we found hypomethylated DMRs being significantly enriched in relapsed patients, in particular in repetitive elements regions of the genome (LINE, SINE, DNA transposon: 38.9% vs 52.4%; p<0.01), supporting an enhanced transposable elements transcription contributing to cancer genomic instability. DMRs clustering analysis correctly divided t(8,21) patients according to their risk of experiencing relapse, independently of their different localization (at promoters, exons or introns), revealing that DNA methylation profiling has a predictive role for identifying patients with worse event-free survival. We then considered the role of methylation over gene expression and found a weak correlation between DMRs (mostly at promoters) and their associated gene levels (14.5% of DMRs with an inverse correlation r <-0.4, p<0.05). To better understand the role of DMRs and transcriptional regulation, we searched for associations between DMRs and chromatin modification patterns. DMRs were enriched at regulatory regions; in particular, we found hypermethylation in promoter and enhancer regions, while hypomethylation was found in repressed chromatin regions (p<0.05). Looking at the transcription factors (TFs) binding sites within the DMRs, we identified that at hypermetylated DMRs the most represented TFs were E2F1 and HDAC1, suggesting they might be almost transcriptional silenced. By contrast, MAFK and FOXA2 binding sites were enriched at hypomethylated sites, suggesting their enhanced activity in relapsed patients as compared to the non-relapsed ones. Finally, we interrogated gene ontology for DMRs-associated genes and deregulated genes found by GEP, showing a significant enrichment for pathways involved in cell adhesion and cytoskeletal organization. Proteome analysis by RPPA validated these pathways being aberrant activated (global test p<0.01) in an independent cohort of t(8;21)-rearranged patients, and supported the ongoing in vitro experiments in t(8;21) cell lines to define candidates genes involved in the pathophysiology of t(8,21) relapse. These data show that the methylation signature may be considered a novel, emerging diagnostic tool making possible to better stratifying t(8,21)-rearranged patients through the identification, already at diagnosis, of those who are prone to relapse . Preliminary data of functional analysis suggest that epigenome of t(8;21) blasts may control cell adhesion properties at bone marrow niche and treatment response, contributing to patients relapse. 1 Pession A, Blood. 2013;122(2):170-8. Disclosures No relevant conflicts of interest to declare.

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Hakimi, A. Ari, Irina Ostrovnaya, Anders Jacobsen, Jonathan A. Coleman, Paul Russo, Roy Mano, Alexander Sankin et al. « Validation and genomic interrogation of the MET variant rs11762213 as a predictor of adverse outcomes in clear cell renal cell carcinoma. » Journal of Clinical Oncology 32, n^o 4_suppl (1 février 2014) : 395. http://dx.doi.org/10.1200/jco.2014.32.4_suppl.395.

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395 Background: The exonic single nucleotide variant rs11762213 located in the METoncogene has recently been identified as a prognostic marker in clear cell renal cell carcinoma (ccRCC) (Schutz et al, Lancet Oncol 2013). We sought to validate this finding and explore the biologic implications using The Cancer Genome Atlas (TCGA) cohort. Methods: The variant call file (VCF) and expression data was available for 272 patients. We then extracted the data for rs11762213 as follows for the "normal" sample: allelic fraction for variant allele > 80% is homozygous variant while allelic fraction for variant allele 30% to 70% is heterozygous. Paired tumor-normal materials, genomic data (whole exome, RNAseq, and DNA methylation) and clinical information were acquired from publically available TCGA datasets. Kaplan-Meier method was used to estimate the survival probabilities. Cancer specific survival (CSS) was analyzed using the competing risk method and Cox proportional hazard regression was used for analysis of time to recurrence. Multivariate competing risk models were also fitted in the TCGA cohort in order to adjust for the Mayo Clinic SSIGN score. Results: Overall, the variant allele of rs11762213 was detected in 10.3% of the cohort and was associated with higher nuclear grade (p=0.03) and trended toward higher clinical stage (p=0.07). After adjusting for the prognostic SSIGN score, the risk allele remained a significant predictor for adverse cancer specific survival (CSS; p<0.0001; Odds Ratio [OR] 3.88, 95% confidence interval [CI] 1.99-7.56) and time to recurrence (TTR; p=0.003; OR 2.97; 95% CI 1.44-6.2). RNA sequencing data for MET did not reveal differences in tumor mRNA expression when stratified by risk allele, but did show differences in the normal kidney expression (p=0.02) in a smaller cohort (n=61). Conclusions: The exonic MET variant rs11762213 is an independent predictor of adverse CSS and TTR in ccRCC and could be integrated into clinical practice for prognostic stratification. Gene expression analysis suggests that inherited variation at MET influences expression of the gene in normal kidney tissue. Further external validation and biological interrogation is necessary.

