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

Related in: MedlinePlus

Transcriptional silencing of CIITA during DC maturation. (A) CIITA mRNA was quantified in Mo-DCs exposed for 24 h to LPS, TNFα, PGN, PAM, pIC or Flagellin. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05 (B) CIITA and IL12B mRNAs were quantified in Mo-DC treated with LPS for the indicated times. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) CIITA and IL12B mRNAs were quantified in Mo-DCs treated for 6 h with the indicated LPS concentrations. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. (D) Nascent CIITA transcripts were quantified in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. The data is representative of four experiments. (E) Binding of Pol-II to CIITA promoter I and a 26 kb upstream region (background control) was assessed by qChIP in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to immature DCs at CIITA promoter I. Statistical significance was derived from three experiments: *, P < 0.05. (F) H4Ac was measured in Mo-DCs activated with LPS for the indicated times at the indicated positions of CIITA. Results are expressed relative to H4Ac at promoter IV in immature DCs. Results are derived from two experiments. The data is representative of four experiments. (G) H4Ac-profiling at the CIITA, HLA-B and IL4 genes was performed by ChIP-chip. H4Ac in untreated Mo-DCs (blue) was determined as the signal ratio between immature DCs (iDC) and input DNA. H4-deacetylation (red) was determined as the signal ratio between iDCs and DCs exposed to LPS for 1 h. Ratios are represented on a log2 scale. Maps of the genes are shown below: the scale in kb and TSSs are indicated. (H) CIITA mRNA was quantified in Mo-DCs treated with LPS for 4 h in the absence or presence of TSA. Results are expressed relative to immature DCs. Results are derived from two experiments. The data is representative of four experiments. 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 1: Transcriptional silencing of CIITA during DC maturation. (A) CIITA mRNA was quantified in Mo-DCs exposed for 24 h to LPS, TNFα, PGN, PAM, pIC or Flagellin. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05 (B) CIITA and IL12B mRNAs were quantified in Mo-DC treated with LPS for the indicated times. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) CIITA and IL12B mRNAs were quantified in Mo-DCs treated for 6 h with the indicated LPS concentrations. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. (D) Nascent CIITA transcripts were quantified in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. The data is representative of four experiments. (E) Binding of Pol-II to CIITA promoter I and a 26 kb upstream region (background control) was assessed by qChIP in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to immature DCs at CIITA promoter I. Statistical significance was derived from three experiments: *, P < 0.05. (F) H4Ac was measured in Mo-DCs activated with LPS for the indicated times at the indicated positions of CIITA. Results are expressed relative to H4Ac at promoter IV in immature DCs. Results are derived from two experiments. The data is representative of four experiments. (G) H4Ac-profiling at the CIITA, HLA-B and IL4 genes was performed by ChIP-chip. H4Ac in untreated Mo-DCs (blue) was determined as the signal ratio between immature DCs (iDC) and input DNA. H4-deacetylation (red) was determined as the signal ratio between iDCs and DCs exposed to LPS for 1 h. Ratios are represented on a log2 scale. Maps of the genes are shown below: the scale in kb and TSSs are indicated. (H) CIITA mRNA was quantified in Mo-DCs treated with LPS for 4 h in the absence or presence of TSA. Results are expressed relative to immature DCs. Results are derived from two experiments. The data is representative of four experiments. All measurements were performed in triplicate for each experiment.

Mentions: CIITA mRNA abundance is decreased during DC maturation (19,21,28). To clarify the mechanism involved, CIITA-silencing was investigated in human monocyte-derived DCs (Mo-DCs). CIITA-silencing was induced by diverse stimuli—including LPS, tumor necrosis factor (TNFα), peptidoglycan (PGN), Pam3CysSerLys4 (PAM), polyinosinic-polycytidylic acid (pIC) and flagellin (Flag)—indicating that it is a general feature of Mo-DC maturation (Figure 1A). Time-course experiments demonstrated that LPS-induced down-regulation of CIITA mRNA was detectable by 1 h and reached baseline levels by 2 h (Figure 1B). This decrease preceded the induction of IL12B (Figure 1B) and IL6 (data not shown) mRNAs. LPS-concentrations as low as 0.25 ng/ml were sufficient to trigger CIITA-silencing, whereas higher concentrations were required for optimal induction of IL12B (Figure 1C) and IL6 (data not shown) mRNAs. Quantification of chromatin-bound nascent transcripts demonstrated that LPS-induced down-regulation of CIITA mRNA resulted from an arrest in transcription that was evident by 15 min and almost complete after 1 h (Figure 1D). Quantitative chromatin-immunoprecipitation (qChIP) experiments revealed a rapid LPS-induced disengagement of RNA-polymerase-II (pol-II) at the DC-specific promoter (pI) of CIITA (Figure 1E).


