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STAT3 acts through pre-existing nucleosome-depleted regions bound by FOS during an epigenetic switch linking inflammation to cancer.

Fleming JD, Giresi PG, Lindahl-Allen M, Krall EB, Lieb JD, Struhl K - Epigenetics Chromatin (2015)

Bottom Line: Transient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors.Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity.These observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 USA.

ABSTRACT

Background: Transient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors.

Results: We show that during the transformation process, nucleosome-depleted regions (defined by formaldehyde-assisted isolation of regulatory elements (FAIRE)) are largely unchanged and that both before and during transformation, STAT3 binds almost exclusively to previously open chromatin regions. Roughly, a third of the transformation-inducible genes require STAT3 for the induction. STAT3 and NF-κB appear to drive the regulation of different gene sets during the transformation process. Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity. Lastly, many STAT3 binding sites are also bound by FOS and the expression of several AP-1 factors is altered during transformation in a STAT3-dependent manner, suggesting that STAT3 may cooperate with AP-1 proteins.

Conclusions: These observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch. In addition, gene expression changes during transformation, whether driven by pre-existing or induced transcription factors, occur largely through pre-existing nucleosome-depleted regions.

No MeSH data available.


Related in: MedlinePlus

Differentially expressed transcriptional regulators during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells done in parallel to RNA samples used for expression microarray analysis. STAT3 protein levels are reduced >25-fold upon siSTAT3 knockdown by 24 h. (B) The numbers of genes differentially up- or downregulated during transformation at 4 and 24 h post ER-Src activation and their dependence on STAT3. (C) Shown are the RNA expression levels at 4 and 24 h post EtOH or TAM treatment in samples transfected with siSCM (scrambled control) or siSTAT3. RNA levels are expressed as fold change over 4-h EtOH- and siSCM-treated samples. All TFs differentially regulated (P value <10−4, >log2 0.5-fold change) upon ER-Src activation at 4 or 24 h post TAM treatment in siSCM-transfected cells. TFs were clustered, and red boxes indicate TFs whose transcriptional response to treatments is correlated. Those TFs known to be involved in tumorigenesis, inflammatory response, metabolic disease, “stemness,” and the circadian rhythm are indicated by colored circles.
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Fig3: Differentially expressed transcriptional regulators during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells done in parallel to RNA samples used for expression microarray analysis. STAT3 protein levels are reduced >25-fold upon siSTAT3 knockdown by 24 h. (B) The numbers of genes differentially up- or downregulated during transformation at 4 and 24 h post ER-Src activation and their dependence on STAT3. (C) Shown are the RNA expression levels at 4 and 24 h post EtOH or TAM treatment in samples transfected with siSCM (scrambled control) or siSTAT3. RNA levels are expressed as fold change over 4-h EtOH- and siSCM-treated samples. All TFs differentially regulated (P value <10−4, >log2 0.5-fold change) upon ER-Src activation at 4 or 24 h post TAM treatment in siSCM-transfected cells. TFs were clustered, and red boxes indicate TFs whose transcriptional response to treatments is correlated. Those TFs known to be involved in tumorigenesis, inflammatory response, metabolic disease, “stemness,” and the circadian rhythm are indicated by colored circles.

Mentions: To identify genes whose expression depends on STAT3 during transformation, we performed microarray analysis in TAM-treated cells in which STAT3 protein (Figure 3A) and RNA levels were reduced more than 10-fold using siRNA (Additional file 1: Figure S5). Approximately one third of genes normally differentially regulated during transformation are dependent on STAT3 (Figure 3B). Genes that show differential upregulation during the transformation correlate both with the number of differential STAT3 sites and with increased STAT3 occupancy during transformation (Additional file 1: Figure S6). These relationships are not observed with genes downregulated during transformation, suggesting that STAT3 directly regulates only genes that are activated during transformation. Interestingly, the p53 motif was found enriched in downregulated genes (Figure 4A), and in this regard, our previous analysis identified p53 as a common node linking inflammatory signals and cancer transformation to metabolic syndrome [11]. The genes encoding p53 and p63 are both upregulated in response to transformation at 4 h, indicating that they are induced early by STAT3, only to be downregulated as transformation progresses (Additional file 1: Figure S8).Figure 3


STAT3 acts through pre-existing nucleosome-depleted regions bound by FOS during an epigenetic switch linking inflammation to cancer.

