Microarray Analysis of LIF/Stat3 Transcriptional Targets in Embryonic Stem Cells
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《干细胞学杂志》
a INSERM U362, Institut Gustave-Roussy, Villejuif, France;
b LGRH-CEA, Evry, France;
c Ludwig Institute for Cancer Research and Christian de Duve Institute of Cellular Pathology University of Louvain, Brussels, Belgium
Key Words. Stat3 ? Self-renewal ? Microarray ? Leukemia inhibitor factor ? grg5
Correspondence: Dalila Sekka?, Ph.D., INSERM U362, Institut Gustave-Roussy. 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France. Telephone: 33-0-142-114-233; Fax: 33-0-142-115-240; e-mail: dsekkai@igr.fr; and Annelise Bennaceur-Griscelli, M.D., Ph.D., INSERM U362, Institut Gustave-Roussy. 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France. Telephone: 33-0-142-114-233; Fax: 33-0-142-115-240; e-mail: abenna@igr.fr
ABSTRACT
Mouse embryonic stem (ES) cells are permanent pluripotent cells derived from the inner cell mass of the blastocyst. These cells undergo self-renewing divisions while retaining the capacity to generate all types of fetal and adult cell lineages and to participate fully in embryonic development when reintroduced into a blastocyst . Mouse ES cells can be propagated in culture in the presence of leukemia inhibitor factor (LIF), which activates the gp130 signaling pathways, resulting in Jak-mediated Stat3 phosphorylation. Several studies have shown that Stat3 is one major player in this process . Other transcriptional regulators, such as Oct3/4 , Nanog , or Id1-3 , also participate in the maintenance of ES cell self-renewal, but Stat3 activation remains prominent because in the absence of LIF stimulation, endogenous expression of these factors is insufficient to sustain ES cell self-renewal. However, very little is known about the downstream target genes of activated Stat3 in ES cells. As their characterization could be of help to understand the molecular mechanisms underlying self-renewal, we used a microarray approach in this study to analyze gene profiling in ES cells with several repetitions of the experiments, allowing a robust statistical analysis of the results. To perform this approach, ES cells were first induced to differentiate upon LIF withdrawal, which led to the disruption of the signaling pathways lying downstream of gp130. To specifically target the stage when ES cells start losing their self-renewal potential without extensive engagement in differentiation, we characterized the earliest stage of appearance of mRNA-encoding germ layer markers and diminution of the ability of ES cells to generate in vitro embryoid bodies and blast cell colonies. In a more specific second strategy, we expressed a dominant-negative Stat3 mutant and identified several potential downstream Stat3 target genes. Comparison of the results obtained in the two independent microarray experiments revealed an overlap of several transcripts that are similarly regulated and are thus likely to participate in the Stat3-mediated self-renewal response.
MATERIALS AND METHODS
Earliest Stages of ES Cell Differentiation
To identify downstream Stat3 target genes specifically involved in self-renewal regulation, we first determined the time window when a consistent decrease in ES cell potential occurred without extensive differentiation. To this end, we used the previously described Gs2 ES cell line expressing the tetracycline regulatable dominant-negative form of Stat3, which induces the differentiation within 6 days . This Stat3F mutant differs from the wild-type protein by a mutation in the Y705 residue (Y705-F) and is believed to act by competitive inhibition of Stat3 phosphorylation at gp130 docking sites and, possibly, by sequestrating Stat3 partners in the cytoplasm. As shown in Figure 1A, Gs2 ES cells cultured under standard condition (+LIF/ON) formed characteristic round-shaped colonies, whereas morphological changes occurred 96 hours after LIF (–LIF) or tetracycline withdrawal (Tet OFF), with the presence of most large flattened differentiated colonies. To determine the time of engagement into differentiation in the two conditions, we also examined the expression of marker genes for the three germ layers . The ectoderm (FGF-5) and parietal and visceral endoderm (GATA-4) markers were detected 48 hours after Stat3F expression and increased during differentiation, whereas the mesoderm marker brachyury appeared later, at 72 hours (Fig. 1B). We also tested the ability of ES cells maintained for 24 or 48 hours without LIF or tetracycline to subsequently form EB3 and BC4 in the absence of LIF and the presence of tetracycline to avoid Stat3F expression during the differentiation step (Table 1). A decrease in BC4 formation was observed when the cells were precultured for 48 hours without tetracycline (55% of the control). More pronounced effects were seen when the cells were LIF-deprived for 48 hours because only 54% (EB3) and 35% (BC4) of the colonies were obtained. Overall, for a given duration of preculture, LIF deprivation is more potent than Stat3F expression for abrogating pluripotency. This feature, consistent with the morphology (Fig. 1A) and the gene expression level of differentiation markers and microarray data (Fig. 1B and see below), may reflect the already described partial inhibition of endogenous Stat3 by Stat3F ( and data not shown).
