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Chromatin-associated protein kinase C-0 regulates an inducible gene expression program and microRNAs in human T lymphocytes

ABSTRACT: 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

ORGANISM(S): Homo sapiens

PROVIDER: GSE26035 | GEO | 2010/12/14

SECONDARY ACCESSION(S): PRJNA135505

REPOSITORIES: GEO

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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.

Project description:The oocyteâs capacity to complete maturation, to succeed fertilization and to reach the blastocyst stage is what defines the oocyteâs competence. The oocyte acquires this competence working closely with somatic cells of the follicle. Cumulus and granulosa cells provided support for the oocyteâs development and conversely the oocyte influence follicular cell growth and differentiation. Existing studies support the idea that follicular-stimulated hormone and luteinizing hormone play an essential role in oocyte competence acquisition through protein kinase A (PKA) and protein kinase C (PKC) signalling in granulosa cells. Therefore, human-like granulosa cells (KGN) were treated with forskolin 10 Î¼M and phorbol 12-myristate 13-acetate 0.1 Î¼M for 24 hours in order to process a transcriptomic analysis of differentially express genes between treatment. Over 2000 genes were founded to be differentially express at cut-off fold change of 1.5 and a p-value of 0.05. Five major upstreams, EGF, TGFB1, VEGF, FGF2 and HGF were founded to play an important role in competence acquisition thought PKA and PKC signalling. Differentially expressed targeted genes of both signalling pathways were classified in seven major ovarian functions such as PTGS2, IL8 and IL6 in inflammation, STAR, CYP11A1, CYP19A1 in steroidogenesis, VEGFC, VEGFA, CXCR4 in angiogenesis, AREG, EGFR, SPRY2 in differentiation, BAX, BCL2L12, CASP1 in apoptosis, CCND1, CCNB1, CCNB2 in division and MMP1, MMP9, TIMP1 in ovulation. Taken together, the results of this study suggest that PKA and PKC signalling potentiate their effects in some functions such as inflammation and apoptosis while some others are more specific to one or the other protein kinase like differentiation, ovulation and angiogenesis that are thought to be more PKC-dependent in human granulosa cells. 8 samples were analysed, 4 controls compared to 4 Phorbol 12-myristate 13-acetate treatments (total of 4 replicates).

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Chromatin-associated protein kinase C-0 regulates an inducible gene expression program and microRNAs in human T lymphocytes

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