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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

Identification of phosphorylated residues on histone H2B. (A) PKC-θ-mediated phosphorylation signals on H2B:21 (residues 21–40 derived from H2B) were detected by PKC-θ microarray profiling. The mean phosphorylation is shown (±s.d.). * denotes a pre-phosphorylated serine and the dotted red line is the background threshold (57500). The location of the histone H2B repression domain (HBR) is marked. (B) PKC-θ phosphorylates H2B Ser32. An in vitro kinase assay was performed by incubating active PKC-θ with either recombinant histones H2B, H3 or H4 or recombinant nucleosomes containing H3.1 or H3.3. Phosphorylated proteins were resolved by SDS-PAGE followed by western blotting for H2B phosphorylation. Negative controls include C1 (no ATP addition), C2 (no PKC addition), and C3 (incubation with PKC-μ). A representative blot of three experiments is shown. (C) PKC-θ phosphorylates H2B Ser36, as assessed by the method shown in B. (D) The Pearson's colocalization coefficient (PCC) was calculated for the fluorescent signal of H2B and PKC-θ as measured by confocal laser scanning microscopy in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations (mean±s.e.m., n=20). **P≤0.01, ****P≤0.0001 (Mann–Whitney test). (E) Representative immunoblot of phosphorylated H2B Ser32 and Ser36 in DMSO and 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (PI) (n=3). (F) Normalized H2B Ser32p densitometry is shown for human CD4+ T cells treated with either the control siRNA (siCtrl) or PKC-θ siRNA (siPKC-θ) (mean±s.e.m., n=3 individuals). ***P≤0.001 (two-tailed Student's t-test). (G) FAIRE chromatin accessibility shown for IL2 and TNF in in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. FAIRE chromatin accessibility is normalized to results for GAPDH (mean±s.e.m., n=3). ***P≤0.001 (two-way ANOVA). (H) FAIRE chromatin accessibility shown for IL2 and other promoters in naïve and memory CD4+ T cells treated with either the control (siCtrl) or the PKC-θ siRNA (siPKC-θ) with or without PMA and Ca2+ ionophore (P/I). FAIRE chromatin accessibility is normalized to GAPDH and expressed as a percentage relative to the stimulated (ST) memory CD4+ T cells treated with the control siRNA (siCtrl) (mean±s.e.m., n=3). *P≤0.05, **P<0.01 (unpaired two-tailed Student's t-test).
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JCS181248F7: Identification of phosphorylated residues on histone H2B. (A) PKC-θ-mediated phosphorylation signals on H2B:21 (residues 21–40 derived from H2B) were detected by PKC-θ microarray profiling. The mean phosphorylation is shown (±s.d.). * denotes a pre-phosphorylated serine and the dotted red line is the background threshold (57500). The location of the histone H2B repression domain (HBR) is marked. (B) PKC-θ phosphorylates H2B Ser32. An in vitro kinase assay was performed by incubating active PKC-θ with either recombinant histones H2B, H3 or H4 or recombinant nucleosomes containing H3.1 or H3.3. Phosphorylated proteins were resolved by SDS-PAGE followed by western blotting for H2B phosphorylation. Negative controls include C1 (no ATP addition), C2 (no PKC addition), and C3 (incubation with PKC-μ). A representative blot of three experiments is shown. (C) PKC-θ phosphorylates H2B Ser36, as assessed by the method shown in B. (D) The Pearson's colocalization coefficient (PCC) was calculated for the fluorescent signal of H2B and PKC-θ as measured by confocal laser scanning microscopy in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations (mean±s.e.m., n=20). **P≤0.01, ****P≤0.0001 (Mann–Whitney test). (E) Representative immunoblot of phosphorylated H2B Ser32 and Ser36 in DMSO and 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (PI) (n=3). (F) Normalized H2B Ser32p densitometry is shown for human CD4+ T cells treated with either the control siRNA (siCtrl) or PKC-θ siRNA (siPKC-θ) (mean±s.e.m., n=3 individuals). ***P≤0.001 (two-tailed Student's t-test). (G) FAIRE chromatin accessibility shown for IL2 and TNF in in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. FAIRE chromatin accessibility is normalized to results for GAPDH (mean±s.e.m., n=3). ***P≤0.001 (two-way ANOVA). (H) FAIRE chromatin accessibility shown for IL2 and other promoters in naïve and memory CD4+ T cells treated with either the control (siCtrl) or the PKC-θ siRNA (siPKC-θ) with or without PMA and Ca2+ ionophore (P/I). FAIRE chromatin accessibility is normalized to GAPDH and expressed as a percentage relative to the stimulated (ST) memory CD4+ T cells treated with the control siRNA (siCtrl) (mean±s.e.m., n=3). *P≤0.05, **P<0.01 (unpaired two-tailed Student's t-test).