Figure 1. Morphological changes and germ layer marker expression on Gs2 embryonic stem cell differentiation. (A): Phase-contrast images of Gs2 embryonic stem cells plated at 5 x 103 cells per cm2 and cultured under standard conditions (leukemia inhibitor factor 100 U/ml and tetracycline 1 μg/ml; control), without Tet (Tet OFF), or without LIF (–LIF) for 96 hours. All photographs are at the same magnification, x 4. (B): Reverse transcription–polymerase chain reaction kinetic analysis of lineage-specific marker expression after Tet removal. As a positive control, cells were cultured for 96 hours without LIF.
Table 1. Decrease in embryonic stem cell–derived EB3 and BC4 formation after a first-step culture in the absence of leukemia inhibitor factor (LIF) or tetracycline (Tet)
Taken together, these data demonstrate that pluripotency is lost by a significant proportion of ES cells after Stat3F expression or, to a greater extend, upon a 48-hour LIF removal. We thus chose to perform gene profiling within the first 48 hours of LIF deprivation or Stat3 activity inhibition.
Microarray Analysis After LIF Deprivation
We first analyzed the modulation of gene expression upon LIF removal for 16, 24, and 48 hours in the presence of tetracycline (Tet ON). For each time point, six independent hybridizations were performed. The data were globally normalized and filtered as described in the experimental procedure section. Among a total of 5,891 genes, 277 were identified as differentially expressed at least at one time point according to a Student’s t-test with a 99% confidence level (p < .01, supplemental online data 1). Kinetic analysis revealed that most of the statistically significant modulations occurred at 48 hours (277 vs. 43 and 46 genes at 16 and 24 hours, respectively). These differentially expressed genes were then grouped according to their expression profile by hierarchical clustering with the Pearson’s correlation. Gene clusters with related expression pattern were easily distinguishable (Fig. 2). mRNAs increasing from 16 to 48 hours were grouped in cluster A, whereas those showing a decrease during this period were grouped in cluster B. Examples of known genes from these two clusters, showing the highest variations (mean of ratio > 1.5), are given in Figure 2. Among those, several genes were expected to vary upon ES cells differentiation. We indeed found a decrease in mRNA levels of osteopontin (spp1) , activin beta-B (Inhbb) , and zinc finger protein 57 (zfp57) and an increase in mesoderm-specific transcript (mest) mRNA. Other genes previously reported to vary (socs3 , zfp42/rex1 , and CD9 ) were also found but are not shown in Figure 2 because of a t-test p value above .01 (.02, .03, and .07, respectively). Interestingly, a decrease in Stat3 and socs3 expression, both described as Stat3 targets, was also found . These observations support evidence for a differentiation of ES cells and validate our approach. Several other genes whose expression has not previously been reported to vary were identified, some of which play an important role in the regulation of cell cycle or differentiation. For instance, mRNA levels of the helix-loop-helix (HLH) transcriptional regulators Id1 and Id2 were found to increase. This was also the case for mRNA-encoding cell-cycle regulators, such as the cyclin-dependent kinase inhibitor p57kip2 and cyclin D3. In addition, this study also pointed to several genes encoding proteins that can interfere with signaling pathways such as transforming growth factor (TGF)-? or Wnt pathway, known to be important for the control of stem cells properties . This is the case for lefty1 (ebaf), a member of the TGF-? superfamily, whose expression increased after LIF removal, and for aes1 (grg5), encoding a groucho-like partner of Tcf/LEF transcription factors, the nuclear mediators of Wnt signaling , whose expression is diminished.
