Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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DNA methylation and gene expression changes during human embryonic stem cell differentiation

ABSTRACT: Investigate the genome-wide DNA methylation and gene expression changes during human embryonic stem cell differentiation. hESCs (H1 and H13) DNA samples for 3 different differentian stage (D0, undifferentiated;D21, 21 days after undirected differentiation; D90, 90 days after undirected differentiation); two-color experiment, paired comparison (differentiated vs undifferentiated; A & B: two biological replicates). Differentian-stage variation in methylation profiling of human DNA samples were detected by MSAM. 2µg of DNA was serially digested with SmaI and XmaI followed by an adaptor ligation and adaptor mediated PCR amplification. Analysis of differentian-stage variation in gene expression of human mRNA samples were implementd following standard Agilent protocol.

ORGANISM(S): Homo sapiens

SUBMITTER: Robert Waterland

PROVIDER: E-GEOD-32371 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Studies in yeast have demonstrated that signalling kinases with well known cytoplasmic functions have a surprisingly active role in the nucleus, where they are tethered to chromatin and modulate gene expression programs. Here we provide evidence for a novel function of the signal transduction kinase, protein kinase C-theta (PKC-0) that physically associates with the proximal regulatory regions of key inducible immune response genes in human T cells. Chromatin-anchored PKC-q forms hitherto undescribed nuclear complexes by interacting with active RNA polymerase II, the histone kinase MSK-1 and the adaptor molecule 14-3-3z. ChIP-on-Chip analysis reveals that PKC-0 binds directly to both the promoter and transcribed regions of genes, as well as to the promoters of microRNA genes implicated in cell migration and invasion. Moreover, enforced expression of these microRNAs is associated with heightened production of mRNAs encoding a distinct subset of inducible immune response genes. Collectively, these data suggest that in addition to its well known role as a cytoplasmic signalling kinase, PKC-0 controls immune gene expression within the nucleus of T cells by participating in chromatin-associated signalling complexes PKC-0 and Pol II ChIP DNA were pooled from five independent ChIP assay experiments that were first individually validated by real-time PCR. Pooled ChIP samples were subsequently amplified based on one round of the whole genome amplification method using the WGA2 kit (Sigma-Aldrich), as described previously (O'Geen, Hollenhorst, Dindot). An alternate random primed and linker-mediated PCR amplification protocol was compared with the WGA2 kit using quantitative real-time PCR, which generated similar yields of amplified material, but failed to preserve the degree of enrichment after the linker-mediated PCR amplification (data not shown). Labelling, hybridization and scanning were performed as described in the Mammalian ChIP-on-chip protocol (version 9.1; Agilent Technologies). Briefly, the control and PKC-0 or Pol II ChIP DNA were labelled with 5 ug cyanine-3 and 5 ug cyanine-5 (Invitrogen BioPrime CGH labeling kit), respectively. Samples were hybridised on each microarray slide for 40 h at 65°C. Agilent human promoter microarrays were utilised comprising of two slides per set defined to cover ~17,000 promoters of human transcripts from -5.5 to +2.5 Kb relative to transcriptional start site. The microarrays were scanned on an Agilent scanner (G2565BA) at 100% PMT gain. Two replicates of each ChIP-on-chip experiment were performed.

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DNA methylation and gene expression changes during human embryonic stem cell differentiation

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