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)

Transcriptional silencing of CIITA during DC maturation. (A) CIITA mRNA was quantified in Mo-DCs exposed for 24 h to LPS, TNFα, PGN, PAM, pIC or Flagellin. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05 (B) CIITA and IL12B mRNAs were quantified in Mo-DC treated with LPS for the indicated times. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) CIITA and IL12B mRNAs were quantified in Mo-DCs treated for 6 h with the indicated LPS concentrations. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. (D) Nascent CIITA transcripts were quantified in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. The data is representative of four experiments. (E) Binding of Pol-II to CIITA promoter I and a 26 kb upstream region (background control) was assessed by qChIP in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to immature DCs at CIITA promoter I. Statistical significance was derived from three experiments: *, P < 0.05. (F) H4Ac was measured in Mo-DCs activated with LPS for the indicated times at the indicated positions of CIITA. Results are expressed relative to H4Ac at promoter IV in immature DCs. Results are derived from two experiments. The data is representative of four experiments. (G) H4Ac-profiling at the CIITA, HLA-B and IL4 genes was performed by ChIP-chip. H4Ac in untreated Mo-DCs (blue) was determined as the signal ratio between immature DCs (iDC) and input DNA. H4-deacetylation (red) was determined as the signal ratio between iDCs and DCs exposed to LPS for 1 h. Ratios are represented on a log2 scale. Maps of the genes are shown below: the scale in kb and TSSs are indicated. (H) CIITA mRNA was quantified in Mo-DCs treated with LPS for 4 h in the absence or presence of TSA. Results are expressed relative to immature DCs. Results are derived from two experiments. The data is representative of four experiments. 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 1: Transcriptional silencing of CIITA during DC maturation. (A) CIITA mRNA was quantified in Mo-DCs exposed for 24 h to LPS, TNFα, PGN, PAM, pIC or Flagellin. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05 (B) CIITA and IL12B mRNAs were quantified in Mo-DC treated with LPS for the indicated times. Results are represented relative to unstimulated DCs. Statistical significance was derived from three experiments: *, P < 0.05, **, P < 0.01, ***, P < 0.001. (C) CIITA and IL12B mRNAs were quantified in Mo-DCs treated for 6 h with the indicated LPS concentrations. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. (D) Nascent CIITA transcripts were quantified in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to unstimulated DCs. Results are derived from two experiments. The data is representative of four experiments. (E) Binding of Pol-II to CIITA promoter I and a 26 kb upstream region (background control) was assessed by qChIP in Mo-DCs exposed to LPS for the indicated times. Results are expressed relative to immature DCs at CIITA promoter I. Statistical significance was derived from three experiments: *, P < 0.05. (F) H4Ac was measured in Mo-DCs activated with LPS for the indicated times at the indicated positions of CIITA. Results are expressed relative to H4Ac at promoter IV in immature DCs. Results are derived from two experiments. The data is representative of four experiments. (G) H4Ac-profiling at the CIITA, HLA-B and IL4 genes was performed by ChIP-chip. H4Ac in untreated Mo-DCs (blue) was determined as the signal ratio between immature DCs (iDC) and input DNA. H4-deacetylation (red) was determined as the signal ratio between iDCs and DCs exposed to LPS for 1 h. Ratios are represented on a log2 scale. Maps of the genes are shown below: the scale in kb and TSSs are indicated. (H) CIITA mRNA was quantified in Mo-DCs treated with LPS for 4 h in the absence or presence of TSA. Results are expressed relative to immature DCs. Results are derived from two experiments. The data is representative of four experiments. All measurements were performed in triplicate for each experiment.
Mentions: CIITA mRNA abundance is decreased during DC maturation (19,21,28). To clarify the mechanism involved, CIITA-silencing was investigated in human monocyte-derived DCs (Mo-DCs). CIITA-silencing was induced by diverse stimuli—including LPS, tumor necrosis factor (TNFα), peptidoglycan (PGN), Pam3CysSerLys4 (PAM), polyinosinic-polycytidylic acid (pIC) and flagellin (Flag)—indicating that it is a general feature of Mo-DC maturation (Figure 1A). Time-course experiments demonstrated that LPS-induced down-regulation of CIITA mRNA was detectable by 1 h and reached baseline levels by 2 h (Figure 1B). This decrease preceded the induction of IL12B (Figure 1B) and IL6 (data not shown) mRNAs. LPS-concentrations as low as 0.25 ng/ml were sufficient to trigger CIITA-silencing, whereas higher concentrations were required for optimal induction of IL12B (Figure 1C) and IL6 (data not shown) mRNAs. Quantification of chromatin-bound nascent transcripts demonstrated that LPS-induced down-regulation of CIITA mRNA resulted from an arrest in transcription that was evident by 15 min and almost complete after 1 h (Figure 1D). Quantitative chromatin-immunoprecipitation (qChIP) experiments revealed a rapid LPS-induced disengagement of RNA-polymerase-II (pol-II) at the DC-specific promoter (pI) of CIITA (Figure 1E).

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
Related in: MedlinePlus