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Werner, Jordan, Hiba Siddiqui, Samiha Syed, Osman Khan, Servando Casillas Garabito et James D. Fackenthal. « Abstract 1735 : DNA demethylating agents have cell type-specific effects on viability and BRCA2 alternative splicing ». Cancer Research 84, n^o 6_Supplement (22 mars 2024) : 1735. http://dx.doi.org/10.1158/1538-7445.am2024-1735.

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Abstract Introduction: BRCA2 is one of two principal tumor suppressors responsible for hereditary breast/ovarian cancer syndrome (HBOC). Patients with strong family histories of breast/ovarian cancer may carry BRCA2 DNA sequence variants of unknown clinical significance (VUSs). To evaluate which of these VUSs may contribute to cancer risk by altering splicing patterns, naturally occurring BRCA2 mRNA alternate splicing events have been extensively characterized, but it is not yet known which if any of these alternate splicing events plays a role in BRCA2 function. One variant lacking exon 3 (∆3), which maintains the full length translational reading frame, lacks sequence encoding EMSY and PALB binding domains as well as transactivation function. Previous work has shown that, while some VUSs associated with increased levels of ∆3 in lymphoblastoid cell lines are not pathogenic, germline deletions that eliminate BRCA2 exon 3 are associated with increased breast cancer risk. It is therefore of interest to characterize the factors that influence the frequency of naturally occurring BRCA2 exon 3-skipping, including cell types and therapies. DNA demethylating agents are used as cancer therapy to promote expression of tumor suppressor genes that may be epigenetically silenced in tumors. However, DNA methylation has been shown to be more frequent in exonic than intronic regions of protein-coding genes, suggesting that methylation may play a role in recruiting spliceosomal components to nascent pre-mRNA. It is therefore of interest to determine whether genomic demethylation affects alternative splicing patterns. Here, we explore whether BRCA2 exon 3 alternative splicing patterns are altered by demethylating agents. Methods: To determine whether DNA demethylating agents can alter the levels of ∆3, MCF7 and/or the non-cancer breast cell line MCF 10A was treated with 5-aza 2’-deoxycytidine (5-AzadC) or 5-Azacytidine (5-AzaC), and isoform-specific RT-PCR was used to compare relative levels of splice junctions containing or skipping exon 3. Results and Conclusions: 1) While the IC50 for both 5-AzadC and 5-AzaC in MCF 10A was similar (less than 1 μM), MCF7 showed considerably more resistance to 5-AzaC than 5-AzadC (IC50 of ≥ 5 μM vs ≤ 0.2 μM). 2) 5-AzaC treatment reduces the proportion of MCF 10A cultures in apoptosis but does not increase the proportion of viable cells, indicating it causes cell death by some other means. 3) While both 5-AzaC and 5-AzadC reduce relative levels of ∆3 in MCF7, 5-azadC does not reduce relative levels of ∆3 in MCF 10A, suggesting there may be a cell type-specific splicing response to genomic demethylation therapies. Citation Format: Jordan Werner, Hiba Siddiqui, Samiha Syed, Osman Khan, Servando Casillas Garabito, James D. Fackenthal. DNA demethylating agents have cell type-specific effects on viability and BRCA2 alternative splicing [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 1735.

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Zhang, Xu, Jihyun Song, Binal N. Shah, Galina Miasnikova, Adelina Sergueeva, Josef T. Prchal et Victor R. Gordeuk. « Hypoxic Response-Dependent Genetic Regulation Revealed By Allele-Specific Expression in Reticulocytes of Chuvash Polycythemia ». Blood 130, Suppl_1 (7 décembre 2017) : 926. http://dx.doi.org/10.1182/blood.v130.suppl_1.926.926.

