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Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes.

Navarro MN, Goebel J, Feijoo-Carnero C, Morrice N, Cantrell DA - Nat. Immunol. (2011)

Bottom Line: A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators.A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus.Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

View Article: PubMed Central - PubMed

Affiliation: The College of Life Sciences, Division of Immunology and Cell Biology, The University of Dundee, Dundee, Scotland, UK.

ABSTRACT
Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

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HDAC7 nuclear exclusion is required for expression of the high affinity IL2 receptor. (a) Relative expression of CD25 mRNA in sorted GFP negative (GFP−) and GFP-HDAC7-ΔP. Data shown are an average of three different experiments ± SEM (*AU= arbitrary units). (b) GFP-HDAC7-ΔP transduced P14-LCMV CTLs were stained for CD25 and analyzed by flow cytometry. GFP positive and negative cells were electronically gated to compare CD25 expression in both populations. (c) GFP-HDAC7 and GFP-HDAC7-ΔP transduced P14-LCMV-CTLs were stained for CD25 and analyzed by flow cytometry. Histograms in b and c are representative of three independently performed experiments. (d,e) P14-LCMV CTLs were stimulated with cognate peptide for 4h before assessing surface CD25 and intracellular IFN-γ expression by flow cytometry. (e) Expression of CD25 was electronically gated as in (d) to compare IFN-γ expression. The percentage of IFN-γ positive cells in both populations is represented as an averaged value of four experiments ± SEM. (f) IFN-γ production was assessed by intracellular staining in P14-LCMV CTLs expressing GFP-HDAC7-ΔP or GFP alone after stimulation with peptide for 4h. Data are representative of three experiments.
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Figure 6: HDAC7 nuclear exclusion is required for expression of the high affinity IL2 receptor. (a) Relative expression of CD25 mRNA in sorted GFP negative (GFP−) and GFP-HDAC7-ΔP. Data shown are an average of three different experiments ± SEM (*AU= arbitrary units). (b) GFP-HDAC7-ΔP transduced P14-LCMV CTLs were stained for CD25 and analyzed by flow cytometry. GFP positive and negative cells were electronically gated to compare CD25 expression in both populations. (c) GFP-HDAC7 and GFP-HDAC7-ΔP transduced P14-LCMV-CTLs were stained for CD25 and analyzed by flow cytometry. Histograms in b and c are representative of three independently performed experiments. (d,e) P14-LCMV CTLs were stimulated with cognate peptide for 4h before assessing surface CD25 and intracellular IFN-γ expression by flow cytometry. (e) Expression of CD25 was electronically gated as in (d) to compare IFN-γ expression. The percentage of IFN-γ positive cells in both populations is represented as an averaged value of four experiments ± SEM. (f) IFN-γ production was assessed by intracellular staining in P14-LCMV CTLs expressing GFP-HDAC7-ΔP or GFP alone after stimulation with peptide for 4h. Data are representative of three experiments.

Mentions: To explore the molecular basis for the role of HDAC7 in CTLs, we used Affymetrix microarray analysis to explore the impact of GFP-HDAC7-ΔP expression on the CTL transcriptional profile. Approximately 12,000 annotated genes were expressed in CTLs, and the impact of returning HDAC7 to the nucleus was a decrease in the expression of less than 2.5% of these and an increase in the expression of another 6% (Fig. 5e). The inhibitory effect of the HDAC7 mutant was thus quite selective and limited to a small subset of the T cell transcriptome (Fig. 5e, Supplementary Table 3). However, one striking observation was that GFP-HDAC7-ΔP selectively repressed expression of mRNA encoding CD25, a key subunit of the high-affinity receptor for IL-2 (Table 7). The selectivity of this effect of GFP-HDAC7-ΔP mutant on the T cell transcriptome was demonstrated by the fact that it down-regulated expression of CD25 mRNA without impacting on expression of mRNA encoding other key cytokine receptors (Table 7). The effect of GFP-HDAC7-ΔP on the expression CD25 was verified by quantitative PCR analysis (Fig. 6a). Moreover, flow cytometric analysis of CD25 protein expression on GFP-HDAC7-ΔP expressing CTLs versus controls cells (GFP-negative or expressing wild-type HDAC7) revealed that expression of the HDAC7 phospho-mutant causes a reduction in CD25 protein expression on CTLs (Fig. 6b,c).


Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes.

