O-GlcNAc and the Epigenetic Regulation of Gene Expression

O-GlcNAcylation is an abundant nutrient-driven modification linked to cellular signaling and regulation of gene expression. Utilizing precursors derived from metabolic flux, O-GlcNAc functions as a homeostatic regulator. The enzymes of O-GlcNAc cycling, OGT and O-GlcNAcase, act in mitochondria, the cytoplasm, and the nucleus in association with epigenetic “writers” and “erasers” of the histone code. Both O-GlcNAc and O-phosphate modify repeats within the RNA polymerase II C-terminal domain (CTD). By communicating with the histone and CTD codes, O-GlcNAc cycling provides a link between cellular metabolic status and the epigenetic machinery. Thus, O-GlcNAcylation is poised to influence trans-generational epigenetic inheritance.     

Epigenetic Regulation Disturbances on Gene Expression in Imprinting Diseases

Molecular Biology - Epigenetic regulation is hereditary and non-hereditary changes in the expression of a particular gene without any corresponding structural changes in its nucleotide sequence....     

Tissue-Specific Effects of Genetic and Epigenetic Variation on Gene Regulation and Splicing

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans’ lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.     

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.     

Cell Density Impacts Epigenetic Regulation of Cytokine-Induced E-Selectin Gene Expression in Vascular Endothelium

Growing evidence suggests that the phenotype of endothelial cells during angiogenesis differs from that of quiescent endothelial cells, although little is known regarding the difference in the susceptibility to inflammation between both the conditions. Here, we assessed the inflammatory response in sparse and confluent endothelial cell monolayers. To obtain sparse and confluent monolayers, human umbilical vein endothelial cells were seeded at a density of 7.3×10³ cells/cm² and 29.2×10³ cells/cm², respectively, followed by culturing for 36 h and stimulation with tumor necrosis factor α. The levels of tumor necrosis factor α-induced E-selectin protein and mRNA expression were higher in the confluent monolayer than in the sparse monolayer. The phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase or nuclear factor-κB activation was not involved in this phenomenon. A chromatin immunoprecipitation assay of the E-selectin promoter using an anti-acetyl-histone H3 antibody showed that the E-selectin promoter was highly and specifically acetylated in the confluent monolayer after tumor necrosis factor α activation. Furthermore, chromatin accessibility real-time PCR showed that the chromatin accessibility at the E-selectin promoter was higher in the confluent monolayer than in the sparse monolayer. Our data suggest that the inflammatory response may change during blood vessel maturation via epigenetic mechanisms that affect the accessibility of chromatin.     

DNA Methylation-Dependent Epigenetic Regulation of Gene Expression in MCF-7 Breast Cancer Cells

To identify epigenetically-regulated genes in breast cancer, MCF-7 cells were exposed to 250nM 5-aza or 5-aza + 50nM TSA for 3 weeks followed by a 5 week recovery period after treatment withdrawal and gene expression patterns were examined by microarray analysis. We identified 20 genes that are associated with a >2-fold increase in expression in response to the demethylating treatment but returned to control levels after treatment withdrawal. RT-PCR verified that the genes identified were expressed at low or undetectable levels in control MCF-7 cells, but increased expression in treated cells. Most of these putative epigentically-regulated genes in MCF-7 cells do not contain CpG islands. In fact, these genes could be classified based upon their promoter CpG features, including genes with: (i) typical CpG features (CpG islands), (ii) intermediate CpG features (weak CpG islands), and (iii) atypical CpG features (no CpG islands). Prototype genes from each class (including CpG-deficient genes) were shown to be methylation-sensitive (subject to CpG methylation and responsive to demethylating agents), suggesting that not all gene targets of DNA methylation in breast cancer will contain a CpG island. Based upon the results of the current study and observations from the literature, we propose expansion of the current model for methylation-dependent regulation of gene expression to include genes lacking typical CpG islands. The expanded model we propose recognizes that all promoter CpG dinucleotides represent legitimate targets for DNA methylation and that the methylation of specific CpG dinucleotides in critical domains of regulatory regions can result in gene silencing.      

Pluripotency,Differentiation, and Reprogramming: A Gene Expression Dynamics Model with Epigenetic Feedback Regulation

Embryonic stem cells exhibit pluripotency: they can differentiate into all types of somatic cells. Pluripotent genes such as Oct4 and Nanog are activated in the pluripotent state, and their expression decreases during cell differentiation. Inversely, expression of differentiation genes such as Gata6 and Gata4 is promoted during differentiation. The gene regulatory network controlling the expression of these genes has been described, and slower-scale epigenetic modifications have been uncovered. Although the differentiation of pluripotent stem cells is normally irreversible, reprogramming of cells can be experimentally manipulated to regain pluripotency via overexpression of certain genes. Despite these experimental advances, the dynamics and mechanisms of differentiation and reprogramming are not yet fully understood. Based on recent experimental findings, we constructed a simple gene regulatory network including pluripotent and differentiation genes, and we demonstrated the existence of pluripotent and differentiated states from the resultant dynamical-systems model. Two differentiation mechanisms, interaction-induced switching from an expression oscillatory state and noise-assisted transition between bistable stationary states, were tested in the model. The former was found to be relevant to the differentiation process. We also introduced variables representing epigenetic modifications, which controlled the threshold for gene expression. By assuming positive feedback between expression levels and the epigenetic variables, we observed differentiation in expression dynamics. Additionally, with numerical reprogramming experiments for differentiated cells, we showed that pluripotency was recovered in cells by imposing overexpression of two pluripotent genes and external factors to control expression of differentiation genes. Interestingly, these factors were consistent with the four Yamanaka factors, Oct4, Sox2, Klf4, and Myc, which were necessary for the establishment of induced pluripotent stem cells. These results, based on a gene regulatory network and expression dynamics, contribute to our wider understanding of pluripotency, differentiation, and reprogramming of cells, and they provide a fresh viewpoint on robustness and control during development.     

人apoA-I分泌型表达调控细胞模型构建

分别从pMD18-T质粒和人基因组DNA扩增出人apoA-I CDS区序列和apoA-I启动子(702bp片段),与pEGFP-N1重组,构成受apoA-I启动子调控的pEGFP-N1质粒和融合蛋白表达质粒,分别转染人肝癌HepG2细胞,以绿色荧光为标志筛选稳定转染系列克隆。用RT-PCR、荧光显微镜、免疫荧光术等鉴定其中一个克隆融合蛋白的表达;分别以胰岛素和葡萄糖刺激物鉴定该克隆的外源apoA-I启动子调控。结果表明:人apoA-I分泌型表达调控肝细胞模型初步建成。