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Abstract Homozygosity for the VHLR200W mutation in Chuvash polycythemia (CP) leads to decreased degradation of the α subunits of hypoxia inducible factor (HIF)-1 and HIF-2 by the hypomorphic variant of VHL, the principal negative regulator of HIFs. The constitutively activated HIFs directly regulate the transcription of a suite of hypoxic responsible genes, including the principal regulators of erythropoiesis, vessel development, and glycolytic metabolism, which further trigger a downstream cascade of gene expression. Besides these transcriptional factors, cis acting elements play an important role in the hypoxic gene regulatory network. To assess the extent of cis regulatory variation in hypoxic gene expression, we compared allele-specific expression (ASE) in purified reticulocytes between VHLR200W homozygote individuals and age- and gender-matched wild type control individuals living at the same altitude of ~200 meters from the Chuvash population. Cell fractions of reticulocytes were purified from 17 VHLR200W homozygotes and 13 wild type individuals. Total RNA was extracted, depleted of ribosomal RNA and hemoglobin transcripts, and reverse transcribed. Strand-specific libraries were constructed for 125 bp paired-end sequencing to 30-45 million read pairs per sample using Illumina HiSeq 2500 platform. The samples were collected and processed in three batches across two years, with VHL genotype randomized in each batch. The sequencing data were mapped to human reference genome and analyzed for differential expression and differential ASE between VHLR200W homozygotes and wild type individuals. At 5% false discovery rate (FDR, i.e., <5 false positives in 100 detected genes), 1,267 genes were differentially expressed with more than 1.2-fold change in CP patients, 703 elevated and 564 decreased. Genes up-regulated in CP were enriched (fold enrichment >5, FDR <0.05) in REACTOME pathways of epigenetic remodeling (Packaging of telomere ends, DNA methylation, HDACs deacetylate histones, PRC2 methylates histones and DNA, Deposition of new CENPA-containing nucleosomes at the centromere, HATs acetylate histones) and oxidative stress induced senescence (DNA damage/telomere stress induced senescence, Senescence-associated secretory phenotype, Oxidative stress induced senescence). Genes decreased in CP were enriched in REACTOME pathways of cell cycle (E2F-enabled inhibition of pre-replication complex formation, Nuclear pore complex disassembly, SUMOylation of DNA replication proteins) and DNA damage repair (Activation of ATR in response to replication stress, SUMOylation of DNA damage response and repair proteins). ASE was analyzed between CP and wild type individuals to assess hypoxic response-dependent genetic effects on gene expression. For the 1,267 genes differentially expressed in the CP, we selected genes containing exonic SNPs with heterozygous alleles for ASE analysis. With a null hypothesis of no cis acting regulation on the gene expression, both alleles are expected to be expressed at the same level, whereas allelic imbalance indicates linked cis regulation. At a given bi-allelic SNP, individuals with ≥2 read counts covering each of the reference and alternative alleles and with ≥20 total counts were included in the analysis. Exonic SNPs with at least one individual in each of the CP and wild type group were further selected to test for differential ASE between the CP and wild type groups, using a generalized linear model. A total of 147 genes passed the filtering and were analyzed, among which 32 were detected to have significant CP-dependent ASE at 5% FDR. Some of these genes may have important roles in hypoxic responses in CP reticulocytes, for example NEIL3, encoding a DNA glycosylase that initiates the first step in base excision repair by cleaving bases damaged by reactive oxygen species, and STOM, encoding an integral membrane protein that localizes to the cell membrane of red blood cells, loss of which is associated with hereditary stomatocytosis. Our study reveals plethora of gene expression changes in CP reticulocytes compared to wild type controls, among which 22% could be regulated by hypoxic response-specific cis genetic variations. These observations indicate the prominence of cis elements in hypoxic response, for which substantial inter-individual differences exist even among a relatively isolated population. Disclosures Gordeuk: Emmaus Life Sciences: Consultancy.

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Pollyea, Daniel A., Aparna Raval, Brenda Kusler, Jason R. Gotlib, Ash A. Alizadeh et Beverly S. Mitchell. « A Novel Missense Mutation In An MDS Patient and Effects on TET2 mRNA Expression and Clinical Outcomes ». Blood 116, n^o 21 (19 novembre 2010) : 1889. http://dx.doi.org/10.1182/blood.v116.21.1889.1889.

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Abstract Abstract 1889 Introduction The ten-eleven translocation 2 (TET2) gene has recently been recognized to be mutated at a relatively high frequency in all myeloid malignancies, representing a compelling unifying feature of these related but phenotypically distinct conditions. The TET2 paralog TET1 catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine; TET2 shares a homologous domain thought to catalyze this conversion, and is hypothesized to act as a tumor suppressor gene (TSG) by regulating DNA methylation and epigenetic control of gene expression at critical loci important for myelopoiesis and leukemogenesis. The prognostic impact of TET2 mutations is largely unknown. The hypomethylating agents 5-azacytidine and decitabine, approved agents with activity in myelodysplastic syndrome (MDS), are incorporated into DNA and/or RNA, inhibiting DNA methyltransferase and preventing promoter methylation and TSG silencing. Given the role of TET2 in epigenetic regulation, the aim of this study was to determine whether TET2 mutations were associated with prognosis in MDS patients (pts) treated with hypomethylating therapies. Methods Bone marrow aspirates from 12 MDS pts who received hypomethylating agents were obtained, and PCR and bidrectional sequencing of TET2 coding exons was performed. RT-PCR was performed in triplicate and analyzed by the comparative Ct method. Results The median age of the 12 pts was 68 (49-80), the median international prognostic scoring system (IPSS) score was 1.75 (0-3) and 7/12 (58%) had normal cytogenetics. Two of 12 (17%) pts had exonic TET2 mutations. Pt 7 exhibited a previously reported heterozygous V1718L missense mutation, while pt 3 had 2 previously unreported heterozygous mutations: a nonsense mutation in exon 3, Q1068X, and a missense mutation in exon 6, R1214V. TET2 mRNA expression from all pt samples that expressed GAPDH was quantified. Pt 3 had decreased TET2 mRNA expression compared to pt 7, MDS patients with wild type TET2 and non-diseased control samples (p=.01). Pts with wild-type TET2 had an overall response rate (defined as complete responses plus partial responses) of 20%, while neither of the patients with TET2 mutations responded to hypomethylating agents. The post-treatment median survival was significantly different between patients harboring TET2 mutations and those with wild type TET2 (log rank test p<.003). Both pts with mutated TET2 ultimately died, 4 months and 6 months after treatment, while 7/10 (70%) pts with wild type TET2 died, with a median time to death or last follow-up of 26 months (95% CI 6.1–47.6) Conclusions We report the existence of two novel mutations in TET2, one of which is a heterozygous nonsense mutation that is predicted to result in a truncated protein lacking the conserved domains. The reduced expression of TET2 observed in this pt may be due to nonsense-mediated decay, a post-transcriptional process that recognizes and degrades mRNA that contains a premature translation termination codon. The adverse association seen in this series between TET2 mutations and clinical outcomes in MDS pts who received hypomethylating drugs merits replication in a larger sample. Disclosures: No relevant conflicts of interest to declare.

