Project description:In this study, MMRd metastatic colorectal cancer (mCRC) patients who failed standard therapies will undergo treatment with pembrolizumab, while RAS-extended mutated MMR-proficient mCRC patients will be tested for o6-methylguanine-DNA-methyltransferase (MGMT) expression (IHC) and then for MGMT promoter methylation. MGMT IHC-negative, promoter methylation positive patients will be treated with temozolomide (TMZ). Patients progressing under temozolomide will be tested for tumor mutational burden (TMB) and proceed to pembrolizumab if TMB is > 20 mutations/Mb. The primary study hypothesis is that tumors with acquired resistance to temozolomide become hypermutated and are sensitive to pembrolizumab.
Project description:<h4><strong>INTRODUCTION:</strong> The identification of frequent acquired mutations shows that patients with oligodendrogliomas have divergent biology with differing prognoses regardless of histological classification. A better understanding of molecular features as well as their metabolic pathways is essential.</h4><h4><strong>OBJECTIVES: </strong>The aim of this study was to examine the relationship between the tumor metabolome, six genomic aberrations (isocitrate dehydrogenase1 [IDH1] mutation, 1p/19q codeletion, tumor protein p53 [TP53] mutation, O<sup>6</sup>-methylguanin-DNA methyltransferase [MGMT] promoter methylation, epidermal growth factor receptor [EGFR] amplification, phosphate and tensin homolog [PTEN] methylation), and the patients' survival time.</h4><h4><strong>METHODS:</strong> We applied <sup>1</sup>H high-resolution magic-angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy to 72 resected oligodendrogliomas.</h4><h4><strong>RESULTS:</strong> The presence of IDH1, TP53, 1p19q codeletion, MGMT promoter methylation reduced the relative risk of death, whereas PTEN methylation and EGFR amplification were associated with poor prognosis. Increased concentration of 2-hydroxyglutarate (2HG), N-acetyl-aspartate (NAA), myo-inositol and the glycerophosphocholine/phosphocholine (GPC/PC) ratio were good prognostic factors. Increasing the concentration of serine, glycine, glutamate and alanine led to an increased relative risk of death.</h4><h4><strong>CONCLUSION:</strong> HRMAS NMR spectroscopy provides accurate information on the metabolomics of oligodendrogliomas, making it possible to find new biomarkers indicative of survival. It enables rapid characterization of intact tissue and could be used as an intraoperative method.</h4>
Project description:Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.
Project description:DNA methylation is an essential epigenetic mark that is required for normal development. Knockout of the DNA methyltransferase enzymes in the mouse hematopoietic compartment reveals that methylation is critical for hematopoietic differentiation. To better understand the role of DNA methylation in hematopoiesis, we characterized genome-wide DNA methylation in primary mouse hematopoietic stem cells (HSC), common myeloid progenitors (CMP), and erythroblasts (ERY). Methyl Binding Domain protein 2 (MBD) enrichment of DNA followed by massively-parallel sequencing (MBD-Seq) was used to map genome-wide DNA methylation. Globally, DNA methylation was most abundant in HSC, with a 40% reduction in CMP, and 67% reduction in ERY. Only 3% of peaks arise during differentiation demonstrating a genome-wide decline in DNA methylation during erythroid development. Analysis of genomic features revealed that 98% of promoter CpG islands are hypomethylated, while 20-25% of non-promoter CpG islands are methylated. Proximal promoter sequences of expressed genes are hypomethylated in all cell types, while gene body methylation positively correlates with gene expression in HSC and CMP. Elevated genome-wide DNA methylation in HSC and the positive association between methylation and gene expression demonstrates that DNA methylation is a mark of cellular plasticity in HSC. Utilizing de novo motif discovery we identified overrepresented transcription factor consensus binding motifs in methylated sequences. Motifs for several ETS transcription factors, including GABPalpha and ELF1 are overrepresented in methylated regions. Our genome-wide survey demonstrates that DNA methylation is markedly altered during myeloid differentiation and identifies critical regions of the genome and transcription factor programs that contribute to hematopoiesis. Examination of changes in methylation profiles during hematopoietic stem cell differentiation
Project description:MOM1 is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we found that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with artificial zinc finger to unmethylated FWA promoter led to establishment of DNA methylation and FWA silencing. This process was blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MORC6. We found that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes was impaired by mutation of MOM1 or mutation of genes encoding the MOM1 interacting PIAL1/2 proteins. In addition to RdDM sites, we identified a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation.
