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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

Distribution of chromatinized PKC-θ in memory CD4+ T cells. (A) Overlapping PKC-θ-bound regions in ex-vivo-derived naïve (TN), effector (TEM), resting (TRM) and activated memory (TAM) CD4+ T cells. Graphs show genomic annotation of unique peaks (outside ring) compared to the peaks common to all subsets (inside ring). (B) PKC-θ peaks in memory CD4+ T cell subsets categorized into intergenic, 3′ untranslated region (UTR), intron, exon, 5′ UTR, promoter and upstream regions according to the nearest ENSEMBL gene, see corresponding colors in A. (C) The percentage of PKC-θ peaks in memory CD4+ T cell subsets and genomic background (Bkgd) annotated according to their chromatin state segmentation: permissive (H3K4me3, H3K27ac and H2K9ac), transcription (H3K36me3), enhancer (H3K4me1, and to different degrees H3K27ac or H3K9ac), repressed (H3K27me3 and/or H3K9me3), heterochromatin (H3K9me3) and quiescent (associated with low levels of histone marks). (D) Chromatin immunoprecipitation (ChIP-PCR) analysis on PKC-θ at the proximal promoters of the transcriptional-memory-responsive genes: IL2, TNF and TNFSF9 and an intronic region of SATB1. ChIP enrichment is shown as a percentage relative to the total input (TI) with background subtraction (mean±s.e.m., n=3). *P≤0.05, ***P≤0.001 (one-tailed Student's t-test).
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JCS181248F2: Distribution of chromatinized PKC-θ in memory CD4+ T cells. (A) Overlapping PKC-θ-bound regions in ex-vivo-derived naïve (TN), effector (TEM), resting (TRM) and activated memory (TAM) CD4+ T cells. Graphs show genomic annotation of unique peaks (outside ring) compared to the peaks common to all subsets (inside ring). (B) PKC-θ peaks in memory CD4+ T cell subsets categorized into intergenic, 3′ untranslated region (UTR), intron, exon, 5′ UTR, promoter and upstream regions according to the nearest ENSEMBL gene, see corresponding colors in A. (C) The percentage of PKC-θ peaks in memory CD4+ T cell subsets and genomic background (Bkgd) annotated according to their chromatin state segmentation: permissive (H3K4me3, H3K27ac and H2K9ac), transcription (H3K36me3), enhancer (H3K4me1, and to different degrees H3K27ac or H3K9ac), repressed (H3K27me3 and/or H3K9me3), heterochromatin (H3K9me3) and quiescent (associated with low levels of histone marks). (D) Chromatin immunoprecipitation (ChIP-PCR) analysis on PKC-θ at the proximal promoters of the transcriptional-memory-responsive genes: IL2, TNF and TNFSF9 and an intronic region of SATB1. ChIP enrichment is shown as a percentage relative to the total input (TI) with background subtraction (mean±s.e.m., n=3). *P≤0.05, ***P≤0.001 (one-tailed Student's t-test).

Mentions: We previously reported the recruitment of PKC-θ to gene promoters in Jurkat T cells (Sutcliffe et al., 2011) and because we showed that PKC-θ is necessary for inducible gene expression in primary human memory CD4+ T cells (Fig. 1C), we next assessed the genome-wide distribution of chromatin-tethered PKC-θ in ex vivo primary human CD4+ T cells. Ex-vivo-derived T cells were sorted into naïve (TN, CD45RO− CD62L+ CD127hi CD25−) and memory CD4+ T cell subsets [effector memory (TEM, CD45RO+ CD62L+/− CD127lo CD25−), resting memory (TRM, CD45RO+ CD62L+/− CD127hi CD25−) and activated memory (TAM, CD45RO+ CD62L+ CD127hi CD25+)], where hi denotes high levels of expression, lo denotes low levels of expression, and +/− either positive or negative expression (Fig. S1E). Sequencing chromatin-immunoprecipitated DNA (ChIP-sequencing) pooled from six individually validated ChIPs generated 2,547,999–4,051,661 uniquely mapped reads. A proportion of PKC-θ binding was common to both naïve and memory T cells. However, the majority of binding was cell type specific, with the most PKC-θ peaks detected in TRM cells followed by TAM cells (Fig. 2A), possibly reflecting chromatin remodeling differences in these T cell subsets. PKC-θ binding was distributed between 3′UTRs and gene promoters, with over half the peaks occurring in introns in all T cell subsets (Fig. 2B).Fig. 2.