Figure 2. Modulation of gene expression upon LIF withdrawal. Gene expression was analyzed after LIF removal for 16, 24, and 48 hours. Statistically significant modulations in gene expression (Student’s t-test, p < .01) were grouped by hierarchical clustering with the Pearson’s correlation. Shown are examples of known genes with variation above 1.5-fold. Color code: yellow, no variation; red, upregulation; green, downregulation.
Microarray Analysis After Expression of a Stat3 Dominant-Negative Form
To analyze variations in gene expression induced by Stat3F (Tet OFF/+LIF), we followed the same time course and performed eight independent hybridizations for each time point (16, 24, and 48 hours). The data were processed as for the first experiment. Six hundred thirty-nine genes were found to be differentially expressed at least at one time point according to a Student’s t-test with a 99% confidence level (p < .01, supplemental online data 2). Kinetic analysis revealed that most of the modulations took place at 48 hours (417 vs. 195 and 116 genes at 16 and 24 hours, respectively). Hierarchical clustering of these genes revealed two clusters of upregulated (C and C’) and one of downregulated (D) genes (Fig. 3). Examples of genes included in these two clusters and displaying a high variation (mean of ratio > 1.5-fold) are shown in Figure 3. With these criteria, we identified 12 downregulated genes, and among them, only 3 are known, namely fibronectin (fn1), asparagine synthetase (asns), and apolipoprotein E (apoE) (Fig. 3).
Figure 3. Modulation of gene expression after Stat3F induction. Expression of Stat3F was induced by tetracycline removal for 16, 24, or 48 hours, and gene expression was analyzed. Statistically significant modulations in gene expression (t-test, p < .01) were grouped by hierarchical clustering with the Pearson’s correlation. Shown are examples of known genes with variation above 1.5-fold. Color code: yellow, no variation; red, upregulation; green, downregulation.
Overlap of the Two Microarray Experiments
LIF-activated gp130 not only delivers a self-renewal signal mediated by Stat3 but also activates other pathways, such as Erk/mitogen-activated protein (MAP) kinase, which, on the contrary, seems to favor ES cell differentiation , implying that genes whose expression is modulated upon LIF withdrawal are not necessarily Stat3-regulated genes involved in the control of ES totipotency. To identify such genes, we crossed the data obtained by the two independent approaches. As mentioned earlier, Stat3F effect on ES differentiation and gene expression is weaker than that observed upon LIF withdrawal; we thus chose to compare the data of the microarray experiments on the basis of the statistical confidence in the modulation of gene expression rather than on the intensity of the observed variations. Comparison of the genes from clusters of upregulated (A, C, C’) and downregulated (B and D) genes obtained in the two experiments (Table 2) revealed an overlap of several similarly regulated genes (hereafter referred to as common genes), including, among others, both upregulated (Id1, Id2, and Lefty1) and downregulated (socs3, Inhbb, and aes1) genes.