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Postnikova, Liubov A., Ekaterina M. Noniashvili, Irina O. Suchkova, Tatyana V. Baranova et Eugene L. Patkin. « The influence of exogenic lactoferrin on DNA methylation in postimplantation mouse embryos developed from zygotes exposed to bisphenol A ». Medical academic journal 22, n^o 4 (1 février 2023) : 45–56. http://dx.doi.org/10.17816/maj109416.

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BACKGROUND: Bisphenol A is a chemical agent ubiquitous in plastic consumer products and a toxin capable of disrupting key epigenetic mechanisms in early embryogenesis. It becomes more and more clear that early development changes in epigenetic pathways caused by exposure to toxic substances are associated with various adult diseases. Therefore the need to identify new agents capable of eliminating epigenetic mechanisms failures caused by the bisphenol A toxin becomes evident. Here we suggest lactoferrin as a normalizer of toxicant-induced epigenomic changes. Currently there is no data on the role of lactoferrin as a normalizer of epigenomic disorders under the influence of toxicants. We assume that in mammalian embryogenesis lactoferrin might function as an epigenetic modulating factor. AIM: The aim of the research is to study effects of lactoferrin on the epigenetic status of postimplantation mouse embryos, exposed to bisphenol A in utero. MATERIALS AND METHODS: In this study, 3 experimental groups of mice and two control group were used. 1. Mice on the first day of pregnancy, injected with 40 mg/kg of body weight of bisphenol A; 2. Mice on the first day of pregnancy, injected with 50 mg/kg of body weight of lactoferrin; 3. Mice on the first day of pregnancy, successively injected with 50 mg/kg body weight of lactoferrin and 40 mg/kg of body weight of bisphenol A. On the 15th day of embryonic development, the level of genome-wide DNA methylation was evaluated in different body parts of the embryos by methyl-sensitive restriction and ImageJ visualization analysis. RESULTS: We demonstrated that in post-implantation mouse embryos, exposure to bisphenol A in the prenatal period caused an increased level of genome-wide DNA methylation. The most prominent effects were observed in brain and abdominal section of the embryos. Together, the present findings confirmed that lactoferrin administration at a dose of 50 mg/kg of body weight resulted in normalization of genome-wide DNA methylation levels after bisphenol A-induced epigenetic alterations. CONCLUSIONS: We assume that lactoferrin may partially neutralize the harmful effects of bisphenol A caused aberrant methylation, and thus can potentially be used as a pharmaceutical product. Factual findings of the present study may help by development of new therapeutic approaches. Nevertheless, further research of the bisphenol A, lactoferrin and lactoferrin + bisphenol A effects on reactive oxygen species and/or antioxidant enzymes is needed.

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He, Shou-Pu, Jun-Ling Sun, Chao Zhang et Xiong-Ming Du. « Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii ». Molecular Biology 45, n^o 2 (avril 2011) : 204–10. http://dx.doi.org/10.1134/s002689331102018x.

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Ridout, Kate E., Pauline Robbe, Doriane Cavalieri, Jennifer Becq, Miao He, Ruth Clifford, Niamh Appleby et al. « Highly Comprehensive Genomic Testing for CLL : WGS, One Key to CLL Patient Stratification ». Blood 132, Supplement 1 (29 novembre 2018) : 3115. http://dx.doi.org/10.1182/blood-2018-99-115935.