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)

Distribution of chromatinized PKC-θ in memory CD4+ T cells. (A) Overlapping PKC-θ-bound regions in ex-vivo-derived naïve (TN), effector (TEM), resting (TRM) and activated memory (TAM) CD4+ T cells. Graphs show genomic annotation of unique peaks (outside ring) compared to the peaks common to all subsets (inside ring). (B) PKC-θ peaks in memory CD4+ T cell subsets categorized into intergenic, 3′ untranslated region (UTR), intron, exon, 5′ UTR, promoter and upstream regions according to the nearest ENSEMBL gene, see corresponding colors in A. (C) The percentage of PKC-θ peaks in memory CD4+ T cell subsets and genomic background (Bkgd) annotated according to their chromatin state segmentation: permissive (H3K4me3, H3K27ac and H2K9ac), transcription (H3K36me3), enhancer (H3K4me1, and to different degrees H3K27ac or H3K9ac), repressed (H3K27me3 and/or H3K9me3), heterochromatin (H3K9me3) and quiescent (associated with low levels of histone marks). (D) Chromatin immunoprecipitation (ChIP-PCR) analysis on PKC-θ at the proximal promoters of the transcriptional-memory-responsive genes: IL2, TNF and TNFSF9 and an intronic region of SATB1. ChIP enrichment is shown as a percentage relative to the total input (TI) with background subtraction (mean±s.e.m., n=3). *P≤0.05, ***P≤0.001 (one-tailed Student's t-test).
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JCS181248F2: Distribution of chromatinized PKC-θ in memory CD4+ T cells. (A) Overlapping PKC-θ-bound regions in ex-vivo-derived naïve (TN), effector (TEM), resting (TRM) and activated memory (TAM) CD4+ T cells. Graphs show genomic annotation of unique peaks (outside ring) compared to the peaks common to all subsets (inside ring). (B) PKC-θ peaks in memory CD4+ T cell subsets categorized into intergenic, 3′ untranslated region (UTR), intron, exon, 5′ UTR, promoter and upstream regions according to the nearest ENSEMBL gene, see corresponding colors in A. (C) The percentage of PKC-θ peaks in memory CD4+ T cell subsets and genomic background (Bkgd) annotated according to their chromatin state segmentation: permissive (H3K4me3, H3K27ac and H2K9ac), transcription (H3K36me3), enhancer (H3K4me1, and to different degrees H3K27ac or H3K9ac), repressed (H3K27me3 and/or H3K9me3), heterochromatin (H3K9me3) and quiescent (associated with low levels of histone marks). (D) Chromatin immunoprecipitation (ChIP-PCR) analysis on PKC-θ at the proximal promoters of the transcriptional-memory-responsive genes: IL2, TNF and TNFSF9 and an intronic region of SATB1. ChIP enrichment is shown as a percentage relative to the total input (TI) with background subtraction (mean±s.e.m., n=3). *P≤0.05, ***P≤0.001 (one-tailed Student's t-test).
Mentions: We previously reported the recruitment of PKC-θ to gene promoters in Jurkat T cells (Sutcliffe et al., 2011) and because we showed that PKC-θ is necessary for inducible gene expression in primary human memory CD4+ T cells (Fig. 1C), we next assessed the genome-wide distribution of chromatin-tethered PKC-θ in ex vivo primary human CD4+ T cells. Ex-vivo-derived T cells were sorted into naïve (TN, CD45RO− CD62L+ CD127hi CD25−) and memory CD4+ T cell subsets [effector memory (TEM, CD45RO+ CD62L+/− CD127lo CD25−), resting memory (TRM, CD45RO+ CD62L+/− CD127hi CD25−) and activated memory (TAM, CD45RO+ CD62L+ CD127hi CD25+)], where hi denotes high levels of expression, lo denotes low levels of expression, and +/− either positive or negative expression (Fig. S1E). Sequencing chromatin-immunoprecipitated DNA (ChIP-sequencing) pooled from six individually validated ChIPs generated 2,547,999–4,051,661 uniquely mapped reads. A proportion of PKC-θ binding was common to both naïve and memory T cells. However, the majority of binding was cell type specific, with the most PKC-θ peaks detected in TRM cells followed by TAM cells (Fig. 2A), possibly reflecting chromatin remodeling differences in these T cell subsets. PKC-θ binding was distributed between 3′UTRs and gene promoters, with over half the peaks occurring in introns in all T cell subsets (Fig. 2B).Fig. 2.

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