Mentions: Given that PKC-θ directly associates with the chromatin template, we used histone peptide microarrays to identify potential PKC-θ substrates. Significant levels of PKC-θ-mediated phosphorylation was detected on H2B- and H2A-derived peptides, particularly those derived from H2B, SKKGFKKAVVKTQKKEGKKR (H2B:11, amino acids 11–31 of the complete H2B sequence), and KTQKKEGKKRKRTRKESYSI (H2B:21, amino acids 21–40 of the complete H2B sequence). The top phosphorylation signal belonged to AQKKDGRKRKRSRKESYSVY (H2B:22, amino acids 22–41 of the complete H2B sequence) (Fig. 7A) containing the RxRxxS recognition pattern (bold) shared by many PKC-θ-targeted substrates such as BAD, NDRG and Rapgef2 (Hornbeck et al., 2012). Here, the highest signal was generated with arginine at residue 27 (Arg27), whereas replacement of alanine 21 with a valine residue substantially reduced phosphorylation to below background, suggesting that Arg27 contributes to optimal phosphorylation whereas Ala21 is crucial for substrate recognition (Fig. 7A). Substrate recognition was also heavily dependent on adjacent histone modifications. For example, butyrylation and propionylation at Lys21, Lys25, Lys28 and Lys29 generally promoted phosphorylation, but malonylation and succinylation at similar residues reduced phosphorylation. Furthermore, lysine methylation exerted status and positional effects on PKC-θ-mediated phosphorylation, such that monomethylation at lysine residues (e.g. Lys20, Lys21, Lys25, Lys28, Lys30, Lys32, Lys34 and Lys35) increased phosphorylation but trimethylated Lys21, Lys25, Lys28 and Lys34 discouraged phosphorylation (Fig. S4H).Fig. 7.


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)