Table 2. Common differentially expressed genes after leukemia inhibitor factor (LIF) removal or STAT3F expression
Validation of Transcript Variations
We further validated the microarray-based findings in Gs2 cell line by real-time RT-PCR with transcripts encoding several common genes. As shown in Figure 4A, Lefty1, p57kip2, Id1, and Id2 mRNA levels increased whereas the expression of aes1 and inhbb was diminished, thus confirming the gene expression evolution drawn from micro array analysis, even though for most of the genes tested, the ratios obtained with RT-PCR assays were higher than those obtained with the chips. We also showed that our results are not restricted to a single ES cell line because aes1 and Id2 mRNAs undergo downregulation and upregulation, respectively, upon LIF withdrawal in IOUD2 and E14 ES cell lines (Fig. 4B).
Figure 4. Validation through real-time reverse transcription–polymerase chain reaction (RT-PCR) of the variation of several common genes. (A): Real-time RT-PCR measurement of mRNA level after Stat3F expression (Tet OFF) or leukemia inhibitor factor (LIF) removal (–LIF) for 48 hours in Gs2 cells. (B): Upregulation and downregulation of, respectively, Id2 and aes1 mRNA level in IOUD2 and E14 ES cells after LIF removal for 48 hours. Data were normalized with gapdh as standard.
Functional Stat3 Sites in the Promoter Region of aes1
Because Stat3 acts mainly as a transactivator, direct Stat3 targets are presumably found among the downregulated group of mRNA. Interestingly, we found three potential STAT consensus sites upstream of the aes1 transcription initiation site: one GAS (TTCCCAGAA; –3485) and two degenerated sites (TTCT-GGGTA, –3455 and TTCACTCCA, –3446) . We assessed the functionality of these Stat3 sites by measuring the transcriptional activation of the luciferase gene under the control of the –3490 to –3419 region from aes1 promoter. Transient transfection of Stat3-overexpressing 293T cells led to twofold to threefold induction of luciferase activity from pAES1 plasmid, whereas no induction was observed when the three Stat3 sites were mutated (pAES1mut) (Fig. 5A). We then further evaluated whether Stat3 specifically binds to the region of the aes1 promoter containing these three sites in ES cells. Measurement of activated Stat3 by Western blot on nuclear extracts have shown that the nucleus of ES cells was cleared from activated Stat3 after 48 hours of LIF deprivation (data not shown). Analysis of the in vivo binding of Stat3 by ChIP assay revealed that Stat3 binds to the promoter of aes1 in LIF-stimulated ES cells (Fig. 5B; lane 1) whereas, as expected, deprivation of LIF for 48 hours abrogated the signal (Fig. 5B; lane 2). The specificity of this signal was confirmed by the absence of amplification when ChIP was performed with an irrelevant antibody (Fig. 5B, lanes 3 and 4). Taken together, these results show that Stat3 is able to bind the promoter of aes1 in ES cells and activates transcription from the Stat sites.
Figure 5. Functionality of the STAT binding sites in the promoter of aes1. (A): 293T cells were cotransfected with aes1 promoter-driven luciferase expression vectors (wild-type, AES1, or mutated, AES1mut) and empty (pRc) or Stat3 expression vector (pRc S3) as indicated. Luciferase activity was measured 48 hours after transfection and normalized with ?-galactosidase activity as an internal control. Values are means ± SE from three independent transfections. (B): Embryonic stem cells were LIF-starved for 48 hours and then restimulated for 15 minutes (lanes 1, 3, and 5). Polymerase chain reaction amplification with primers toward part of the aes1 promoter containing the putative STAT3 binding sites was performed on chromatin sample (INPUT, positive control) or on Stat3-immunoprecipitated or irrelevant immunoglobulin G-immunoprecipitated (negative control) chromatin samples (ChIP) as indicated.
DISCUSSION
We thank Prof. A. Smith for the gift of the Gs2 cells, J. Bromberg for pRcCMV and pRcCMV-Stat3 expression vector, and Edwige Leclercq and Marie-Hélène Courtier for helpful technical assistance with the RT-PCR experiments. D.S. is a recipient of a fellowship from Institut Gustave-Roussy. This work was supported in part by a grant from Association Contre le Cancer (ARC 4728).
