Unknown,Transcriptomics,Genomics,Proteomics

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Zic3 ChIP-on-chip in mouse ES cells

ABSTRACT: The transcription factor Zic3 is required for maintenance of embryonic stem (ES) cell pluripotency (Lim LS et al, Mol Biol Cell. 2007;18:1348-1358). By genome-wide chromatin immunoprecipitation (ChIP-chip) in ES cells, we have identified 379 direct Zic3 targets, many of which are functionally associated with pluripotency, cell cycle, proliferation, oncogenesis and early embryogenesis. E14 cells were cultured to a density of 1 x 108 cells for each IP. Two biological replicates were performed per experiment. Cells were cross-linked for 10 minutes at room temperature with 1% (w/v) formaldehyde and the reaction subsequently quenched with 125mM glycine. Nuclear fractions were isolated and the DNA sheared to average lengths of 200-500 bp. For analysis on mouse promoter arrays, purified ChIP material was processed according to the Agilent ChIP-on-chip protocol, and labeled DNA was hybridized to Agilent mouse promoter ChIP-on-chip arrays for 40 hours at 65oC (G4490A; Agilent Technologies, Santa Clara, CA). Chips were washed and scanned as per manufacturer’s protocol and the results were processed with Agilent's ChIP Analytics software v1.3. A p-value cutoff <0.001 was specified in our analysis. To further minimize false positives, we applied a “neighborhood voting” algorithm [2] to filter for high confidence Zic3-enriched sites, wherein binding was considered genuine only in the presence of a second, significantly enriched, neighboring probe (p < 0.005).

ORGANISM(S): Mus musculus

SUBMITTER: Linda Lim

PROVIDER: E-GEOD-22195 | 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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Zic3 ChIP-on-chip in mouse ES cells

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