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Abstract Background Chronic Lymphocytic Leukemia (CLL) is characterised by a highly heterogeneous natural history and treatment response. Indeed, 50% of immunoglobulin heavy chain variable region (IgHV) hypermutated patients have an excellent progression free survival (PFS) after chemoimmunotherapy. Conversely, 25% of FCR treated patients relapse within 24 months (high risk CLL). Recent studies have shown that complex karyotype with or without TP53 disruption predicts for relapse after BCL2 therapy and BTK inhibitors. However, TP53 is the only marker for which routine testing is available. Overall, nearly 80% of patients relapsing after frontline FCR do not present a known poor risk genomic marker. Additional candidate genomic predictors of poor outcome including mutations in coding regions of NOTCH1, SF3B1 and RPS15, non-coding regions of NOTCH1 and enhancer regions of PAX5, telomere length, IgHV status, and DNA Damage Repair (DDR) germline mutations including TP53 and ATM have been reported in CLL. Further, the role of mutational signatures and regions of kataegis also merit additional investigation in progressive CLL. Evaluating all candidate predictors requires complex time consuming, multi-modality testing outside the scope of routine clinical diagnostic practice, however, in isolation, each has low predictive value. Here, we show preliminary data on a novel patient stratification method based on whole genome sequencing (WGS) data incorporating multiple genomic features in a single test. Patients and Methods Tumor (peripheral blood) and germline (saliva) samples were collected from 321 patients from 6 UK trials via the Genomics England CLL pilot: ARCTIC (n=61), AdMIRe (n=64), CLL 210 (n=30), CLEAR (n=12), RIAltO (n=88) and FLAIR (n=66). We performed WGS on the HiSeqX (Illumina). After read alignment, we detected somatic variants using Strelka 2.4.7 for small variants detection (SNV and InDels), Manta 0.28.0 for structural variant (SV) detection, and Canvas 1.3.1 for copy number variant (CNV) detection (Illumina). Non-coding regions were annotated with information from primary CLL, CLL cell lines and B-cell ENCODE databases. Mutational signatures and putative regions of kataegis were calculated based on Alexandrov et al. (Nature, 2013) and Lawrence et al. (Nature, 2013). Telomere lengths were assessed using Telomerecat. Data aggregation was performed using contingency tables combined with non-negative matrix factorization. Results Mean coverage was 94.2X for tumor and 28.5X for germline samples. We found a median of 9172 SNPs/sample after filtering and 2348 indels/sample across 321 patients. High risk CLL was enriched for genomic complexity and poor prognostic mutations. The most frequently mutated genes were SF3B1 (17%), TP53 (13%), NOTCH1 (12%), IGLL5 (12%), and ATM (11%). Analysis of non-coding regions using DNA methylation markers, ATAC-seq and Hi-C revealed potential candidate regions associated with early relapse. Using CNA and SV data, we identified interesting patterns of genomic complexity and structural variants, including a trend towards enrichment of del8p in Relapse/Refractory and FCR non-responders. Additionally, we investigated mutation signatures and kataegis across coding and non-coding regions of the genome. We correlated exonic regions of DDR genes in germline data with clinical outcomes and extended this to genes mutated in both tumor and germline data, termed germline-tumor double-hits. We examined the relationship between the Alexandrov hypermutation signature, IgHV status (determined by % homology to the reference genome) and PFS, and combined mutational density at the Ig locus with mutation signature aiming to predict IgHV status. Finally, we produced a binary contingency matrix, using non-negative matrix factorization to cluster the samples. This method highlighted patient groups with shared genomic profiles. Conclusion We present preliminary data on a patient stratification method derived from WGS of 321 paired germline and CLL trial samples. Our predictive signature includes driver gene mutations, CNAs, IgHV status, genomic complexity, telomere length, overall mutation burden and genes with germline-tumor double-hits. Our comprehensive, NGS-based patient stratification attempts to predict patient outcome in a single sequencing run. Disclosures Becq: Illumina: Employment. He:Illumina: Employment. Ross:Illumina: Employment. Bentley:Illumina: Employment. Pettitt:Celgene: Research Funding; Gilead: Research Funding; Roche: Research Funding; GSK/Novartis: Research Funding; Napp: Research Funding; AstraZeneca: Research Funding; Chugai: Research Funding. Hillmen:Novartis: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Research Funding; Celgene: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria.

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Visconte, Valeria, Bartlomiej P. Przychodzen, Steffan T. Nawrocki, Swapna Thota, Kevin R. Kelly, Bhumika Patel, Cassandra M. Hirsch et al. « Genetic and Epigenetic Defects in the Autophagy Machinery in Myelodysplastic Syndromes ». Blood 128, n^o 22 (2 décembre 2016) : 4301. http://dx.doi.org/10.1182/blood.v128.22.4301.4301.