DISCLOSURES
The authors indicate no potential conflicts of interest.
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b LGRH-CEA, Evry, France;
c Ludwig Institute for Cancer Research and Christian de Duve Institute of Cellular Pathology University of Louvain, Brussels, Belgium
Key Words. Stat3 ? Self-renewal ? Microarray ? Leukemia inhibitor factor ? grg5
Correspondence: Dalila Sekka?, Ph.D., INSERM U362, Institut Gustave-Roussy. 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France. Telephone: 33-0-142-114-233; Fax: 33-0-142-115-240; e-mail: dsekkai@igr.fr; and Annelise Bennaceur-Griscelli, M.D., Ph.D., INSERM U362, Institut Gustave-Roussy. 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France. Telephone: 33-0-142-114-233; Fax: 33-0-142-115-240; e-mail: abenna@igr.fr
ABSTRACT
Mouse embryonic stem (ES) cells are permanent pluripotent cells derived from the inner cell mass of the blastocyst. These cells undergo self-renewing divisions while retaining the capacity to generate all types of fetal and adult cell lineages and to participate fully in embryonic development when reintroduced into a blastocyst . Mouse ES cells can be propagated in culture in the presence of leukemia inhibitor factor (LIF), which activates the gp130 signaling pathways, resulting in Jak-mediated Stat3 phosphorylation. Several studies have shown that Stat3 is one major player in this process . Other transcriptional regulators, such as Oct3/4 , Nanog , or Id1-3 , also participate in the maintenance of ES cell self-renewal, but Stat3 activation remains prominent because in the absence of LIF stimulation, endogenous expression of these factors is insufficient to sustain ES cell self-renewal. However, very little is known about the downstream target genes of activated Stat3 in ES cells. As their characterization could be of help to understand the molecular mechanisms underlying self-renewal, we used a microarray approach in this study to analyze gene profiling in ES cells with several repetitions of the experiments, allowing a robust statistical analysis of the results. To perform this approach, ES cells were first induced to differentiate upon LIF withdrawal, which led to the disruption of the signaling pathways lying downstream of gp130. To specifically target the stage when ES cells start losing their self-renewal potential without extensive engagement in differentiation, we characterized the earliest stage of appearance of mRNA-encoding germ layer markers and diminution of the ability of ES cells to generate in vitro embryoid bodies and blast cell colonies. In a more specific second strategy, we expressed a dominant-negative Stat3 mutant and identified several potential downstream Stat3 target genes. Comparison of the results obtained in the two independent microarray experiments revealed an overlap of several transcripts that are similarly regulated and are thus likely to participate in the Stat3-mediated self-renewal response.
MATERIALS AND METHODS
Earliest Stages of ES Cell Differentiation
To identify downstream Stat3 target genes specifically involved in self-renewal regulation, we first determined the time window when a consistent decrease in ES cell potential occurred without extensive differentiation. To this end, we used the previously described Gs2 ES cell line expressing the tetracycline regulatable dominant-negative form of Stat3, which induces the differentiation within 6 days . This Stat3F mutant differs from the wild-type protein by a mutation in the Y705 residue (Y705-F) and is believed to act by competitive inhibition of Stat3 phosphorylation at gp130 docking sites and, possibly, by sequestrating Stat3 partners in the cytoplasm. As shown in Figure 1A, Gs2 ES cells cultured under standard condition (+LIF/ON) formed characteristic round-shaped colonies, whereas morphological changes occurred 96 hours after LIF (–LIF) or tetracycline withdrawal (Tet OFF), with the presence of most large flattened differentiated colonies. To determine the time of engagement into differentiation in the two conditions, we also examined the expression of marker genes for the three germ layers . The ectoderm (FGF-5) and parietal and visceral endoderm (GATA-4) markers were detected 48 hours after Stat3F expression and increased during differentiation, whereas the mesoderm marker brachyury appeared later, at 72 hours (Fig. 1B). We also tested the ability of ES cells maintained for 24 or 48 hours without LIF or tetracycline to subsequently form EB3 and BC4 in the absence of LIF and the presence of tetracycline to avoid Stat3F expression during the differentiation step (Table 1). A decrease in BC4 formation was observed when the cells were precultured for 48 hours without tetracycline (55% of the control). More pronounced effects were seen when the cells were LIF-deprived for 48 hours because only 54% (EB3) and 35% (BC4) of the colonies were obtained. Overall, for a given duration of preculture, LIF deprivation is more potent than Stat3F expression for abrogating pluripotency. This feature, consistent with the morphology (Fig. 1A) and the gene expression level of differentiation markers and microarray data (Fig. 1B and see below), may reflect the already described partial inhibition of endogenous Stat3 by Stat3F ( and data not shown).
