Limits...
Extensive remodeling of DC function by rapid maturation-induced transcriptional silencing.

Seguín-Estévez Q, Dunand-Sauthier I, Lemeille S, Iseli C, Ibberson M, Ioannidis V, Schmid CD, Rousseau P, Barras E, Geinoz A, Xenarios I, Acha-Orbea H, Reith W - Nucleic Acids Res. (2014)

Bottom Line: This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation.The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function.Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, University of Geneva Medical School, CH-1211 Geneva, Switzerland.

Show MeSH
CIITA silencing is a conserved primary response mediated by the p38 and ERK pathways. (A) CIITA mRNA was quantified in immature and 4 h-LPS-treated Mo-DCs in the presence of the indicated concentrations of cycloheximide (CHX), Jun kinase inhibitor SP600125, p38 inhibitor SB202190, NF-κB inhibitor lactacystin, ERK inhibitor U0126 and U0126 + SB202190. Results are represented relative to immature DCs. Results are derived from two experiments. (B) CIITA mRNA was quantified in DC2114 cells exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Results are derived from two experiments. (C) Nascent CIITA RNA was quantified in DC2114 cells. exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (D) CIITA and Il6 mRNAs were quantified in DC2114 cells treated for 6 h with the indicated concentrations of CpG. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01. (E) H4Ac was measured in DC2114 cells activated with CpG for the indicated times at the indicated positions of the CIIta gene. Results are expressed relative to H4Ac at promoter IV in immature DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. All measurements were performed in triplicate for each experiment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150779&req=5

Figure 2: CIITA silencing is a conserved primary response mediated by the p38 and ERK pathways. (A) CIITA mRNA was quantified in immature and 4 h-LPS-treated Mo-DCs in the presence of the indicated concentrations of cycloheximide (CHX), Jun kinase inhibitor SP600125, p38 inhibitor SB202190, NF-κB inhibitor lactacystin, ERK inhibitor U0126 and U0126 + SB202190. Results are represented relative to immature DCs. Results are derived from two experiments. (B) CIITA mRNA was quantified in DC2114 cells exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Results are derived from two experiments. (C) Nascent CIITA RNA was quantified in DC2114 cells. exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (D) CIITA and Il6 mRNAs were quantified in DC2114 cells treated for 6 h with the indicated concentrations of CpG. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01. (E) H4Ac was measured in DC2114 cells activated with CpG for the indicated times at the indicated positions of the CIIta gene. Results are expressed relative to H4Ac at promoter IV in immature DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. All measurements were performed in triplicate for each experiment.

Mentions: The rapid kinetics of CIITA silencing suggested that it is a primary response triggered by pre-existing signal-transduction pathways. This was confirmed by the finding that it was not abrogated by the protein-synthesis inhibitor cycloheximide (Figure 2A). Selective inhibitors were used to identify signal-transduction pathways mediating CIITA silencing (Figure 2A). Efficacy of the inhibitors was controlled by qRT-PCR experiments examining the expression of genes induced via the targeted pathways and western-blot experiments examining phosphorylation of signaling intermediates (data not shown). Inhibitors of NF-κB activation, including lactacystine (Figure 2A) and MG-132 (data not shown) had no impact on CIITA silencing. Inhibitors of the c-Jun N-terminal kinase JNK (SP600125), p38 (SB202190) and extracellular signal-regulated kinases ERK (U0126) MAPK pathways also had no impact when added individually. However, CIITA silencing was completely abrogated by blocking both the p38 and ERK pathways (Figure 2A).


Extensive remodeling of DC function by rapid maturation-induced transcriptional silencing.

Seguín-Estévez Q, Dunand-Sauthier I, Lemeille S, Iseli C, Ibberson M, Ioannidis V, Schmid CD, Rousseau P, Barras E, Geinoz A, Xenarios I, Acha-Orbea H, Reith W - Nucleic Acids Res. (2014)

CIITA silencing is a conserved primary response mediated by the p38 and ERK pathways. (A) CIITA mRNA was quantified in immature and 4 h-LPS-treated Mo-DCs in the presence of the indicated concentrations of cycloheximide (CHX), Jun kinase inhibitor SP600125, p38 inhibitor SB202190, NF-κB inhibitor lactacystin, ERK inhibitor U0126 and U0126 + SB202190. Results are represented relative to immature DCs. Results are derived from two experiments. (B) CIITA mRNA was quantified in DC2114 cells exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Results are derived from two experiments. (C) Nascent CIITA RNA was quantified in DC2114 cells. exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (D) CIITA and Il6 mRNAs were quantified in DC2114 cells treated for 6 h with the indicated concentrations of CpG. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01. (E) H4Ac was measured in DC2114 cells activated with CpG for the indicated times at the indicated positions of the CIIta gene. Results are expressed relative to H4Ac at promoter IV in immature DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. All measurements were performed in triplicate for each experiment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150779&req=5

Figure 2: CIITA silencing is a conserved primary response mediated by the p38 and ERK pathways. (A) CIITA mRNA was quantified in immature and 4 h-LPS-treated Mo-DCs in the presence of the indicated concentrations of cycloheximide (CHX), Jun kinase inhibitor SP600125, p38 inhibitor SB202190, NF-κB inhibitor lactacystin, ERK inhibitor U0126 and U0126 + SB202190. Results are represented relative to immature DCs. Results are derived from two experiments. (B) CIITA mRNA was quantified in DC2114 cells exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Results are derived from two experiments. (C) Nascent CIITA RNA was quantified in DC2114 cells. exposed to CpG for the indicated times. Results are presented relative unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (D) CIITA and Il6 mRNAs were quantified in DC2114 cells treated for 6 h with the indicated concentrations of CpG. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01. (E) H4Ac was measured in DC2114 cells activated with CpG for the indicated times at the indicated positions of the CIIta gene. Results are expressed relative to H4Ac at promoter IV in immature DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. All measurements were performed in triplicate for each experiment.
Mentions: The rapid kinetics of CIITA silencing suggested that it is a primary response triggered by pre-existing signal-transduction pathways. This was confirmed by the finding that it was not abrogated by the protein-synthesis inhibitor cycloheximide (Figure 2A). Selective inhibitors were used to identify signal-transduction pathways mediating CIITA silencing (Figure 2A). Efficacy of the inhibitors was controlled by qRT-PCR experiments examining the expression of genes induced via the targeted pathways and western-blot experiments examining phosphorylation of signaling intermediates (data not shown). Inhibitors of NF-κB activation, including lactacystine (Figure 2A) and MG-132 (data not shown) had no impact on CIITA silencing. Inhibitors of the c-Jun N-terminal kinase JNK (SP600125), p38 (SB202190) and extracellular signal-regulated kinases ERK (U0126) MAPK pathways also had no impact when added individually. However, CIITA silencing was completely abrogated by blocking both the p38 and ERK pathways (Figure 2A).

Bottom Line: This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation.The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function.Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, University of Geneva Medical School, CH-1211 Geneva, Switzerland.

Show MeSH