Navarro MN, Goebel J, Feijoo-Carnero C, Morrice N, Cantrell DA - Nat. Immunol. (2011)

HDAC7 nuclear exclusion is required for expression of the high affinity IL2 receptor. (a) Relative expression of CD25 mRNA in sorted GFP negative (GFP−) and GFP-HDAC7-ΔP. Data shown are an average of three different experiments ± SEM (*AU= arbitrary units). (b) GFP-HDAC7-ΔP transduced P14-LCMV CTLs were stained for CD25 and analyzed by flow cytometry. GFP positive and negative cells were electronically gated to compare CD25 expression in both populations. (c) GFP-HDAC7 and GFP-HDAC7-ΔP transduced P14-LCMV-CTLs were stained for CD25 and analyzed by flow cytometry. Histograms in b and c are representative of three independently performed experiments. (d,e) P14-LCMV CTLs were stimulated with cognate peptide for 4h before assessing surface CD25 and intracellular IFN-γ expression by flow cytometry. (e) Expression of CD25 was electronically gated as in (d) to compare IFN-γ expression. The percentage of IFN-γ positive cells in both populations is represented as an averaged value of four experiments ± SEM. (f) IFN-γ production was assessed by intracellular staining in P14-LCMV CTLs expressing GFP-HDAC7-ΔP or GFP alone after stimulation with peptide for 4h. Data are representative of three experiments.
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Figure 6: HDAC7 nuclear exclusion is required for expression of the high affinity IL2 receptor. (a) Relative expression of CD25 mRNA in sorted GFP negative (GFP−) and GFP-HDAC7-ΔP. Data shown are an average of three different experiments ± SEM (*AU= arbitrary units). (b) GFP-HDAC7-ΔP transduced P14-LCMV CTLs were stained for CD25 and analyzed by flow cytometry. GFP positive and negative cells were electronically gated to compare CD25 expression in both populations. (c) GFP-HDAC7 and GFP-HDAC7-ΔP transduced P14-LCMV-CTLs were stained for CD25 and analyzed by flow cytometry. Histograms in b and c are representative of three independently performed experiments. (d,e) P14-LCMV CTLs were stimulated with cognate peptide for 4h before assessing surface CD25 and intracellular IFN-γ expression by flow cytometry. (e) Expression of CD25 was electronically gated as in (d) to compare IFN-γ expression. The percentage of IFN-γ positive cells in both populations is represented as an averaged value of four experiments ± SEM. (f) IFN-γ production was assessed by intracellular staining in P14-LCMV CTLs expressing GFP-HDAC7-ΔP or GFP alone after stimulation with peptide for 4h. Data are representative of three experiments.
Mentions: To explore the molecular basis for the role of HDAC7 in CTLs, we used Affymetrix microarray analysis to explore the impact of GFP-HDAC7-ΔP expression on the CTL transcriptional profile. Approximately 12,000 annotated genes were expressed in CTLs, and the impact of returning HDAC7 to the nucleus was a decrease in the expression of less than 2.5% of these and an increase in the expression of another 6% (Fig. 5e). The inhibitory effect of the HDAC7 mutant was thus quite selective and limited to a small subset of the T cell transcriptome (Fig. 5e, Supplementary Table 3). However, one striking observation was that GFP-HDAC7-ΔP selectively repressed expression of mRNA encoding CD25, a key subunit of the high-affinity receptor for IL-2 (Table 7). The selectivity of this effect of GFP-HDAC7-ΔP mutant on the T cell transcriptome was demonstrated by the fact that it down-regulated expression of CD25 mRNA without impacting on expression of mRNA encoding other key cytokine receptors (Table 7). The effect of GFP-HDAC7-ΔP on the expression CD25 was verified by quantitative PCR analysis (Fig. 6a). Moreover, flow cytometric analysis of CD25 protein expression on GFP-HDAC7-ΔP expressing CTLs versus controls cells (GFP-negative or expressing wild-type HDAC7) revealed that expression of the HDAC7 phospho-mutant causes a reduction in CD25 protein expression on CTLs (Fig. 6b,c).

Bottom Line: A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators.A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus.Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

View Article: PubMed Central - PubMed

Affiliation: The College of Life Sciences, Division of Immunology and Cell Biology, The University of Dundee, Dundee, Scotland, UK.

ABSTRACT
Here we report an unbiased analysis of the cytotoxic T lymphocyte (CTL) serine-threonine phosphoproteome by high-resolution mass spectrometry. We identified approximately 2,000 phosphorylations in CTLs, of which approximately 450 were controlled by T cell antigen receptor (TCR) signaling. A significantly overrepresented group of molecules identified included transcription activators, corepressors and chromatin regulators. A focus on chromatin regulators showed that CTLs had high expression of the histone deacetylase HDAC7 but continually phosphorylated and exported this transcriptional repressor from the nucleus. Dephosphorylation of HDAC7 resulted in its accumulation in the nucleus and suppressed expression of genes encoding key cytokines, cytokine receptors and adhesion molecules that determine CTL function. Screening of the CTL phosphoproteome has thus identified intrinsic pathways of serine-threonine phosphorylation that target chromatin regulators and determine the CTL functional program.

Show MeSH
Related in: MedlinePlus