Figure 1. Morphological changes and germ layer marker expression on Gs2 embryonic stem cell differentiation. (A): Phase-contrast images of Gs2 embryonic stem cells plated at 5 x 103 cells per cm2 and cultured under standard conditions (leukemia inhibitor factor 100 U/ml and tetracycline 1 μg/ml; control), without Tet (Tet OFF), or without LIF (–LIF) for 96 hours. All photographs are at the same magnification, x 4. (B): Reverse transcription–polymerase chain reaction kinetic analysis of lineage-specific marker expression after Tet removal. As a positive control, cells were cultured for 96 hours without LIF.
Table 1. Decrease in embryonic stem cell–derived EB3 and BC4 formation after a first-step culture in the absence of leukemia inhibitor factor (LIF) or tetracycline (Tet)
Taken together, these data demonstrate that pluripotency is lost by a significant proportion of ES cells after Stat3F expression or, to a greater extend, upon a 48-hour LIF removal. We thus chose to perform gene profiling within the first 48 hours of LIF deprivation or Stat3 activity inhibition.
Microarray Analysis After LIF Deprivation
We first analyzed the modulation of gene expression upon LIF removal for 16, 24, and 48 hours in the presence of tetracycline (Tet ON). For each time point, six independent hybridizations were performed. The data were globally normalized and filtered as described in the experimental procedure section. Among a total of 5,891 genes, 277 were identified as differentially expressed at least at one time point according to a Student’s t-test with a 99% confidence level (p < .01, supplemental online data 1). Kinetic analysis revealed that most of the statistically significant modulations occurred at 48 hours (277 vs. 43 and 46 genes at 16 and 24 hours, respectively). These differentially expressed genes were then grouped according to their expression profile by hierarchical clustering with the Pearson’s correlation. Gene clusters with related expression pattern were easily distinguishable (Fig. 2). mRNAs increasing from 16 to 48 hours were grouped in cluster A, whereas those showing a decrease during this period were grouped in cluster B. Examples of known genes from these two clusters, showing the highest variations (mean of ratio > 1.5), are given in Figure 2. Among those, several genes were expected to vary upon ES cells differentiation. We indeed found a decrease in mRNA levels of osteopontin (spp1) , activin beta-B (Inhbb) , and zinc finger protein 57 (zfp57) and an increase in mesoderm-specific transcript (mest) mRNA. Other genes previously reported to vary (socs3 , zfp42/rex1 , and CD9 ) were also found but are not shown in Figure 2 because of a t-test p value above .01 (.02, .03, and .07, respectively). Interestingly, a decrease in Stat3 and socs3 expression, both described as Stat3 targets, was also found . These observations support evidence for a differentiation of ES cells and validate our approach. Several other genes whose expression has not previously been reported to vary were identified, some of which play an important role in the regulation of cell cycle or differentiation. For instance, mRNA levels of the helix-loop-helix (HLH) transcriptional regulators Id1 and Id2 were found to increase. This was also the case for mRNA-encoding cell-cycle regulators, such as the cyclin-dependent kinase inhibitor p57kip2 and cyclin D3. In addition, this study also pointed to several genes encoding proteins that can interfere with signaling pathways such as transforming growth factor (TGF)-? or Wnt pathway, known to be important for the control of stem cells properties . This is the case for lefty1 (ebaf), a member of the TGF-? superfamily, whose expression increased after LIF removal, and for aes1 (grg5), encoding a groucho-like partner of Tcf/LEF transcription factors, the nuclear mediators of Wnt signaling , whose expression is diminished.
