Project description:Background: Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications such as DNA methylation and the noncoding RNA-mediated mechanism. This results in the senescence of MSCs. We compared differential methylation patterns of genes and miRNAs between early-passage and late-passage cells and estimated the relationship between senescence and DNA methylation patterns. Genomic DNA of MSCs cultured at passage 5 (P5) and passage 15 (P15) was enriched using methylated DNA immunoprecipitation assays, and DNA methylation changes due to long-term culture of MSCs were analyzed using Human 2.1M Deluxe Promoter Arrays (NimbleGen). Results: When we analyzed the methylation differences between P5 and P15 more than twice, 3,338 genes showed more than two-fold higher methylation at P5 than P15, whereas 4,670 genes showed more than two-fold higher methylation at P15 than P5. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. Conclusion: These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.
Project description:MicroRNAs (miRNAs) regulate a spectrum of development and defense response processes in plants. The brown planthopper (BPH) is the most devastating insect pest of rice, BPH resistance gene BPH15 has a strong resistance to BPH. In this study, we analyzed six miRNA profiles of BPH15 introgression line (P15) and susceptible recipient line (PC) in 3 time points (0h, 6h and 48h) after BPH attacked, and identified 464 known miRNAs and 183 novel miRNAs. Before BPH feeding we identified 23 miRNAs expressed differently in P15 and PC. Then after BPH feeding, 104 miRNAs were found expressed differently in P15, and 80 miRNAs were found expressed differently in PC. Among them, miR167, miR444d, miR1846e, miR3979-3p, miR531 were found to be involved in BPH stress response. The response to BPH of P15 was much wider and more rapidly than PC. The levels of a subset of miRNAs were confirmed by qRT-PCR. The targets of miRNAs were predicted and validated by gene expression profiling. Additionally, the target genes of 2 differently expressed miRNAs (miR160f-5p and miR167a-5p) were confirmed by detecting YFP fluorescence and western blotting. Our data provide an important basis for evaluating the role of miRNA in the regulation of BPH interactions in rice.
Project description:Aging is associated with an abnormal increase of DNA methylation in human gene promoters, including in bone marrow stem cells. DNA methylation patterns are further perturbed in hematological malignancies such as acute myeloid leukemia (AML) but the physiological significance of such epigenetic changes is unknown. Using epigenetic editing of human stem/progenitor cells (HSPCs), we show that p15 methylation affects hematopoiesis in vivo. We edited the CDKN2B (p15) promoter and ARF (p14) using dCas9-3A3L and observed DNA methylation spreading beyond the gRNA location. We find that despite a transient delivery system, DNA methylation is maintained during myeloid differentiation in vitro, and hypermethylation of the p15 promoter reduces gene expression. In vivo, edited human HSPCs can engraft the bone marrow of mice and targeted DNA methylation is maintained in HSPCs long term. Moreover, epigenetic changes are conserved and inherited in both myeloid and lymphoid lineages. Although the proportion of myeloid (CD33+) and lymphoid (CD19+) cells is unaffected, monocyte (CD14+) populations decreased and granulocytes (CD66b+) increased in mice engrafted with p15 hypermethylated HSPCs. Monocytes derived from p15 hypermethylated HSPCs appear to be activated and show increased inflammatory transcriptional programs. We believe these findings have clinical relevance since we found p15 promoter methylation in the peripheral blood of patients with clonal hematopoiesis. Our study shows DNA methylation can be targeted and maintained in human HSPCs and demonstrated functional relevance of aberrant DNA methylation on the p15 locus. As such, other ageing associated aberrant DNA methylation may impact hematopoiesis in vivo.
Project description:ChIP-seq of H3K27ac in P15 sciatic nerve was used to identify actively engaged enhancers in Schwann cells, which constitute the majority of nuclei in peripheral nerve.
Project description:Whole livers were collected from mice at the age of day 15 (P15). Total RNA was purified using Isogen. Gene expression was measured in livers at the age of 15 days (P15).