Novel O-GlcNAcylation on Thr71 of histone H4 preferentially localizes to active gene regions
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ABSTRACT: We report genome-wide distribution of newly discovered O-GlcNAcylated H4 at threonine 71 (H4T71Gc) in mouse embryonic stem cells (mESCs, J1 line) cultured in 25 mM glucose (mESCs-25mMG) or 1 mM glucose (mESCs-1mMG) condition, human embryonic stem cells (hESCs, H9) and human induced-pluripotent stem cells (hiPSCs, 201B7). We found that H4T71Gc preferentially localize to gene regions, especially those with relatively high expression levels. Interestingly, the localized gene region of H4T71Gc changes in response to changes in extracellular glucose concentration. This study using ChIP-seq analysis provides genomic distribution of novel histone O-GlcNAcylation in mouse and human pluripotent stem cells.
Project description:We report genome-wide distribution of O-GlcNAcylated H2A at serine 40 (H2AS40Gc) in mouse embryonic stem cells (mESCs, J1 line) cultured in 25 mM glucose (HG-mESCs) or 1 mM glucose (LG-mESCs) condition. We found that H2AS40Gc was mainly located at genic area, positively correlated with the gene expression both in HG- and LG-mESCs. Interestingly, H2AS40Gc localization was overlapped with H2AX, γH2AX and O-GlcNAc transferase (Ogt), and varied by extracellular glucose concentration. This study using ChIP-seq and RNA-seq analysis provides genomic distribution of newly O-GlcNAc histone modification in mESCs.
Project description:J1 mouse embryonic stem cells (mESCs) and NIH-3T3 fibroblasts were grown in standard media conditions. Hybridized cells were fixed with 4% paraformaldehyde; permeabilized in 70% ethanol; incubated for 12 hours at 30C in an RNA preserving hybridization (RPH) buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K).
Project description:To know exactly how SB431542 contribute to mouse embryonic stem cells (mESCs) undifferentiated state maintenance, microarray experiment for DMSO mock treated and SB431542 treated J1 mESCs was performed J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented without or with SB431542 for 24 hours, then total RNA was extracted for analysis.
Project description:To know exactly how SB431542 contribute to mouse embryonic stem cells (mESCs) undifferentiated state maintenance, microarray experiment for DMSO mock treated and SB431542 treated J1 mESCs was performed
Project description:PD0325901 is involved in improving reprogramming efficiency during inducing pluripotent stem cells (iPSC) or maintain a blastocyst-like state in embryonic stem cells (ESC). However, the knowledge about this small molecule regulating miRNAs in ESC was limited. To understand the role of miRNAs during PD03-induced ESC maintenance and gain an insight how PD0325901 regulates miRNAs expression; we performed small RNA sequencing using Illumina HiSeq 2000 under the compounds treatment. The data show the miRNAs regulated by PD0325901. J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented with PD0325901 for 24 hours, J1 treated with DMSO was set as control. Then total RNA was extracted for analysis.
Project description:J1 mouse embryonic stem cells (mESCs) and NIH-3T3 fibroblasts were grown in standard media conditions. Hybridized cells were fixed with 4% paraformaldehyde; incubated for 12 hours at 30C in an RNA preserving hybridization (RPH) buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C in with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM pH 8.0 Tris, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K). one replicate per sample
Project description:We established induced pluripotent stem cells (iPSC) from centrenarians by retroviral transduction of primary human fibroblasts. To show the similarity between 201B7 iPSC and 100-1 #16 iPSC (induced pluripotent stem cells from centenarian), this experiment was designed. Undifferentiated 201B7 iPSC, and 100-1 #16 iPSCs were collected. Then, they were applied in this experiment.
Project description:We report genome-wide distribution of germ cell-specific linker histone variant H1T in cancer cell line AGS, MDA-MB-231, and mouse embryonic stem cells (mESCs). We found that H1T expressed not only testis but also non-germ cells such as cancer cells and pluripotent stem cells and showed the biased distribution at rDNA repeat unit. Moreover, on the rDNA region, H1T regulated the chromatin structure and pre-rRNA expression. This study using ChIP-seq analysis provides genomic distribution of H1T in non-germinal cells. ChIP-seq analysis of linker histone H1T in AGS, MDA-MB-231 and mESCs
Project description:We established induced pluripotent stem cells (iPSC) from centrenarians by retroviral transduction of primary human fibroblasts. To show the similarity between 201B7 iPSC and 100-1 #16 iPSC (induced pluripotent stem cells from centenarian), this experiment was designed.
Project description:It has been demonstrated that vitamin C enhances reprogramming efficiency during inducing pluripotent stem cells, however, the underlying mechanisms are not fully understood. To find the downstream target genes of vitamin C and investigate the mechanism of vitamin C on reprogramming promotion, we performed Microarray analyses to identify its downstream targets. Retinoic acid (RA), a stimulus molecule for cellular differentiation, is set as negative control. The data show the genes regulated by vitamin C or RA. J1 mESCs maintained in medium containing 1000 U/mL LIF and supplemented without or with vitamin C or RA for 24 hours, then total RNA was extracted for analysis.