Chromatin-associated protein kinase C-0 regulates an inducible gene expression program and microRNAs in human T lymphocytes
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE26035 | GEO | 2010/12/14
SECONDARY ACCESSION(S): PRJNA135505
REPOSITORIES: GEO
Similar Datasets
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.
2010-12-14 | E-GEOD-26035 | biostudies-arrayexpress
Project description:Adaptive immune responses to infection result in the formation of memory T and B cells that respond more rapidly and robustly to reinfections, providing the basis of the immunological memory targeted by vaccines. Underlying the enhanced responsiveness of memory cells is their ability to rapidly up-regulate the transcription of key effector genes at a higher level compared to naïve cells (termed transcriptional memory). While transcriptionally permissive histone modifications are known to provide chromatin structures that facilitate transcriptional memory, the molecular mechanisms that underpin this process still remain elusive. The role of the cytoplasmic signalling kinase, PKC-θ in T cell signalling cascades is well established, however PKC-θ has also recently been described as a chromatin-modifying enzyme. We have previously demonstrated that PKC-θ forms a part of an active transcription complex that docks at the IL2 gene - a gene which displays transcriptional memory. In this study, we perturbed PKC-θ expression using approaches such as siRNA knockdown in primary human naïve and memory CD4+ T cells to show that transcriptional memory is highly dependent on PKC-θ, particularly in memory CD4+ T cells. Chromatin-tethered PKC-θ was immuno-precipitated with high levels of the activating marks, H3K4me3 and H3K9ac and low levels of the repressive mark, H3K27me3 at the IL2 promoter in ex vivo-derived, activated memory CD4+ T cells. Further ChIP-sequencing analysis revealed that PKC-θ was prevalent at transcriptionally permissive domains such as promoters, upstream regions and exons but also at intronic regions containing putative enhancers annotated to a subset of genes preferentially expressed in memory CD4+ T cells. Collectively, these data argue that chromatin-tethered PKC-θ can directly regulate genes to establish and maintain a permissive chromatin state that facilitates transcriptional memory in human CD4+ T cells.
2016-05-01 | GSE61241 | GEO
Project description:Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-q) regulates EMT by acting as a critical chromatin-anchored switch for inducible genes via TGF-M-NM-2 and the key inflammatory regulatory protein, NFkB. Chromatinized PKC-q exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-q-sensitive genes that are directly tethered to PKC-q in the mesenchymal state. Collectively, we show that crosstalk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer. 2 biological samples were analysed, Immunoprecipitated and total input samples were obtained from each biological treatment. 2 Technical replicates were performed (samples from the sample lib prep were run on two different lanes).
2014-06-22 | E-GEOD-53320 | biostudies-arrayexpress
Project description:Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) regulates EMT by acting as a critical chromatin-anchored switch for inducible genes. Genome-wide transcriptome analysis identifies a unique cohort of inducible PKC-θ-sensitive genes in MCF7 cells stimulated with phorbol ester. MCF7 Cells treated with mock or siRNA against PKCtheta were left unstimulated or stimulated with PMA. No replicates.
2014-06-22 | E-GEOD-53267 | biostudies-arrayexpress
Project description:Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-q) regulates EMT by acting as a critical chromatin-anchored switch for inducible genes via TGF-β and the key inflammatory regulatory protein, NFkB. Chromatinized PKC-q exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-q-sensitive genes that are directly tethered to PKC-q in the mesenchymal state. Collectively, we show that crosstalk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer.
2014-06-22 | GSE53320 | GEO
Project description:Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) regulates EMT by acting as a critical chromatin-anchored switch for inducible genes. Genome-wide transcriptome analysis identifies a unique cohort of inducible PKC-θ-sensitive genes in MCF7 cells stimulated with phorbol ester.
2014-06-22 | GSE53267 | GEO
Project description:Aberrant TGFbeta signalling is a hallmark of epithelial derived tumours. Signalling patterns can depend on the membrane trafficking and internalization of the TGFbeta receptors. Protein kinase C (PKC), particularly the atypical PKC isoforms, alter the trafficking of TGFbeta receptors and can alter TGFbeta induced gene expression. We used microarrays to detail the programme of gene expression underlying TGFbeta induction between control or aPKC silenced A549 cells.
2011-01-29 | GSE26241 | GEO
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 μM and phorbol 12-myristate 13-acetate 0.1 μM 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 Forskolin treatments (total of 4 replicates).
2016-07-20 | E-GEOD-84541 | biostudies-arrayexpress
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 μM and phorbol 12-myristate 13-acetate 0.1 μM 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).
2016-07-20 | E-GEOD-84542 | biostudies-arrayexpress
Project description:While Argonaute (AGO) proteins play a major role in transcriptional gene silencing (TGS) in many organisms, their role in the nucleus of somatic mammalian cells remains elusive. Here, we have purified AGO1 and AGO2 chromatin-embedded complexes, and found these proteins associated with previously described partners, but also with chromatin modifiers and, rather unexpectedly, with different splicing factors. Using the CD44 gene as a model for alternative splicing, we show that both AGO1 and AGO2 are required for Protein Kinase C (PKC)-dependent variant exon inclusion. AGO proteins facilitate the spliceosome recruitment and modulate the elongation rate of RNA polymerase II (RNAPII). The recruitment of AGO proteins to CD44 transcribed region is dependent on both the endonuclease Dicer and the chromodomain-containing protein HP1g, and results in locally increased levels of histone H3 lysine 9 (H3K9) methylation on variant exons. Genome wide analysis of splicing in either AGO2 or Dicer null cells showed that the two proteins have similar effects on many splicing events. Finally, sRNAs associated with nuclear AGO2 are mostly in sense orientation relative to protein-coding genes, supporting a role for intragenic antisense non-coding RNAs in the recruitment AGO and splicing factors. Together, our data demonstrate for the first time that the endogenous RNAi pathway is involved in alternative splicing decisions, unravelling a new model in which AGO proteins couple RNAPII elongation and chromatin modification. Deep sequencing of small RNAs (approx. 15-80 nucleotides) bound to either cytoplasmic or chromatin-associated AGO2 complex.
2012-09-10 | E-GEOD-39748 | biostudies-arrayexpress