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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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Effect of RNAi silencing of IRF1, IRF2, USF1 and USF2 on CEACAM1 expression in MDA-MB-468 cells. A. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and IRF1, IRF2, USF1, and USF2 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for IRF1, IRF2, USF1, and USF2 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane. B. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and CEACAM-1 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for CEACAM1 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane.
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Figure 6: Effect of RNAi silencing of IRF1, IRF2, USF1 and USF2 on CEACAM1 expression in MDA-MB-468 cells. A. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and IRF1, IRF2, USF1, and USF2 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for IRF1, IRF2, USF1, and USF2 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane. B. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and CEACAM-1 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for CEACAM1 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane.

Mentions: We conclude from the above analyses that IRF1 and USF1 are critical transcription factors in the regulation of CEACAM1 in the breast cell lines analyzed. Since the MDA-MB-468 cells have intermediate levels of CEACAM1 mRNA expression, lower than MCF-10A and higher than MCF7 cells (Figure 1), we predicted that they will be most sensitive to alterations in the levels of these critical transcription factors. In order to test this prediction, we transfected these cells with RNAi oligos to IRF1 and USF1 plus RNAi to the related transcription factors IRF2 and USF2 that bind to the analogous sites in the CEACAM1 promoter. Several RNAi oligos plus non-specific RNAi were tested to confirm the ability of RNAi to silence their specific targets at mRNA (Table 2) and the protein level (Figure 6A). Compared to the controls that included no treatment, lipofectamine only, or unspecific RNAi, we found a dramatic down-regulation of CEACAM1 protein expression by RNAi to IRF1, IRF2, and USF1, but not to USF2 (Figure 6B). These results confirm our prediction that IRF1 and USF1 critically regulate the expression of CEACAM1, and further, add a role for IRF2. This is especially interesting since in other systems, IRF-2 has been shown to antagonize IRF1 [36]. The implications of this finding will be discussed later.


Regulation of CEACAM1 transcription in human breast epithelial cells.

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

Effect of RNAi silencing of IRF1, IRF2, USF1 and USF2 on CEACAM1 expression in MDA-MB-468 cells. A. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and IRF1, IRF2, USF1, and USF2 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for IRF1, IRF2, USF1, and USF2 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane. B. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and CEACAM-1 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for CEACAM1 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane.
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Figure 6: Effect of RNAi silencing of IRF1, IRF2, USF1 and USF2 on CEACAM1 expression in MDA-MB-468 cells. A. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and IRF1, IRF2, USF1, and USF2 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for IRF1, IRF2, USF1, and USF2 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane. B. MDA-MB-468 cells were treated with the following controls: U (untreated), lipofectamine (no RNAi), or non-specific RNAi (-) and CEACAM-1 levels measured by western blot analysis after 72 hrs. Cells treated with RNAi for IRF1 (3 oligos), IRF2 (3 oligos), USF1 (3 oligos), or USF2 (2 oligos) were analyzed for CEACAM1 expression by western blot analysis after 72 hrs. Lysates were probed with antibody to β-actin to ensure equal protein loading in each lane.
Mentions: We conclude from the above analyses that IRF1 and USF1 are critical transcription factors in the regulation of CEACAM1 in the breast cell lines analyzed. Since the MDA-MB-468 cells have intermediate levels of CEACAM1 mRNA expression, lower than MCF-10A and higher than MCF7 cells (Figure 1), we predicted that they will be most sensitive to alterations in the levels of these critical transcription factors. In order to test this prediction, we transfected these cells with RNAi oligos to IRF1 and USF1 plus RNAi to the related transcription factors IRF2 and USF2 that bind to the analogous sites in the CEACAM1 promoter. Several RNAi oligos plus non-specific RNAi were tested to confirm the ability of RNAi to silence their specific targets at mRNA (Table 2) and the protein level (Figure 6A). Compared to the controls that included no treatment, lipofectamine only, or unspecific RNAi, we found a dramatic down-regulation of CEACAM1 protein expression by RNAi to IRF1, IRF2, and USF1, but not to USF2 (Figure 6B). These results confirm our prediction that IRF1 and USF1 critically regulate the expression of CEACAM1, and further, add a role for IRF2. This is especially interesting since in other systems, IRF-2 has been shown to antagonize IRF1 [36]. The implications of this finding will be discussed later.

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