Figure 2. Modulation of gene expression upon LIF withdrawal. Gene expression was analyzed after LIF removal for 16, 24, and 48 hours. Statistically significant modulations in gene expression (Student’s t-test, p < .01) were grouped by hierarchical clustering with the Pearson’s correlation. Shown are examples of known genes with variation above 1.5-fold. Color code: yellow, no variation; red, upregulation; green, downregulation.
Microarray Analysis After Expression of a Stat3 Dominant-Negative Form
To analyze variations in gene expression induced by Stat3F (Tet OFF/+LIF), we followed the same time course and performed eight independent hybridizations for each time point (16, 24, and 48 hours). The data were processed as for the first experiment. Six hundred thirty-nine genes were found to be differentially expressed at least at one time point according to a Student’s t-test with a 99% confidence level (p < .01, supplemental online data 2). Kinetic analysis revealed that most of the modulations took place at 48 hours (417 vs. 195 and 116 genes at 16 and 24 hours, respectively). Hierarchical clustering of these genes revealed two clusters of upregulated (C and C’) and one of downregulated (D) genes (Fig. 3). Examples of genes included in these two clusters and displaying a high variation (mean of ratio > 1.5-fold) are shown in Figure 3. With these criteria, we identified 12 downregulated genes, and among them, only 3 are known, namely fibronectin (fn1), asparagine synthetase (asns), and apolipoprotein E (apoE) (Fig. 3).
Figure 3. Modulation of gene expression after Stat3F induction. Expression of Stat3F was induced by tetracycline removal for 16, 24, or 48 hours, and gene expression was analyzed. Statistically significant modulations in gene expression (t-test, p < .01) were grouped by hierarchical clustering with the Pearson’s correlation. Shown are examples of known genes with variation above 1.5-fold. Color code: yellow, no variation; red, upregulation; green, downregulation.
Overlap of the Two Microarray Experiments
LIF-activated gp130 not only delivers a self-renewal signal mediated by Stat3 but also activates other pathways, such as Erk/mitogen-activated protein (MAP) kinase, which, on the contrary, seems to favor ES cell differentiation , implying that genes whose expression is modulated upon LIF withdrawal are not necessarily Stat3-regulated genes involved in the control of ES totipotency. To identify such genes, we crossed the data obtained by the two independent approaches. As mentioned earlier, Stat3F effect on ES differentiation and gene expression is weaker than that observed upon LIF withdrawal; we thus chose to compare the data of the microarray experiments on the basis of the statistical confidence in the modulation of gene expression rather than on the intensity of the observed variations. Comparison of the genes from clusters of upregulated (A, C, C’) and downregulated (B and D) genes obtained in the two experiments (Table 2) revealed an overlap of several similarly regulated genes (hereafter referred to as common genes), including, among others, both upregulated (Id1, Id2, and Lefty1) and downregulated (socs3, Inhbb, and aes1) genes.
Table 2. Common differentially expressed genes after leukemia inhibitor factor (LIF) removal or STAT3F expression
Validation of Transcript Variations
We further validated the microarray-based findings in Gs2 cell line by real-time RT-PCR with transcripts encoding several common genes. As shown in Figure 4A, Lefty1, p57kip2, Id1, and Id2 mRNA levels increased whereas the expression of aes1 and inhbb was diminished, thus confirming the gene expression evolution drawn from micro array analysis, even though for most of the genes tested, the ratios obtained with RT-PCR assays were higher than those obtained with the chips. We also showed that our results are not restricted to a single ES cell line because aes1 and Id2 mRNAs undergo downregulation and upregulation, respectively, upon LIF withdrawal in IOUD2 and E14 ES cell lines (Fig. 4B).
