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Regulation of CEACAM1 transcription in human breast epithelial cells.

Gencheva M, Chen CJ, Nguyen T, Shively JE - BMC Mol. Biol. (2010)

Bottom Line: As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells.The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure.Our data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.

ABSTRACT

Background: Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast).

Results: Using in vivo footprinting and chromatin immunoprecipitation experiments we show that the CEACAM1 proximal promoter in breast cells is bound in its active state by SP1, USF1/USF2, and IRF1/2. When down-regulated the CEACAM1 promoter remains accessible to USF2 and partially accessible to USF1. Interferon-γ up-regulates CEACAM1 mRNA by a mechanism involving further induction of IRF-1 and USF1 binding at the promoter. As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells. The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure.

Conclusions: Our data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.

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Induction of CEACAM1 expression in MCF7 cells with IFN γ. A. Top, RT-PCR with total RNA isolated from MCF7 cells, either untreated (-) or treated for 6 h with 500 U/ml of IFN γ (+). Bottom, CEACAM1 mRNA levels were monitored by real time PCR and normalized to GAPDH (triplicates ± SD). B. Total cellular protein lysates from MDA-MB-468, MCF10A and MCF7 cells either untreated (-) or treated (+) with 500 u/ml IFN γ were subjected to Western blot and probed with antibodies to IRF1, IRF2, and CEACAM1. β-actin was used as a loading control. Closed and open circle indicate two CEACAM1 isoforms with a different migration from MDA-MB-468 and MCF10A cells, respectively. C. Chromatin immunoprecipitation of CEACAM1 promoter DNA from MCF7 cells untreated (-) or treated with 500 u/ml of IFN γ for 6 h. Antibodies to USF1, USF2, IRF1 and a control IgG were used, numbers refer to percent of input.
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Figure 4: Induction of CEACAM1 expression in MCF7 cells with IFN γ. A. Top, RT-PCR with total RNA isolated from MCF7 cells, either untreated (-) or treated for 6 h with 500 U/ml of IFN γ (+). Bottom, CEACAM1 mRNA levels were monitored by real time PCR and normalized to GAPDH (triplicates ± SD). B. Total cellular protein lysates from MDA-MB-468, MCF10A and MCF7 cells either untreated (-) or treated (+) with 500 u/ml IFN γ were subjected to Western blot and probed with antibodies to IRF1, IRF2, and CEACAM1. β-actin was used as a loading control. Closed and open circle indicate two CEACAM1 isoforms with a different migration from MDA-MB-468 and MCF10A cells, respectively. C. Chromatin immunoprecipitation of CEACAM1 promoter DNA from MCF7 cells untreated (-) or treated with 500 u/ml of IFN γ for 6 h. Antibodies to USF1, USF2, IRF1 and a control IgG were used, numbers refer to percent of input.

Mentions: To verify binding of a transcription factor at the IRF-1 binding site, we performed ChIP with an antibody to IRF-1, as well as an antibody to IRF-2. IRF-2 is a well-studied repressor recognizing consensus sites common to the IRF group of proteins [31], thus making it a candidate for modulation of CEACAM1 expression, perhaps opposing IRF-1. While IRF1 binding was evident in MCF10A and MDA-MB-468 cells, there was a very low IRF-1 ChIP signal in MCF7 cells (Figure 3C). On the other hand, strong IRF-2 binding to the CEACAM1 promoter was detected only in the MDA-MB-468 cells. Western blot analysis demonstrated that IRF2 is expressed in both MCF10A and MCF7 cells, but weakly in MDA-MB-468 cells (Figure 4B). Our data is consistent with the footprinting results that show no IRF1 binding at the ISRE site in MCF-7 cells. The possible role for IRF-2 as a transcriptional repressor is unlikely since it was detected only in the ChIP analysis on MDA-MB-468 cells that are able to express CEACAM1.


Regulation of CEACAM1 transcription in human breast epithelial cells.

Gencheva M, Chen CJ, Nguyen T, Shively JE - BMC Mol. Biol. (2010)

Induction of CEACAM1 expression in MCF7 cells with IFN γ. A. Top, RT-PCR with total RNA isolated from MCF7 cells, either untreated (-) or treated for 6 h with 500 U/ml of IFN γ (+). Bottom, CEACAM1 mRNA levels were monitored by real time PCR and normalized to GAPDH (triplicates ± SD). B. Total cellular protein lysates from MDA-MB-468, MCF10A and MCF7 cells either untreated (-) or treated (+) with 500 u/ml IFN γ were subjected to Western blot and probed with antibodies to IRF1, IRF2, and CEACAM1. β-actin was used as a loading control. Closed and open circle indicate two CEACAM1 isoforms with a different migration from MDA-MB-468 and MCF10A cells, respectively. C. Chromatin immunoprecipitation of CEACAM1 promoter DNA from MCF7 cells untreated (-) or treated with 500 u/ml of IFN γ for 6 h. Antibodies to USF1, USF2, IRF1 and a control IgG were used, numbers refer to percent of input.
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Figure 4: Induction of CEACAM1 expression in MCF7 cells with IFN γ. A. Top, RT-PCR with total RNA isolated from MCF7 cells, either untreated (-) or treated for 6 h with 500 U/ml of IFN γ (+). Bottom, CEACAM1 mRNA levels were monitored by real time PCR and normalized to GAPDH (triplicates ± SD). B. Total cellular protein lysates from MDA-MB-468, MCF10A and MCF7 cells either untreated (-) or treated (+) with 500 u/ml IFN γ were subjected to Western blot and probed with antibodies to IRF1, IRF2, and CEACAM1. β-actin was used as a loading control. Closed and open circle indicate two CEACAM1 isoforms with a different migration from MDA-MB-468 and MCF10A cells, respectively. C. Chromatin immunoprecipitation of CEACAM1 promoter DNA from MCF7 cells untreated (-) or treated with 500 u/ml of IFN γ for 6 h. Antibodies to USF1, USF2, IRF1 and a control IgG were used, numbers refer to percent of input.
Mentions: To verify binding of a transcription factor at the IRF-1 binding site, we performed ChIP with an antibody to IRF-1, as well as an antibody to IRF-2. IRF-2 is a well-studied repressor recognizing consensus sites common to the IRF group of proteins [31], thus making it a candidate for modulation of CEACAM1 expression, perhaps opposing IRF-1. While IRF1 binding was evident in MCF10A and MDA-MB-468 cells, there was a very low IRF-1 ChIP signal in MCF7 cells (Figure 3C). On the other hand, strong IRF-2 binding to the CEACAM1 promoter was detected only in the MDA-MB-468 cells. Western blot analysis demonstrated that IRF2 is expressed in both MCF10A and MCF7 cells, but weakly in MDA-MB-468 cells (Figure 4B). Our data is consistent with the footprinting results that show no IRF1 binding at the ISRE site in MCF-7 cells. The possible role for IRF-2 as a transcriptional repressor is unlikely since it was detected only in the ChIP analysis on MDA-MB-468 cells that are able to express CEACAM1.

Bottom Line: As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells.The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure.Our data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.

ABSTRACT

Background: Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast).

Results: Using in vivo footprinting and chromatin immunoprecipitation experiments we show that the CEACAM1 proximal promoter in breast cells is bound in its active state by SP1, USF1/USF2, and IRF1/2. When down-regulated the CEACAM1 promoter remains accessible to USF2 and partially accessible to USF1. Interferon-γ up-regulates CEACAM1 mRNA by a mechanism involving further induction of IRF-1 and USF1 binding at the promoter. As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells. The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure.

Conclusions: Our data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.

Show MeSH
Related in: MedlinePlus