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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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Chromatin structure at the CEACAM1 promoter in MDA-MB-468, MCF10A and MCF7 cells. A. Chromatin immunoprecipitations of CEACAM1 promoter DNA from MDA-MB-468, MCF10A and MCF7 cells with antibodies detecting acetylated Lys9 and Lys 18 of histone H3 (H3Ac), trimethylated Lys 9 of histone H3 (H3KMe), and trimethylated Lys 27 of histone H3 (H3K27Me). IgG was used as negative control; In: input DNA. B. MCF7 cells were treated with either DMSO or Trichostatin A for 0 h, 6 h, and 24 h, respectively, and total RNA was isolated and subjected to RT-PCR with primers amplifying CEACAM1 (top). GAPDH was used as a loading control. Bottom, CEACAM1 mRNA levels were quantified by real time PCR, using GAPDH for normalization.
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Figure 5: Chromatin structure at the CEACAM1 promoter in MDA-MB-468, MCF10A and MCF7 cells. A. Chromatin immunoprecipitations of CEACAM1 promoter DNA from MDA-MB-468, MCF10A and MCF7 cells with antibodies detecting acetylated Lys9 and Lys 18 of histone H3 (H3Ac), trimethylated Lys 9 of histone H3 (H3KMe), and trimethylated Lys 27 of histone H3 (H3K27Me). IgG was used as negative control; In: input DNA. B. MCF7 cells were treated with either DMSO or Trichostatin A for 0 h, 6 h, and 24 h, respectively, and total RNA was isolated and subjected to RT-PCR with primers amplifying CEACAM1 (top). GAPDH was used as a loading control. Bottom, CEACAM1 mRNA levels were quantified by real time PCR, using GAPDH for normalization.

Mentions: In order to determine whether chromatin structure plays a role in modulating CEACAM1 transcription, we monitored the promoter region for histone modifications. First, we used an antibody which recognizes acetylated lysine 9 and lysine 18 of histone H3, marks associated with actively transcribed genes, and probed the CEACAM1 promoter by ChIP in MDA-MB-468, MCF10A and MCF7 cells. While both MDA-MB-468 and MCF10A cells exhibited a strong signal for acetylated histone H3, in MCF7 cells the CEACAM1 promoter showed significantly decreased acetylation, in agreement with the CEACAM1 expression pattern in these cell lines (Figure 5A). Since a hypoacetylated promoter can be activated by histone deacetylase inhibitors, we treated MCF7 cells with 1 μM Trichostatin A for 0 h, 6 h, and 24 h, respectively and monitored CEACAM1 mRNA levels by RT-PCR. Trichostatin A treatment induced a modest increase in CEACAM1 mRNA levels (Figure 5B), suggesting that apart from reduced acetylation there are other factors contributing to CEACAM1 down-regulation. We next performed ChIP with antibodies to trimethyl-histone H3 Lys 9, a well studied histone modification linked to condensed chromatin structure and gene silencing [33]. Neither MDA-MB-468 nor MCF10A cells showed H3 Lys9 trimethylation at the CEACAM1 promoter; for MCF7 cells the signal was also essentially negative. We also performed ChIP to detect histone H3 lysine 27 trimethylation at the CEACAM1 promoter, another mark of silenced chromatin [34,35]. Unexpectedly, all three cell lines exhibited strong H3K27 trimethylation at the CEACAM1 promoter region (Figure 5A). Thus, it is unlikely that the role of the H3K27 mark on the CEACAM1 promoter is solely down-regulation of gene expression. It is also unlikely that H3K27 trimethylation is responsible for CEACAM1 down-regulation in MCF7 cells.


Regulation of CEACAM1 transcription in human breast epithelial cells.

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

Chromatin structure at the CEACAM1 promoter in MDA-MB-468, MCF10A and MCF7 cells. A. Chromatin immunoprecipitations of CEACAM1 promoter DNA from MDA-MB-468, MCF10A and MCF7 cells with antibodies detecting acetylated Lys9 and Lys 18 of histone H3 (H3Ac), trimethylated Lys 9 of histone H3 (H3KMe), and trimethylated Lys 27 of histone H3 (H3K27Me). IgG was used as negative control; In: input DNA. B. MCF7 cells were treated with either DMSO or Trichostatin A for 0 h, 6 h, and 24 h, respectively, and total RNA was isolated and subjected to RT-PCR with primers amplifying CEACAM1 (top). GAPDH was used as a loading control. Bottom, CEACAM1 mRNA levels were quantified by real time PCR, using GAPDH for normalization.
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Figure 5: Chromatin structure at the CEACAM1 promoter in MDA-MB-468, MCF10A and MCF7 cells. A. Chromatin immunoprecipitations of CEACAM1 promoter DNA from MDA-MB-468, MCF10A and MCF7 cells with antibodies detecting acetylated Lys9 and Lys 18 of histone H3 (H3Ac), trimethylated Lys 9 of histone H3 (H3KMe), and trimethylated Lys 27 of histone H3 (H3K27Me). IgG was used as negative control; In: input DNA. B. MCF7 cells were treated with either DMSO or Trichostatin A for 0 h, 6 h, and 24 h, respectively, and total RNA was isolated and subjected to RT-PCR with primers amplifying CEACAM1 (top). GAPDH was used as a loading control. Bottom, CEACAM1 mRNA levels were quantified by real time PCR, using GAPDH for normalization.
Mentions: In order to determine whether chromatin structure plays a role in modulating CEACAM1 transcription, we monitored the promoter region for histone modifications. First, we used an antibody which recognizes acetylated lysine 9 and lysine 18 of histone H3, marks associated with actively transcribed genes, and probed the CEACAM1 promoter by ChIP in MDA-MB-468, MCF10A and MCF7 cells. While both MDA-MB-468 and MCF10A cells exhibited a strong signal for acetylated histone H3, in MCF7 cells the CEACAM1 promoter showed significantly decreased acetylation, in agreement with the CEACAM1 expression pattern in these cell lines (Figure 5A). Since a hypoacetylated promoter can be activated by histone deacetylase inhibitors, we treated MCF7 cells with 1 μM Trichostatin A for 0 h, 6 h, and 24 h, respectively and monitored CEACAM1 mRNA levels by RT-PCR. Trichostatin A treatment induced a modest increase in CEACAM1 mRNA levels (Figure 5B), suggesting that apart from reduced acetylation there are other factors contributing to CEACAM1 down-regulation. We next performed ChIP with antibodies to trimethyl-histone H3 Lys 9, a well studied histone modification linked to condensed chromatin structure and gene silencing [33]. Neither MDA-MB-468 nor MCF10A cells showed H3 Lys9 trimethylation at the CEACAM1 promoter; for MCF7 cells the signal was also essentially negative. We also performed ChIP to detect histone H3 lysine 27 trimethylation at the CEACAM1 promoter, another mark of silenced chromatin [34,35]. Unexpectedly, all three cell lines exhibited strong H3K27 trimethylation at the CEACAM1 promoter region (Figure 5A). Thus, it is unlikely that the role of the H3K27 mark on the CEACAM1 promoter is solely down-regulation of gene expression. It is also unlikely that H3K27 trimethylation is responsible for CEACAM1 down-regulation in MCF7 cells.

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