Figure 4. Validation through real-time reverse transcription–polymerase chain reaction (RT-PCR) of the variation of several common genes. (A): Real-time RT-PCR measurement of mRNA level after Stat3F expression (Tet OFF) or leukemia inhibitor factor (LIF) removal (–LIF) for 48 hours in Gs2 cells. (B): Upregulation and downregulation of, respectively, Id2 and aes1 mRNA level in IOUD2 and E14 ES cells after LIF removal for 48 hours. Data were normalized with gapdh as standard.
Functional Stat3 Sites in the Promoter Region of aes1
Because Stat3 acts mainly as a transactivator, direct Stat3 targets are presumably found among the downregulated group of mRNA. Interestingly, we found three potential STAT consensus sites upstream of the aes1 transcription initiation site: one GAS (TTCCCAGAA; –3485) and two degenerated sites (TTCT-GGGTA, –3455 and TTCACTCCA, –3446) . We assessed the functionality of these Stat3 sites by measuring the transcriptional activation of the luciferase gene under the control of the –3490 to –3419 region from aes1 promoter. Transient transfection of Stat3-overexpressing 293T cells led to twofold to threefold induction of luciferase activity from pAES1 plasmid, whereas no induction was observed when the three Stat3 sites were mutated (pAES1mut) (Fig. 5A). We then further evaluated whether Stat3 specifically binds to the region of the aes1 promoter containing these three sites in ES cells. Measurement of activated Stat3 by Western blot on nuclear extracts have shown that the nucleus of ES cells was cleared from activated Stat3 after 48 hours of LIF deprivation (data not shown). Analysis of the in vivo binding of Stat3 by ChIP assay revealed that Stat3 binds to the promoter of aes1 in LIF-stimulated ES cells (Fig. 5B; lane 1) whereas, as expected, deprivation of LIF for 48 hours abrogated the signal (Fig. 5B; lane 2). The specificity of this signal was confirmed by the absence of amplification when ChIP was performed with an irrelevant antibody (Fig. 5B, lanes 3 and 4). Taken together, these results show that Stat3 is able to bind the promoter of aes1 in ES cells and activates transcription from the Stat sites.
Figure 5. Functionality of the STAT binding sites in the promoter of aes1. (A): 293T cells were cotransfected with aes1 promoter-driven luciferase expression vectors (wild-type, AES1, or mutated, AES1mut) and empty (pRc) or Stat3 expression vector (pRc S3) as indicated. Luciferase activity was measured 48 hours after transfection and normalized with ?-galactosidase activity as an internal control. Values are means ± SE from three independent transfections. (B): Embryonic stem cells were LIF-starved for 48 hours and then restimulated for 15 minutes (lanes 1, 3, and 5). Polymerase chain reaction amplification with primers toward part of the aes1 promoter containing the putative STAT3 binding sites was performed on chromatin sample (INPUT, positive control) or on Stat3-immunoprecipitated or irrelevant immunoglobulin G-immunoprecipitated (negative control) chromatin samples (ChIP) as indicated.
DISCUSSION
We thank Prof. A. Smith for the gift of the Gs2 cells, J. Bromberg for pRcCMV and pRcCMV-Stat3 expression vector, and Edwige Leclercq and Marie-Hélène Courtier for helpful technical assistance with the RT-PCR experiments. D.S. is a recipient of a fellowship from Institut Gustave-Roussy. This work was supported in part by a grant from Association Contre le Cancer (ARC 4728).
DISCLOSURES
The authors indicate no potential conflicts of interest.
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