Limits...
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

STAT3 binding profiles before and during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells. (B) Distribution of STAT3 occupancy at RefSeq gene features. “All STAT3” represents all treatments/time points, i.e., cumulative. “Differential” refers to STAT3 occupancy in transformed cells only. (C) Occupancy of STAT3 DNA binding site motifs by STAT3 protein, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. Data was from all STAT3 conditions/time points. (D) Gene ontology terms associated with transformation differential STAT3 ChIP-seq sites and the overlap between at 4, 12, and 36 h post TAM treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: STAT3 binding profiles before and during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells. (B) Distribution of STAT3 occupancy at RefSeq gene features. “All STAT3” represents all treatments/time points, i.e., cumulative. “Differential” refers to STAT3 occupancy in transformed cells only. (C) Occupancy of STAT3 DNA binding site motifs by STAT3 protein, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. Data was from all STAT3 conditions/time points. (D) Gene ontology terms associated with transformation differential STAT3 ChIP-seq sites and the overlap between at 4, 12, and 36 h post TAM treatment.

Mentions: STAT3 is critical for oncogenic transformation, and the STAT3 motif is overrepresented within FAIRE regions associated with differentially regulated genes (Figure 1C, and see below). STAT3 RNA levels increase modestly by ~50% during transformation (Additional file 1: Figure S5), and STAT3 activity, as measured by Tyr705 phosphorylation, increases ~4-fold (Figure 2A). Using chromatin immunoprecipitation (ChIP)-seq, we identify 26,783 STAT3 binding sites (P value <10−9) in non-transformed ER-Src cells, presumably representing a basal level of ER-Src signaling and hence STAT3 function. Upon transformation, STAT3-bound sites (as defined by the same statistical cutoff) more than double at 4 h (77,262), 12 h (67,015), and 36 h (74,584) post induction. In accord with STAT3 binding in macrophages [20], most STAT3 sites are located within introns and regions located away from RefSeq gene features, with only 7% of STAT3 binding sites located less than 2,500 bp upstream of a RefSeq TSS (Figure 2B). Approximately 15,000 genes contain at least one STAT3 binding site within their putative regulatory DNA domain as defined by GREAT (Methods), and only 3% of STAT3 sites are not associated with a putative regulatory DNA domain.All STAT3 binding during the transformation process is restricted almost exclusively to sites within FAIRE regions that existed prior to induction of transformation. Further, FAIRE regions containing the STAT3 motif (Figure 2C) are exceptionally well occupied by STAT3. At a motif quality score of 14 (near-perfect canonical motif), 80% of STAT3 motifs within FAIRE regions are bound by STAT3, in contrast to 8% of motifs with a quality score of 14 outside of FAIRE regions (Figure 2C). Thus, binding of STAT3 to its motif is largely limited to nucleosome-depleted genomic loci.Figure 2


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)

STAT3 binding profiles before and during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells. (B) Distribution of STAT3 occupancy at RefSeq gene features. “All STAT3” represents all treatments/time points, i.e., cumulative. “Differential” refers to STAT3 occupancy in transformed cells only. (C) Occupancy of STAT3 DNA binding site motifs by STAT3 protein, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. Data was from all STAT3 conditions/time points. (D) Gene ontology terms associated with transformation differential STAT3 ChIP-seq sites and the overlap between at 4, 12, and 36 h post TAM treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: STAT3 binding profiles before and during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells. (B) Distribution of STAT3 occupancy at RefSeq gene features. “All STAT3” represents all treatments/time points, i.e., cumulative. “Differential” refers to STAT3 occupancy in transformed cells only. (C) Occupancy of STAT3 DNA binding site motifs by STAT3 protein, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. Data was from all STAT3 conditions/time points. (D) Gene ontology terms associated with transformation differential STAT3 ChIP-seq sites and the overlap between at 4, 12, and 36 h post TAM treatment.
Mentions: STAT3 is critical for oncogenic transformation, and the STAT3 motif is overrepresented within FAIRE regions associated with differentially regulated genes (Figure 1C, and see below). STAT3 RNA levels increase modestly by ~50% during transformation (Additional file 1: Figure S5), and STAT3 activity, as measured by Tyr705 phosphorylation, increases ~4-fold (Figure 2A). Using chromatin immunoprecipitation (ChIP)-seq, we identify 26,783 STAT3 binding sites (P value <10−9) in non-transformed ER-Src cells, presumably representing a basal level of ER-Src signaling and hence STAT3 function. Upon transformation, STAT3-bound sites (as defined by the same statistical cutoff) more than double at 4 h (77,262), 12 h (67,015), and 36 h (74,584) post induction. In accord with STAT3 binding in macrophages [20], most STAT3 sites are located within introns and regions located away from RefSeq gene features, with only 7% of STAT3 binding sites located less than 2,500 bp upstream of a RefSeq TSS (Figure 2B). Approximately 15,000 genes contain at least one STAT3 binding site within their putative regulatory DNA domain as defined by GREAT (Methods), and only 3% of STAT3 sites are not associated with a putative regulatory DNA domain.All STAT3 binding during the transformation process is restricted almost exclusively to sites within FAIRE regions that existed prior to induction of transformation. Further, FAIRE regions containing the STAT3 motif (Figure 2C) are exceptionally well occupied by STAT3. At a motif quality score of 14 (near-perfect canonical motif), 80% of STAT3 motifs within FAIRE regions are bound by STAT3, in contrast to 8% of motifs with a quality score of 14 outside of FAIRE regions (Figure 2C). Thus, binding of STAT3 to its motif is largely limited to nucleosome-depleted genomic loci.Figure 2

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