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AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

Tullai JW, Tacheva S, Owens LJ, Graham JR, Cooper GM - PLoS ONE (2011)

Bottom Line: Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells.Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.

Methodology/principal findings: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.

Conclusions/significance: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

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Analysis of JunD binding to predicted AP-1 sites by chromatin immunoprecipitation.Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with an anti-JunD antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for normal rabbit IgG. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1) relative to the transcription start site. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. * indicates greater than 3-fold binding compared to MYOG in both untreated and PDGF treated samples; # indicates greater than 3-fold binding compared to MYOG in the PDGF treated sample only.
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pone-0020150-g003: Analysis of JunD binding to predicted AP-1 sites by chromatin immunoprecipitation.Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with an anti-JunD antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for normal rabbit IgG. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1) relative to the transcription start site. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. * indicates greater than 3-fold binding compared to MYOG in both untreated and PDGF treated samples; # indicates greater than 3-fold binding compared to MYOG in the PDGF treated sample only.

Mentions: JunD and JunB AP-1 family members have weaker activation domains and can potentially act as repressors that antagonize or inhibit the binding of activating family members [15], [16]. We therefore hypothesized that gene induction through AP-1 may be the result of JunD and/or JunB being displaced by an activating family member such as c-Jun. To test this, we conducted ChIP analysis for both JunB and JunD at the predicted AP-1 sites illustrated in Figure 1. JunB ChIP experiments did not show binding or recruitment greater than that of the negative control promoter, MYOG, or than that of the IgG control (data not shown). In contrast, ChIP analysis for JunD indicated that for many of the genes, JunD was bound to the upstream regions (Figure 3). In all, 8 out of 12 genes showed JunD binding greater than 3-fold as compared to the negative control promoter, MYOG. For seven of the genes showing JunD binding (BHLHB2, CTGF, CYR61, FOSB, NR4A1, PLAU, and RGS2), the relative binding of the PDGF and untreated (NT) samples were not significantly different, but both higher than that of the negative control, MYOG, and of the control IgG sample. The eighth gene, NR4A2, showed increased binding of JunD upon PDGF stimulation. Binding of JunD to these 8 genes corresponded to 100% overlap with those sites to which we demonstrated c-Jun binding (see Figure 2B).


AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

Tullai JW, Tacheva S, Owens LJ, Graham JR, Cooper GM - PLoS ONE (2011)

Analysis of JunD binding to predicted AP-1 sites by chromatin immunoprecipitation.Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with an anti-JunD antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for normal rabbit IgG. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1) relative to the transcription start site. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. * indicates greater than 3-fold binding compared to MYOG in both untreated and PDGF treated samples; # indicates greater than 3-fold binding compared to MYOG in the PDGF treated sample only.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102068&req=5

pone-0020150-g003: Analysis of JunD binding to predicted AP-1 sites by chromatin immunoprecipitation.Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with an anti-JunD antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for normal rabbit IgG. The numbers in parentheses refer to the 5′-most position of the putative AP-1 binding site (see Figure 1) relative to the transcription start site. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. * indicates greater than 3-fold binding compared to MYOG in both untreated and PDGF treated samples; # indicates greater than 3-fold binding compared to MYOG in the PDGF treated sample only.
Mentions: JunD and JunB AP-1 family members have weaker activation domains and can potentially act as repressors that antagonize or inhibit the binding of activating family members [15], [16]. We therefore hypothesized that gene induction through AP-1 may be the result of JunD and/or JunB being displaced by an activating family member such as c-Jun. To test this, we conducted ChIP analysis for both JunB and JunD at the predicted AP-1 sites illustrated in Figure 1. JunB ChIP experiments did not show binding or recruitment greater than that of the negative control promoter, MYOG, or than that of the IgG control (data not shown). In contrast, ChIP analysis for JunD indicated that for many of the genes, JunD was bound to the upstream regions (Figure 3). In all, 8 out of 12 genes showed JunD binding greater than 3-fold as compared to the negative control promoter, MYOG. For seven of the genes showing JunD binding (BHLHB2, CTGF, CYR61, FOSB, NR4A1, PLAU, and RGS2), the relative binding of the PDGF and untreated (NT) samples were not significantly different, but both higher than that of the negative control, MYOG, and of the control IgG sample. The eighth gene, NR4A2, showed increased binding of JunD upon PDGF stimulation. Binding of JunD to these 8 genes corresponded to 100% overlap with those sites to which we demonstrated c-Jun binding (see Figure 2B).

Bottom Line: Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells.Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.

Methodology/principal findings: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.

Conclusions/significance: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

Show MeSH
Related in: MedlinePlus