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Abstract Autophagy is a degradation process for the turnover of damaged organelles and long-lived proteins that also plays an important role during erythropoiesis. Accordingly, knockout of the essential autophagy gene Atg7 in mice leads to clinico-morphologic features of MDS. To date, no study has determined the prevalence and impact of defects (mutations, aberrant expression) in the autophagy machinery in MDS. We interrogated the occurrence of alterations in 180 autophagy genes by analyzing WES of patients with MDS (N=120). For comparison, we analyzed results from other hematologic neoplasms (N=103) and TCGA (N=202). We detected somatic mutations in autophagy genes in 40/425 patients (9%). Mutations were enriched in MDS (12%;14/120) and prevalent in higher risk MDS patients (30%;12/40). Mutations were found in: ATG2A, ATG4C, ATG14, ATG16L1, BCL2, CDKN2AIPNL, COG8, DNM1L, DNM2, GYS1, HIF1A, KIF1B, LAMP2, MLST8, MTOR, NOD2, PIK3CB, PIK3C2A/B, PIK3C2G, PPP2R2A/B, PPP2R3A, PRKAA1/2, PRKACB, PRKAG1/G2, PTPN2, RICTOR, RPTOR, SEC22B, SMURF1, SQSTM1, STAT3, SUPT20H, TAB2, TNFSF10/13B, ULK4, USP10, VPS11/33B, VTI1A, WDFY3/4, WAC. Twenty-five mutations had a cut-off >20% VAF. Most patients (31/40;78%) had a sole mutation, 4 patients carried 2 mutations each (ULK4, WDFY3), (KIF1B, SEC22B), (VIT1A, TAB2), (DNM2, WAC). PRKACB mutations were found in 3 patients. NOD2, PTPN2, PRKAG1, SEC22B, ULK4, VPS11 and WAC mutations were found in 2 patients. inces autophagy genes has been Loss of function mutations were observed in ULK4, NOD2 and WAC. Four genes mapped to commonly deleted regions e.g., 5q (CDKN2AIPNL, SQSTM1) or 7q (SMURF1, PRKAG2) and coincided with haploinsufficient expression, while 3 genes had hemizygous configuration (SMURF1, PPP2R3A, PIK3C2G). Reactome analysis clustered the mutations in effectors/inhibitors and early stage. The analysis of 263,973 germline variants detected that PIK3C2G, NOD2 and HIF1A were also associated with exonic germline variants predicted to be significantly deleterious. Mutations or aberrant expression of core components of the autophagy network have been associated with poor outcomes in multiple diseases. In our cohort, 21 patients died. Most (57%;21/37) of patients had abnormal karyotype with 4 patients having complex karyotype including -17 and -7. Among the cases with abnormal karyotype, 4 cases had del(5q). Mutant patients had worse survival trending toward significance compared to WT patients (MUTvs. WT=20 vs. 90; median 14 vs. 20 months; LogR=.09). Among disease groups, autophagy mutations were associated with significantly inferior survival in MDS (MUT vs. WT=13 vs. 61; 17 vs. 35 months; LogR=.018) and MDS/MPN (MUT vs. WT=4 vs. 31; 12 vs. 30 months; LogR=.037). Seven mutant patients who received hypomethylating agents had no response. Comparative analyses (Sanger, TruSeq, WES, TCGA) identified that autophagy gene mutations were significantly associated with TET2 (28%;11/40; P=.02) among other mutations [RUNX1, STAG2 (20%;8/40), SRSF2 (18%;7/40), DNMT3A, ASXL1 (15%;6/40)]. Clonal hierarchy showed that autophagy gene mutations were mainly secondary events, were ancestral events in 7 (ATG2A, DNML1, PRKACB, PRKAG1, PTPN2, SEC22B, STAT3) and co-dominant in 2 patients (NOD2, MLST8). When autophagy genes mutations were secondary, the most represented ancestral mutationswere in splicing factors (N=9; SRSF2, PRPF8, U2AF1) and DNA methylation (N=4; TET2, DNMT3A). RNA sequencing determined that changes in autophagy gene expression are overrepresented in specific MDS subtypes with distinct mutational profiles. The expression levels of 2 ULK family members commonly elevated during erythroid maturation were found in SF3B1K700E compared to SF3B1WT MDS patients (N=6; ULK1, FC=2; ULK3, FC=4; P=.05). Erythroid cells of these SF3B1K700E patients showed increased autophagosomes compared to SF3B1WT cells. In vivo administration of the mTOR inhibitor, temsirolimus (10 mg/kg i.p. 5d/week for 2 wk) improved the erythropoiesis of a transgenic Sf3b1 mouse model by increasing CD71+ cells (10-20% vs. 5%) and ameliorating anemia (Hgb: 7.9 vs. 6.6 g/dL; P=.07; MCV: 43.5 vs. 42.4 fL; P=.08). In sum, defects in autophagy genes are present in MDS, co-occur with other mutations and impact survival. Changes in expression levels of autophagy genes may be associated with MDS phenotypes and modulated by autophagy inducing drugs as evidenced in models of SF3B1 mutations. Disclosures Kelly: Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Speakers Bureau. Maciejewski:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Speakers Bureau.

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Ribeiro, André Mauric F., Leticia P. Sanglard, Hiruni R. Wijesena, Daniel C. Ciobanu, Steve Horvath et Matthew L. Spangler. « DNA methylation profile in beef cattle is influenced by additive genetics and age ». Scientific Reports 12, n^o 1 (14 juillet 2022). http://dx.doi.org/10.1038/s41598-022-16350-9.