Identification of phosphorylated residues on histone H2B. (A) PKC-θ-mediated phosphorylation signals on H2B:21 (residues 21–40 derived from H2B) were detected by PKC-θ microarray profiling. The mean phosphorylation is shown (±s.d.). * denotes a pre-phosphorylated serine and the dotted red line is the background threshold (57500). The location of the histone H2B repression domain (HBR) is marked. (B) PKC-θ phosphorylates H2B Ser32. An in vitro kinase assay was performed by incubating active PKC-θ with either recombinant histones H2B, H3 or H4 or recombinant nucleosomes containing H3.1 or H3.3. Phosphorylated proteins were resolved by SDS-PAGE followed by western blotting for H2B phosphorylation. Negative controls include C1 (no ATP addition), C2 (no PKC addition), and C3 (incubation with PKC-μ). A representative blot of three experiments is shown. (C) PKC-θ phosphorylates H2B Ser36, as assessed by the method shown in B. (D) The Pearson's colocalization coefficient (PCC) was calculated for the fluorescent signal of H2B and PKC-θ as measured by confocal laser scanning microscopy in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations (mean±s.e.m., n=20). **P≤0.01, ****P≤0.0001 (Mann–Whitney test). (E) Representative immunoblot of phosphorylated H2B Ser32 and Ser36 in DMSO and 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (PI) (n=3). (F) Normalized H2B Ser32p densitometry is shown for human CD4+ T cells treated with either the control siRNA (siCtrl) or PKC-θ siRNA (siPKC-θ) (mean±s.e.m., n=3 individuals). ***P≤0.001 (two-tailed Student's t-test). (G) FAIRE chromatin accessibility shown for IL2 and TNF in in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. FAIRE chromatin accessibility is normalized to results for GAPDH (mean±s.e.m., n=3). ***P≤0.001 (two-way ANOVA). (H) FAIRE chromatin accessibility shown for IL2 and other promoters in naïve and memory CD4+ T cells treated with either the control (siCtrl) or the PKC-θ siRNA (siPKC-θ) with or without PMA and Ca2+ ionophore (P/I). FAIRE chromatin accessibility is normalized to GAPDH and expressed as a percentage relative to the stimulated (ST) memory CD4+ T cells treated with the control siRNA (siCtrl) (mean±s.e.m., n=3). *P≤0.05, **P<0.01 (unpaired two-tailed Student's t-test).
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JCS181248F7: Identification of phosphorylated residues on histone H2B. (A) PKC-θ-mediated phosphorylation signals on H2B:21 (residues 21–40 derived from H2B) were detected by PKC-θ microarray profiling. The mean phosphorylation is shown (±s.d.). * denotes a pre-phosphorylated serine and the dotted red line is the background threshold (57500). The location of the histone H2B repression domain (HBR) is marked. (B) PKC-θ phosphorylates H2B Ser32. An in vitro kinase assay was performed by incubating active PKC-θ with either recombinant histones H2B, H3 or H4 or recombinant nucleosomes containing H3.1 or H3.3. Phosphorylated proteins were resolved by SDS-PAGE followed by western blotting for H2B phosphorylation. Negative controls include C1 (no ATP addition), C2 (no PKC addition), and C3 (incubation with PKC-μ). A representative blot of three experiments is shown. (C) PKC-θ phosphorylates H2B Ser36, as assessed by the method shown in B. (D) The Pearson's colocalization coefficient (PCC) was calculated for the fluorescent signal of H2B and PKC-θ as measured by confocal laser scanning microscopy in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations (mean±s.e.m., n=20). **P≤0.01, ****P≤0.0001 (Mann–Whitney test). (E) Representative immunoblot of phosphorylated H2B Ser32 and Ser36 in DMSO and 1 μM C27-treated Jurkat T cells with or without PMA and Ca2+ ionophore (PI) (n=3). (F) Normalized H2B Ser32p densitometry is shown for human CD4+ T cells treated with either the control siRNA (siCtrl) or PKC-θ siRNA (siPKC-θ) (mean±s.e.m., n=3 individuals). ***P≤0.001 (two-tailed Student's t-test). (G) FAIRE chromatin accessibility shown for IL2 and TNF in in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. FAIRE chromatin accessibility is normalized to results for GAPDH (mean±s.e.m., n=3). ***P≤0.001 (two-way ANOVA). (H) FAIRE chromatin accessibility shown for IL2 and other promoters in naïve and memory CD4+ T cells treated with either the control (siCtrl) or the PKC-θ siRNA (siPKC-θ) with or without PMA and Ca2+ ionophore (P/I). FAIRE chromatin accessibility is normalized to GAPDH and expressed as a percentage relative to the stimulated (ST) memory CD4+ T cells treated with the control siRNA (siCtrl) (mean±s.e.m., n=3). *P≤0.05, **P<0.01 (unpaired two-tailed Student's t-test).
Mentions: Given that PKC-θ directly associates with the chromatin template, we used histone peptide microarrays to identify potential PKC-θ substrates. Significant levels of PKC-θ-mediated phosphorylation was detected on H2B- and H2A-derived peptides, particularly those derived from H2B, SKKGFKKAVVKTQKKEGKKR (H2B:11, amino acids 11–31 of the complete H2B sequence), and KTQKKEGKKRKRTRKESYSI (H2B:21, amino acids 21–40 of the complete H2B sequence). The top phosphorylation signal belonged to AQKKDGRKRKRSRKESYSVY (H2B:22, amino acids 22–41 of the complete H2B sequence) (Fig. 7A) containing the RxRxxS recognition pattern (bold) shared by many PKC-θ-targeted substrates such as BAD, NDRG and Rapgef2 (Hornbeck et al., 2012). Here, the highest signal was generated with arginine at residue 27 (Arg27), whereas replacement of alanine 21 with a valine residue substantially reduced phosphorylation to below background, suggesting that Arg27 contributes to optimal phosphorylation whereas Ala21 is crucial for substrate recognition (Fig. 7A). Substrate recognition was also heavily dependent on adjacent histone modifications. For example, butyrylation and propionylation at Lys21, Lys25, Lys28 and Lys29 generally promoted phosphorylation, but malonylation and succinylation at similar residues reduced phosphorylation. Furthermore, lysine methylation exerted status and positional effects on PKC-θ-mediated phosphorylation, such that monomethylation at lysine residues (e.g. Lys20, Lys21, Lys25, Lys28, Lys30, Lys32, Lys34 and Lys35) increased phosphorylation but trimethylated Lys21, Lys25, Lys28 and Lys34 discouraged phosphorylation (Fig. S4H).Fig. 7.

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