Project description:We report the generation and analysis of high-throughput DNA methylation profiles at nucleotide resolution in a subset of targeted gene trap mouse mutants. Using high-throughput sequencing of bisulfite treated DNA, we generated DNA methylation percentage for CpG islands, and LacZ (reporter) gene in mice with the apparent silencing of the targeted gene promoter reflected by reduced reporter mRNA level. These results were contrasted with findings for a set of mutants with no silencing or CpG methylation following targeted mutagenesis using the same gene trap vector. Our findings supports the hypothesis that presence of the exogenous DNA in the targeting vector may influence the expression of genes in close proximity or may lead to promoter silencing of the target where the promoter is marked by CpG methylation. Examination of CpG methylation profiles in Knock-out and wild type mice We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. CpG Islands (samples labeled as CpG) and trans gene (samples labeled as LacZ) were amplified after Bisulfite treatment. Please note that the same gDNA was used to amplify CpG Island (Gene_CpG_KO ) and LacZ (Gene_LacZ_KO) reporter for the same gene. PCR product of amplification was gel separated, isolated and pooled. After libraries were prepared and sequenced, the alignment was performed. CpG island and LacZ alignments were done separately resulting in three different Processed Data files per gene investigated: Gene_CpG_KO, Gene_LacZ_KO and Gene_CpG_WT. LacZ reference is included in the submission, but is also available for download from KOMP Phenotype website (www.kompphenotype.org). Also please note that the libraries were prepared using Illumina TruSeq RNA Sample Prep Kit starting from adapter binding step as samples were double stranded Bisulfite treated DNA amplicons. So the library preparation was done as with RNASeq, but samples investigated were bisulfite treated.
Project description:Aberrant DNA hypermethylation is a major epigenetic mechanism to inactivate tumor suppressor genes in cancer, and Epstein-Barr virus (EBV) positive gastric cancer is known as the most frequently hypermethylated tumor among whole human malignancies. We here performed comprehensive analysis of epigenomic alteration during EBV infection, by Infinium HumanMethylation 450K BeadChip for DNA methylation and ChIP-sequencing for histone modification alteration. While 7,727 genes showed increase of DNA methylation in a promoter region, roughly half of these were “DNA methylation-sensitive” genes including anti-oncogenic genes e.g. CDKN2A and CDH1, which acquired DNA methylation in the whole promoter regions, thus leading to gene repression. Another half were “DNA methylation-resistant” genes including DNA repair genes, where DNA methylation is acquired in the surrounding of promoter regions but unmethylated status is protected in the vicinity of transcription start site, with gene expression retained. Histone modification alteration also occurred dynamically during EBV infection, in coordinated manner with DNA methylation alteration. DNA methylation-sensitive genes significantly correlated with loss of H3K27me3 pre-marks or decrease of active histone marks e.g. H3K4me3 and H3K27ac, and apoptosis-related genes were significantly enriched in these epigenetically repressed genes. Gain of active histone marks significantly correlated with DNA methylation-resistant genes, and genes related to mitotic cell cycle and DNA repair were significantly enriched in these epigenetically activated genes. Orchestrated epigenetic alterations play an important role in gene regulation during EBV infection, and histone modification status in promoter regions significantly associated with acquisition of de novo DNA methylation or protection of unmethylated status at TSS.
Project description:DNA methylation at proximal promoters facilitates lineage restriction by silencing cell-type specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such non-proximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent non-proximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, non-promoter DNA methylation by Dnmt3a may be utilized for maintaining active chromatin states of genes critical for development.