Fleming JD, Giresi PG, Lindahl-Allen M, Krall EB, Lieb JD, Struhl K - Epigenetics Chromatin (2015)

Differentially expressed transcriptional regulators during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells done in parallel to RNA samples used for expression microarray analysis. STAT3 protein levels are reduced >25-fold upon siSTAT3 knockdown by 24 h. (B) The numbers of genes differentially up- or downregulated during transformation at 4 and 24 h post ER-Src activation and their dependence on STAT3. (C) Shown are the RNA expression levels at 4 and 24 h post EtOH or TAM treatment in samples transfected with siSCM (scrambled control) or siSTAT3. RNA levels are expressed as fold change over 4-h EtOH- and siSCM-treated samples. All TFs differentially regulated (P value <10−4, >log2 0.5-fold change) upon ER-Src activation at 4 or 24 h post TAM treatment in siSCM-transfected cells. TFs were clustered, and red boxes indicate TFs whose transcriptional response to treatments is correlated. Those TFs known to be involved in tumorigenesis, inflammatory response, metabolic disease, “stemness,” and the circadian rhythm are indicated by colored circles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362815&req=5

Fig3: Differentially expressed transcriptional regulators during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells done in parallel to RNA samples used for expression microarray analysis. STAT3 protein levels are reduced >25-fold upon siSTAT3 knockdown by 24 h. (B) The numbers of genes differentially up- or downregulated during transformation at 4 and 24 h post ER-Src activation and their dependence on STAT3. (C) Shown are the RNA expression levels at 4 and 24 h post EtOH or TAM treatment in samples transfected with siSCM (scrambled control) or siSTAT3. RNA levels are expressed as fold change over 4-h EtOH- and siSCM-treated samples. All TFs differentially regulated (P value <10−4, >log2 0.5-fold change) upon ER-Src activation at 4 or 24 h post TAM treatment in siSCM-transfected cells. TFs were clustered, and red boxes indicate TFs whose transcriptional response to treatments is correlated. Those TFs known to be involved in tumorigenesis, inflammatory response, metabolic disease, “stemness,” and the circadian rhythm are indicated by colored circles.
Mentions: To identify genes whose expression depends on STAT3 during transformation, we performed microarray analysis in TAM-treated cells in which STAT3 protein (Figure 3A) and RNA levels were reduced more than 10-fold using siRNA (Additional file 1: Figure S5). Approximately one third of genes normally differentially regulated during transformation are dependent on STAT3 (Figure 3B). Genes that show differential upregulation during the transformation correlate both with the number of differential STAT3 sites and with increased STAT3 occupancy during transformation (Additional file 1: Figure S6). These relationships are not observed with genes downregulated during transformation, suggesting that STAT3 directly regulates only genes that are activated during transformation. Interestingly, the p53 motif was found enriched in downregulated genes (Figure 4A), and in this regard, our previous analysis identified p53 as a common node linking inflammatory signals and cancer transformation to metabolic syndrome [11]. The genes encoding p53 and p63 are both upregulated in response to transformation at 4 h, indicating that they are induced early by STAT3, only to be downregulated as transformation progresses (Additional file 1: Figure S8).Figure 3

Bottom Line: Transient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors.Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity.These observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 USA.

ABSTRACT

Background: Transient induction of the Src oncoprotein in a non-transformed breast cell line can initiate an epigenetic switch to a cancer cell via a positive feedback loop that involves activation of the signal transducer and activator of transcription 3 protein (STAT3) and NF-κB transcription factors.

Results: We show that during the transformation process, nucleosome-depleted regions (defined by formaldehyde-assisted isolation of regulatory elements (FAIRE)) are largely unchanged and that both before and during transformation, STAT3 binds almost exclusively to previously open chromatin regions. Roughly, a third of the transformation-inducible genes require STAT3 for the induction. STAT3 and NF-κB appear to drive the regulation of different gene sets during the transformation process. Interestingly, STAT3 directly regulates the expression of NFKB1, which encodes a subunit of NF-κB, and IL6, a cytokine that stimulates STAT3 activity. Lastly, many STAT3 binding sites are also bound by FOS and the expression of several AP-1 factors is altered during transformation in a STAT3-dependent manner, suggesting that STAT3 may cooperate with AP-1 proteins.

Conclusions: These observations uncover additional complexities to the inflammatory feedback loop that are likely to contribute to the epigenetic switch. In addition, gene expression changes during transformation, whether driven by pre-existing or induced transcription factors, occur largely through pre-existing nucleosome-depleted regions.

No MeSH data available.


Related in: MedlinePlus