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AbstractDNA methylation (DNAm) has been considered a promising indicator of biological age in mammals and could be useful to increase the accuracy of phenotypic prediction in livestock. The objectives of this study were to estimate the heritability and age effects of site-specific DNAm (DNAm level) and cumulative DNAm across all sites (DNAm load) in beef cattle. Blood samples were collected from cows ranging from 217 to 3,192 days (0.6 to 8.7 years) of age (n = 136). All animals were genotyped, and DNAm was obtained using the Infinium array HorvathMammalMethylChip40. Genetic parameters for DNAm were obtained from an animal model based on the genomic relationship matrix, including the fixed effects of age and breed composition. Heritability estimates of DNAm levels ranged from 0.18 to 0.72, with a similar average across all regions and chromosomes. Heritability estimate of DNAm load was 0.45. The average age effect on DNAm level varied among genomic regions. The DNAm level across the genome increased with age in the promoter and 5′ UTR and decreased in the exonic, intronic, 3′ UTR, and intergenic regions. In addition, DNAm level increased with age in regions enriched in CpG and decreased in regions deficient in CpG. Results suggest DNAm profiles are influenced by both genetics and the environmental effect of age in beef cattle.

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Wu, Hao, Wendi Zhou, Haijun Liu, Xudai Cui, Wenkui Ma, Haixin Wu, Guangdong Li et al. « Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned, ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer ». Journal of Animal Science and Biotechnology 13, n^o 1 (25 novembre 2022). http://dx.doi.org/10.1186/s40104-022-00764-6.

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Abstract Background SCNT (somatic cell nuclear transfer) is of great significance to biological research and also to the livestock breeding. However, the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods. This indicates the potential epigenetic variations between them. DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences. In this study, ASMT (acetylserotonin-O-methyltransferase) ovarian overexpression transgenic goat was produced by using SCNT. To investigate whether there are epigenetic differences between cloned and WT (wild type) goats, WGBS (whole-genome bisulfite sequencing) was used to measure the whole-genome methylation of these animals. Results It is observed that the different mCpG sites are mainly present in the intergenic and intronic regions between cloned and WT animals, and their CG-type methylation sites are strongly correlated. DMR (differentially methylated region) lengths are located around 1000 bp, mainly distributed in the exonic, intergenic and intronic functional domains. A total of 56 and 36 DMGs (differentially methylated genes) were identified by GO and KEGG databases, respectively. Functional annotation showed that DMGs were enriched in biological-process, cellular-component, molecular-function and other signaling pathways. A total of 10 identical genes related to growth and development were identified in GO and KEGG databases. Conclusion The differences in methylation genes among the tested animals have been identified. A total of 10 DMGs associated with growth and development were identified between cloned and WT animals. The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT. These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology, particularly in goats.

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Nagae, Genta, Shogo Yamamoto, Masashi Fujita, Takanori Fujita, Aya Nonaka, Takayoshi Umeda, Shiro Fukuda et al. « Genetic and epigenetic basis of hepatoblastoma diversity ». Nature Communications 12, n^o 1 (20 septembre 2021). http://dx.doi.org/10.1038/s41467-021-25430-9.

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AbstractHepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their “cell of origin” derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.

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Meulemans, Laëtitia, Stéphanie Baert Desurmont, Marie-Christine Waill, Gaia Castelain, Audrey Killian, Julie Hauchard, Thierry Frebourg et al. « Comprehensive RNA and protein functional assessments contribute to the clinical interpretation of MSH2 variants causing in-frame splicing alterations ». Journal of Medical Genetics, 16 septembre 2022, jmedgenet—2022–108576. http://dx.doi.org/10.1136/jmg-2022-108576.

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BackgroundSpliceogenic variants in disease-causing genes are often presumed pathogenic since most induce frameshifts resulting in loss of function. However, it was recently shown in cancer predisposition genes that some may trigger in-frame anomalies that preserve function. Here, we addressed this question by using MSH2, a DNA mismatch repair gene implicated in Lynch syndrome, as a model system.MethodsEighteen MSH2 variants, mostly localised within canonical splice sites, were analysed by using minigene splicing assays. The impact of the resulting protein alterations was assessed in a methylation tolerance-based assay. Clinicopathological characteristics of variant carriers were collected.ResultsThree in-frame RNA biotypes were identified based on variant-induced spliceogenic outcomes: exon skipping (E3, E4, E5 and E12), segmental exonic deletions (E7 and E15) and intronic retentions (I3, I6, I12 and I13). The 10 corresponding protein isoforms exhibit either large deletions (49–93 amino acids (aa)), small deletions (12 or 16 aa) or insertions (3–10 aa) within different functional domains. We showed that all these modifications abrogate MSH2 function, in agreement with the clinicopathological features of variant carriers.ConclusionAltogether, these data demonstrate that MSH2 function is intolerant to in-frame indels caused by the spliceogenic variants analysed in this study, supporting their pathogenic nature. This work stresses the importance of combining complementary RNA and protein approaches to ensure accurate clinical interpretation of in-frame spliceogenic variants.

