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Regulatory network analyses reveal genome-wide potentiation of LIF signaling by glucocorticoids and define an innate cell defense response.

Langlais D, Couture C, Balsalobre A, Drouin J - PLoS Genet. (2008)

Bottom Line: Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling.Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes.Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada.

ABSTRACT
While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response.

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Related in: MedlinePlus

Targets of LIF/STAT3 action.A) The time course of STAT3 activation (phospho- STAT3) was determined in AtT-20 cells following treatment with LIF (10 ng/ml). Western blot analysis of P-STAT3 is compared to total STAT3 protein. B) Time course of STAT3 occupancy on the promoter of known STAT3 target genes determined by ChIP and QPCR. C) Chromosomal distribution of genomic binding sites for STAT3 determined by ChIP-chip analysis of LIF-treated (20 min) AtT-20 cells. Triplicate ChIP samples were analyzed on Affymetrix Mouse Tiling 2.0R Array Sets. Raw data were extracted with GCOS software (Affymetrix) and were analyzed using the MAT software package. STAT3 enrichment peaks were selected on the basis of a P value threshold of 10−5. Redundant sequence filtering led to the removal of 74 sequences, thus yielding a final count of 3449 STAT3 binding sites. The list of these sites is presented in Table S1. The tiling array results were validated by QPCR analysis of independent ChIPs for 42 loci distributed randomly throughout all chromosomes; all 42 were confirmed. The same loci were used for further studies in Figure 3. FDR, calculated false discovery rate.
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pgen-1000224-g001: Targets of LIF/STAT3 action.A) The time course of STAT3 activation (phospho- STAT3) was determined in AtT-20 cells following treatment with LIF (10 ng/ml). Western blot analysis of P-STAT3 is compared to total STAT3 protein. B) Time course of STAT3 occupancy on the promoter of known STAT3 target genes determined by ChIP and QPCR. C) Chromosomal distribution of genomic binding sites for STAT3 determined by ChIP-chip analysis of LIF-treated (20 min) AtT-20 cells. Triplicate ChIP samples were analyzed on Affymetrix Mouse Tiling 2.0R Array Sets. Raw data were extracted with GCOS software (Affymetrix) and were analyzed using the MAT software package. STAT3 enrichment peaks were selected on the basis of a P value threshold of 10−5. Redundant sequence filtering led to the removal of 74 sequences, thus yielding a final count of 3449 STAT3 binding sites. The list of these sites is presented in Table S1. The tiling array results were validated by QPCR analysis of independent ChIPs for 42 loci distributed randomly throughout all chromosomes; all 42 were confirmed. The same loci were used for further studies in Figure 3. FDR, calculated false discovery rate.

Mentions: In order to assess the cellular response to LIF/STAT3, the time course of STAT3 activation in response to LIF in AtT-20 cells, a model of mouse pituitary corticotroph cells, was determined by Western blot analysis of phospho-STAT3 (Figure 1A). This analysis indicated a peak of phospho-STAT3 at about 20 minutes following LIF treatment. In principle, activated phospho-STAT3 should lead to promoter occupancy of STAT3 target genes and thus the time course of promoter recruitment was assessed by chromatin immunoprecipitation (ChIP) in AtT-20 cells for a panel of STAT3 target genes (Figure 1B). For most of these genes, maximal promoter occupancy was achieved between 10 and 20 minutes after LIF stimulation.


Regulatory network analyses reveal genome-wide potentiation of LIF signaling by glucocorticoids and define an innate cell defense response.

Langlais D, Couture C, Balsalobre A, Drouin J - PLoS Genet. (2008)

Targets of LIF/STAT3 action.A) The time course of STAT3 activation (phospho- STAT3) was determined in AtT-20 cells following treatment with LIF (10 ng/ml). Western blot analysis of P-STAT3 is compared to total STAT3 protein. B) Time course of STAT3 occupancy on the promoter of known STAT3 target genes determined by ChIP and QPCR. C) Chromosomal distribution of genomic binding sites for STAT3 determined by ChIP-chip analysis of LIF-treated (20 min) AtT-20 cells. Triplicate ChIP samples were analyzed on Affymetrix Mouse Tiling 2.0R Array Sets. Raw data were extracted with GCOS software (Affymetrix) and were analyzed using the MAT software package. STAT3 enrichment peaks were selected on the basis of a P value threshold of 10−5. Redundant sequence filtering led to the removal of 74 sequences, thus yielding a final count of 3449 STAT3 binding sites. The list of these sites is presented in Table S1. The tiling array results were validated by QPCR analysis of independent ChIPs for 42 loci distributed randomly throughout all chromosomes; all 42 were confirmed. The same loci were used for further studies in Figure 3. FDR, calculated false discovery rate.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2562516&req=5

pgen-1000224-g001: Targets of LIF/STAT3 action.A) The time course of STAT3 activation (phospho- STAT3) was determined in AtT-20 cells following treatment with LIF (10 ng/ml). Western blot analysis of P-STAT3 is compared to total STAT3 protein. B) Time course of STAT3 occupancy on the promoter of known STAT3 target genes determined by ChIP and QPCR. C) Chromosomal distribution of genomic binding sites for STAT3 determined by ChIP-chip analysis of LIF-treated (20 min) AtT-20 cells. Triplicate ChIP samples were analyzed on Affymetrix Mouse Tiling 2.0R Array Sets. Raw data were extracted with GCOS software (Affymetrix) and were analyzed using the MAT software package. STAT3 enrichment peaks were selected on the basis of a P value threshold of 10−5. Redundant sequence filtering led to the removal of 74 sequences, thus yielding a final count of 3449 STAT3 binding sites. The list of these sites is presented in Table S1. The tiling array results were validated by QPCR analysis of independent ChIPs for 42 loci distributed randomly throughout all chromosomes; all 42 were confirmed. The same loci were used for further studies in Figure 3. FDR, calculated false discovery rate.
Mentions: In order to assess the cellular response to LIF/STAT3, the time course of STAT3 activation in response to LIF in AtT-20 cells, a model of mouse pituitary corticotroph cells, was determined by Western blot analysis of phospho-STAT3 (Figure 1A). This analysis indicated a peak of phospho-STAT3 at about 20 minutes following LIF treatment. In principle, activated phospho-STAT3 should lead to promoter occupancy of STAT3 target genes and thus the time course of promoter recruitment was assessed by chromatin immunoprecipitation (ChIP) in AtT-20 cells for a panel of STAT3 target genes (Figure 1B). For most of these genes, maximal promoter occupancy was achieved between 10 and 20 minutes after LIF stimulation.

Bottom Line: Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling.Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes.Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Génétique Moléculaire, Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada.

ABSTRACT
While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response.

Show MeSH
Related in: MedlinePlus