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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 c-Jun binding and recruitment to predicted AP-1 sites by chromatin immunoprecipitation.A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. 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. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).
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pone-0020150-g002: Analysis of c-Jun binding and recruitment to predicted AP-1 sites by chromatin immunoprecipitation.A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. 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. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).

Mentions: Our previous computational analysis identified a total of 22 conserved, putative AP-1 sites across all 12 of the genes that were induced by inhibition of GSK-3 (Figure 1). The AP-1 family of transcription factors is made up of three Jun family members (c-Jun, JunB, and JunD) and four Fos family members (c-Fos, FosB, Fra-1 and Fra-2). AP-1 is a dimeric transcription complex that is formed from Jun-Jun family homodimers, or Jun-Fos family heterodimers [15], [16]. It is the particular combination of the dimers that determines the transcriptional activity. FosB, c-Fos, and c-Jun are activators, whereas JunD, JunB, Fra-1 and Fra-2 have weaker transactivation domains and can act as repressors by competing for c-Fos, FosB and c-Jun binding. To determine which family members are present in quiescent T98G cells, immunoblots for all members were conducted. As expected, c-Fos, FosB, Fra-1 and Fra-2 were not detected in the quiescent cells, as they require growth factor signaling for their induction [17], [18], [19], [20] (data not shown). c-Jun, JunD and JunB were all detected by immunoblots, although JunB was only weakly detected (Figure 2A). c-Jun, JunD and JunB therefore were pursued in subsequent chromatin immunoprecipitation (ChIP) experiments.


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 c-Jun binding and recruitment to predicted AP-1 sites by chromatin immunoprecipitation.A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. 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. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).
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pone-0020150-g002: Analysis of c-Jun binding and recruitment to predicted AP-1 sites by chromatin immunoprecipitation.A. Quiescent T98G cell extracts were immunoblotted with anti-cJun, anti-JunB and anti-JunD antibodies. Images were taken from different parts of the same autoradiography film, and therefore have identical exposures. Sizes indicated are in kDa. As expected, JunD has two protein products which run as a 40–50 kDa doublet B. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated (NT), and then chromatin was immunoprecipitated with anti-c-Jun antibody or normal rabbit IgG. Only the PDGF-stimulated samples are plotted for the normal rabbit IgG immunoprecipitates. 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. When more than one position is listed, this refers to all possible sites that would be detected within the resolution limits of the ChIP PCR amplicon (approximately ±250 nucleotides). For those genes with multiple putative AP-1 sites, only one representative site (if all were negative for a given gene) or that site which indicated binding are shown for clarity. For a complete list of all tested sites, see Table S1. Data are presented as percent input averaged from 4 separate experiments ± S.E. MYOG served as a negative control promoter. Asterisks indicate greater than 3-fold binding as compared to MYOG in both untreated and PDGF treated samples C. Recruitment of c-Jun upon stimulation with PDGF or direct inhibition of GSK-3 with SB-216763. Quiescent T98G cells were treated with PDGF for 30 minutes or left untreated, or treated with SB-216763 for 1 hour or with DMSO vehicle control. Only those genes that initially showed c-Jun binding (panel A) are shown. Data are presented as fold change over untreated (for PDGF) or fold change over DMSO vehicle control (for SB-216763), and are averaged from 4 separate experiments ±S.E. No significant change was observed in the normal rabbit IgG or MYOG samples (not shown).
Mentions: Our previous computational analysis identified a total of 22 conserved, putative AP-1 sites across all 12 of the genes that were induced by inhibition of GSK-3 (Figure 1). The AP-1 family of transcription factors is made up of three Jun family members (c-Jun, JunB, and JunD) and four Fos family members (c-Fos, FosB, Fra-1 and Fra-2). AP-1 is a dimeric transcription complex that is formed from Jun-Jun family homodimers, or Jun-Fos family heterodimers [15], [16]. It is the particular combination of the dimers that determines the transcriptional activity. FosB, c-Fos, and c-Jun are activators, whereas JunD, JunB, Fra-1 and Fra-2 have weaker transactivation domains and can act as repressors by competing for c-Fos, FosB and c-Jun binding. To determine which family members are present in quiescent T98G cells, immunoblots for all members were conducted. As expected, c-Fos, FosB, Fra-1 and Fra-2 were not detected in the quiescent cells, as they require growth factor signaling for their induction [17], [18], [19], [20] (data not shown). c-Jun, JunD and JunB were all detected by immunoblots, although JunB was only weakly detected (Figure 2A). c-Jun, JunD and JunB therefore were pursued in subsequent chromatin immunoprecipitation (ChIP) experiments.

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