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AHLAWAT, SONIKA, NEHA SAROVA, REKHA SHARMA, REENA ARORA et M. S. TANTIA. « Promoter DNA methylation and expression analysis of PIWIL1 gene in purebred and crossbred cattle bulls ». Indian Journal of Animal Sciences 89, n^o 7 (26 juillet 2019). http://dx.doi.org/10.56093/ijans.v89i7.92014.

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Major credit for India being the largest producer of milk in the world, goes to crossbred cows produced by inseminating low-producing indigenous cattle with semen from high producing exotic bulls. However, over the years, the policy of crossbreeding has been confronted with a major problem of subfertility in crossbred male progenies, culminating into disposal of a major fraction of mature bulls. Many studies have demonstrated relationship between epigenetic alterations and male fertility across different species. PIWIL1 is an important candidate gene for spermatogenesis and germ line development. Negative correlation between DNA methylation and expression of this gene has been highlighted in inter species hybrids of cattle and yaks. The present study envisaged elucidating promoter methylation status and expression profile of PIWIL1 gene in exotic Holstein Friesian cattle, indigenous Sahiwal cattle and their crossbreds with varying semen motility parameters. Semen samples were collected from bulls for isolation of DNA and RNA from spermatozoa. Bisulfite converted DNA was used to amplify promoter of PIWIL1 gene using methylation specific primers. The amplified products were sequenced after cloning in pTZ57R/ T vector. The degree of methylation of the PIWIL1 promoter region was significantly higher in poor motility crossbred bulls (7.17%) as compared to good motility crossbreds (1.02%), Sahiwal (1.02%) and Holstein Friesian bulls (0.77%). PIWIL1 expression was 1.75, 1.71 and 1.59 folds higher in HF, Sahiwal and good motility crossbreds, respectively as compared to poor motility crossbreds.

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Cui, Can, Zhen Wang, Yingjuan Su et Ting Wang. « Antioxidant Regulation and DNA Methylation Dynamics During Mikania micrantha Seed Germination Under Cold Stress ». Frontiers in Plant Science 13 (8 avril 2022). http://dx.doi.org/10.3389/fpls.2022.856527.

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As a primary goal, adaptation to cold climate could expand an invasion range of exotic plants. Here, we aimed to explore the regulation strategy of M. micrantha seed development under cold stress through molecular physiology and multi-omics analysis. Significant increase of hydrogen peroxide, malondialdehyde, and electrolyte leakage observed under cold stress revealed that oxidative damage within M. micrantha seed cells was induced in the initial germination phase. Proteomic data underscored an activation of antioxidant activity to maintain redox homeostasis, with a cluster of antioxidant proteins identified. Genomic-wide transcriptome, in combination with time-series whole-genome bisulfite sequencing mining, elucidated that seven candidate genes, which were the target of DNA demethylation-dependent ROS scavenging, were possibly associated with an M. micrantha germ break. Progressive gain of CHH context DNA methylation identified in an early germination phrase suggested a role of a DNA methylation pathway, while an active DNA demethylation pathway was also initiated during late seed development, which was in line with the expression trend of methylation and demethylation-related genes verified through qRT-PCR. These data pointed out that cold-dependent DNA demethylation and an antioxidant regulatory were involved together in restoring seed germination. The expression level of total 441 genes presented an opposite trend to the methylation divergence, while the expression of total 395 genes was proved to be negatively associated with their methylation levels. These data provided new insights into molecular reprograming events during M. micrantha seed development.

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Liu, Ling, Hai Nguyen, Urmi Das, Samuel Ogunsola, Jiankun Yu, Lei Lei, Matthew Kung, Shervin Pejhan, Mojgan Rastegar et Jiuyong Xie. « Epigenetic control of adaptive or homeostatic splicing during interval-training activities ». Nucleic Acids Research, 25 avril 2024. http://dx.doi.org/10.1093/nar/gkae311.

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Abstract Interval-training activities induce adaptive cellular changes without altering their fundamental identity, but the precise underlying molecular mechanisms are not fully understood. In this study, we demonstrate that interval-training depolarization (ITD) of pituitary cells triggers distinct adaptive or homeostatic splicing responses of alternative exons. This occurs while preserving the steady-state expression of the Prolactin and other hormone genes. The nature of these splicing responses depends on the exon's DNA methylation status, the methyl-C-binding protein MeCP2 and its associated CA-rich motif-binding hnRNP L. Interestingly, the steady expression of the Prolactin gene is also reliant on MeCP2, whose disruption leads to exacerbated multi-exon aberrant splicing and overexpression of the hormone gene transcripts upon ITD, similar to the observed hyperprolactinemia or activity-dependent aberrant splicing in Rett Syndrome. Therefore, epigenetic control is crucial for both adaptive and homeostatic splicing and particularly the steady expression of the Prolactin hormone gene during ITD. Disruption in this regulation may have significant implications for the development of progressive diseases.

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Articles de revues sur le sujet « Exonic DNA methylation »

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