Dataset Information

Nuclear PKCq binding to DNA in T lymphocytes.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE61241 | GEO | 2016/05/01

SECONDARY ACCESSION(S): PRJNA260577

REPOSITORIES: GEO

ACCESS DATA

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.

Dataset Information

Nuclear PKCq binding to DNA in T lymphocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure