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Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Li J, Hardy K, Phetsouphanh C, Tu WJ, Sutcliffe EL, McCuaig R, Sutton CR, Zafar A, Munier CM, Zaunders JJ, Xu Y, Theodoratos A, Tan A, Lim PS, Knaute T, Masch A, Zerweck J, Brezar V, Milburn PJ, Dunn J, Casarotto MG, Turner SJ, Seddiki N, Kelleher AD, Rao S - J. Cell. Sci. (2016)

Bottom Line: Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation.This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood.Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Education, Science, Technology & Mathematics, University of Canberra, Canberra, Australian Capital Territory 2617, Australia Department of Microbiology & Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria 3010, Australia.

No MeSH data available.


Related in: MedlinePlus

Direct regulation of gene expression by nuclear PKC-θ. (A) Representative western blot of HA-tagged PKC-θ protein (HAPKC-θ) levels in non-stimulated (NS) and PMA and Ca2+ ionophore (P/I)-activated Jurkat T cells transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. (B) Representative confocal microscopy images showing subcellular localization of PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids. (C) Quantification of microscopy to show the ratio of nuclear to cytoplasmic-located HA-tagged PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids (mean±s.e.m., n=3 repeats). ***P<0.0001 (Mann–Whitney test). (D) Gene expression of transcriptional-memory-responsive genes in Jurkat T cells transfected with vector only (VO), wild-type (WT) and cytoplasm-restricted PKC-θ mutant plasmids (NLS) (mean±s.e.m., n=3) with the percentage inhibition calculated between the WT and NLS during primary (1°) activation for TNF and secondary (2°) activation for IL2, IL8, IL3, IFNG, CSF2, CCL4L1 and TNFSF9. NS, non-stimulated cells. *P≤0.05, **P≤0.01 and ***P≤0.001 (two-way ANOVA).
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JCS181248F5: Direct regulation of gene expression by nuclear PKC-θ. (A) Representative western blot of HA-tagged PKC-θ protein (HAPKC-θ) levels in non-stimulated (NS) and PMA and Ca2+ ionophore (P/I)-activated Jurkat T cells transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. (B) Representative confocal microscopy images showing subcellular localization of PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids. (C) Quantification of microscopy to show the ratio of nuclear to cytoplasmic-located HA-tagged PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids (mean±s.e.m., n=3 repeats). ***P<0.0001 (Mann–Whitney test). (D) Gene expression of transcriptional-memory-responsive genes in Jurkat T cells transfected with vector only (VO), wild-type (WT) and cytoplasm-restricted PKC-θ mutant plasmids (NLS) (mean±s.e.m., n=3) with the percentage inhibition calculated between the WT and NLS during primary (1°) activation for TNF and secondary (2°) activation for IL2, IL8, IL3, IFNG, CSF2, CCL4L1 and TNFSF9. NS, non-stimulated cells. *P≤0.05, **P≤0.01 and ***P≤0.001 (two-way ANOVA).

Mentions: To distinguish the cytoplasmic and nuclear roles of PKC-θ in transcriptional memory responses, a HA-tagged wild-type PKC-θ (HAPKC or WT) or a PKC-θ-NLS mutant (NLS-PKC) was expressed in Jurkat T cells. Although the total HAPKC-θ protein expression was similar for all PKC-θ constructs (Fig. 5A), a significant proportion of NLS-PKC-θ was restricted to the cytoplasm (Fig. 5B,C). Having a mutated NLS significantly reduced the expression of certain effector genes, such as TNF, during primary activation, but this inhibition was particularly evident during re-stimulation for IL2, IL8, IL3, CSF2, CCL4L1, IFNG and TNFSF9 (Fig. 5D). Using this novel PKC-NLS mutant, we showed that nuclear translocation of PKC-θ is required for optimal transcriptional memory responses.Fig. 5.


Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Li J, Hardy K, Phetsouphanh C, Tu WJ, Sutcliffe EL, McCuaig R, Sutton CR, Zafar A, Munier CM, Zaunders JJ, Xu Y, Theodoratos A, Tan A, Lim PS, Knaute T, Masch A, Zerweck J, Brezar V, Milburn PJ, Dunn J, Casarotto MG, Turner SJ, Seddiki N, Kelleher AD, Rao S - J. Cell. Sci. (2016)

Direct regulation of gene expression by nuclear PKC-θ. (A) Representative western blot of HA-tagged PKC-θ protein (HAPKC-θ) levels in non-stimulated (NS) and PMA and Ca2+ ionophore (P/I)-activated Jurkat T cells transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. (B) Representative confocal microscopy images showing subcellular localization of PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids. (C) Quantification of microscopy to show the ratio of nuclear to cytoplasmic-located HA-tagged PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids (mean±s.e.m., n=3 repeats). ***P<0.0001 (Mann–Whitney test). (D) Gene expression of transcriptional-memory-responsive genes in Jurkat T cells transfected with vector only (VO), wild-type (WT) and cytoplasm-restricted PKC-θ mutant plasmids (NLS) (mean±s.e.m., n=3) with the percentage inhibition calculated between the WT and NLS during primary (1°) activation for TNF and secondary (2°) activation for IL2, IL8, IL3, IFNG, CSF2, CCL4L1 and TNFSF9. NS, non-stimulated cells. *P≤0.05, **P≤0.01 and ***P≤0.001 (two-way ANOVA).
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Related In: Results  -  Collection

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JCS181248F5: Direct regulation of gene expression by nuclear PKC-θ. (A) Representative western blot of HA-tagged PKC-θ protein (HAPKC-θ) levels in non-stimulated (NS) and PMA and Ca2+ ionophore (P/I)-activated Jurkat T cells transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. (B) Representative confocal microscopy images showing subcellular localization of PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids. (C) Quantification of microscopy to show the ratio of nuclear to cytoplasmic-located HA-tagged PKC-θ in Jurkat T cells transfected with WT or cytoplasm-restricted PKC-θ (NLS) plasmids (mean±s.e.m., n=3 repeats). ***P<0.0001 (Mann–Whitney test). (D) Gene expression of transcriptional-memory-responsive genes in Jurkat T cells transfected with vector only (VO), wild-type (WT) and cytoplasm-restricted PKC-θ mutant plasmids (NLS) (mean±s.e.m., n=3) with the percentage inhibition calculated between the WT and NLS during primary (1°) activation for TNF and secondary (2°) activation for IL2, IL8, IL3, IFNG, CSF2, CCL4L1 and TNFSF9. NS, non-stimulated cells. *P≤0.05, **P≤0.01 and ***P≤0.001 (two-way ANOVA).
Mentions: To distinguish the cytoplasmic and nuclear roles of PKC-θ in transcriptional memory responses, a HA-tagged wild-type PKC-θ (HAPKC or WT) or a PKC-θ-NLS mutant (NLS-PKC) was expressed in Jurkat T cells. Although the total HAPKC-θ protein expression was similar for all PKC-θ constructs (Fig. 5A), a significant proportion of NLS-PKC-θ was restricted to the cytoplasm (Fig. 5B,C). Having a mutated NLS significantly reduced the expression of certain effector genes, such as TNF, during primary activation, but this inhibition was particularly evident during re-stimulation for IL2, IL8, IL3, CSF2, CCL4L1, IFNG and TNFSF9 (Fig. 5D). Using this novel PKC-NLS mutant, we showed that nuclear translocation of PKC-θ is required for optimal transcriptional memory responses.Fig. 5.

Bottom Line: Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation.This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood.Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Education, Science, Technology & Mathematics, University of Canberra, Canberra, Australian Capital Territory 2617, Australia Department of Microbiology & Immunology, The Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria 3010, Australia.

No MeSH data available.


Related in: MedlinePlus