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Analysis of Response Elements Involved in the Regulation of the Human Neonatal Fc Receptor Gene (FCGRT).

Mikulska JE - PLoS ONE (2015)

Bottom Line: Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter.In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation.EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.

ABSTRACT
Human epithelial, endothelial and PMA-differentiated THP-1 cell lines were used as model systems to study the transcriptional regulation of the human FCGRT gene encoding the alpha chain of hFcRn. The data obtained from site-directed mutagenesis in transient transfection experiments indicate that the Sp1 sites at positions -641, -635, and -313, CF1/YY1 elements at positions -586 and -357, and the AP-1 motif at -276 within the-660/-233 fragment of the human FCGRT promoter (hFCGRT) participate in the regulation of human FCGRT in all selected cell lines. However, their individual contribution to promoter activity is not equivalent. The Sp1 binding site at -313 and the AP-1 site at -276 are critical for the activity of the hFCGRT promoter in epithelial and endothelial cells. Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter. In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation. EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression.

No MeSH data available.


Related in: MedlinePlus

Effect of mutation in the C/EBPβ binding site on the hFCGRT promoter activity in Lu 106, Caco-2, and HSkMEC cell lines.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B and C) Summarized results of luciferase activity tests. The pGL3-711(mC/EBPβ-1) mutant construct, containing a mutation in the C/EBPβ sequence at position -497, was prepared as described in Materials and Methods. Transfection and normalization were performed as described in the legend to Fig 8. Twenty-four hours after transfection, the cells were exposed to PMA(100 ng/ml) (B) and LPS (5 μg/ml) (C) for 6 hours and harvested for luciferase assays. The cells after transfection with the wild-type pGL3-711(WT) construct were cultured for 6 hours in media without LPS and PMA prior to harvesting (control). The promoter activity of the mutant constructs and pGL3-basic plasmid is represented as a percentage of the normalized activity of the wild-type promoter construct in the presence of PMA (pGL3-711(WT)PMA construct) or LPS (pGL3-711(WT)LPS construct), which is defined as 100%. Data shown are means ± SD of three to six independent experiments performed in duplicate. Significant reduction in the promoter activity of the mutant construct compared to wild-type construct, *P < 0.01 vs. pGL3-711(WT)PMA and pGL3-711(WT)LPS. **–denotes a significant difference (**P < 0.01) between the promoter activity of the wild-type construct in stimulated (PMA or LPS) and unstimulated cells.
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pone.0135141.g011: Effect of mutation in the C/EBPβ binding site on the hFCGRT promoter activity in Lu 106, Caco-2, and HSkMEC cell lines.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B and C) Summarized results of luciferase activity tests. The pGL3-711(mC/EBPβ-1) mutant construct, containing a mutation in the C/EBPβ sequence at position -497, was prepared as described in Materials and Methods. Transfection and normalization were performed as described in the legend to Fig 8. Twenty-four hours after transfection, the cells were exposed to PMA(100 ng/ml) (B) and LPS (5 μg/ml) (C) for 6 hours and harvested for luciferase assays. The cells after transfection with the wild-type pGL3-711(WT) construct were cultured for 6 hours in media without LPS and PMA prior to harvesting (control). The promoter activity of the mutant constructs and pGL3-basic plasmid is represented as a percentage of the normalized activity of the wild-type promoter construct in the presence of PMA (pGL3-711(WT)PMA construct) or LPS (pGL3-711(WT)LPS construct), which is defined as 100%. Data shown are means ± SD of three to six independent experiments performed in duplicate. Significant reduction in the promoter activity of the mutant construct compared to wild-type construct, *P < 0.01 vs. pGL3-711(WT)PMA and pGL3-711(WT)LPS. **–denotes a significant difference (**P < 0.01) between the promoter activity of the wild-type construct in stimulated (PMA or LPS) and unstimulated cells.

Mentions: The activity of the hFCGRT promoter was increased 2-fold in Caco-2, Lu 106 and HSkMEC cell lines stimulated by PMA (Fig 11B) and LPS (Fig 11C) in comparison with unstimulated cells. When the C/EBPβ site at -497 was mutated [pGL3-711(mC/EBPβ-1) construct], promoter activity was reduced by almost 50%, reaching a level comparable to that driven by the wild-type pGL3-711(WT) construct in untreated cells.


Analysis of Response Elements Involved in the Regulation of the Human Neonatal Fc Receptor Gene (FCGRT).

Mikulska JE - PLoS ONE (2015)

Effect of mutation in the C/EBPβ binding site on the hFCGRT promoter activity in Lu 106, Caco-2, and HSkMEC cell lines.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B and C) Summarized results of luciferase activity tests. The pGL3-711(mC/EBPβ-1) mutant construct, containing a mutation in the C/EBPβ sequence at position -497, was prepared as described in Materials and Methods. Transfection and normalization were performed as described in the legend to Fig 8. Twenty-four hours after transfection, the cells were exposed to PMA(100 ng/ml) (B) and LPS (5 μg/ml) (C) for 6 hours and harvested for luciferase assays. The cells after transfection with the wild-type pGL3-711(WT) construct were cultured for 6 hours in media without LPS and PMA prior to harvesting (control). The promoter activity of the mutant constructs and pGL3-basic plasmid is represented as a percentage of the normalized activity of the wild-type promoter construct in the presence of PMA (pGL3-711(WT)PMA construct) or LPS (pGL3-711(WT)LPS construct), which is defined as 100%. Data shown are means ± SD of three to six independent experiments performed in duplicate. Significant reduction in the promoter activity of the mutant construct compared to wild-type construct, *P < 0.01 vs. pGL3-711(WT)PMA and pGL3-711(WT)LPS. **–denotes a significant difference (**P < 0.01) between the promoter activity of the wild-type construct in stimulated (PMA or LPS) and unstimulated cells.
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Related In: Results  -  Collection

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pone.0135141.g011: Effect of mutation in the C/EBPβ binding site on the hFCGRT promoter activity in Lu 106, Caco-2, and HSkMEC cell lines.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B and C) Summarized results of luciferase activity tests. The pGL3-711(mC/EBPβ-1) mutant construct, containing a mutation in the C/EBPβ sequence at position -497, was prepared as described in Materials and Methods. Transfection and normalization were performed as described in the legend to Fig 8. Twenty-four hours after transfection, the cells were exposed to PMA(100 ng/ml) (B) and LPS (5 μg/ml) (C) for 6 hours and harvested for luciferase assays. The cells after transfection with the wild-type pGL3-711(WT) construct were cultured for 6 hours in media without LPS and PMA prior to harvesting (control). The promoter activity of the mutant constructs and pGL3-basic plasmid is represented as a percentage of the normalized activity of the wild-type promoter construct in the presence of PMA (pGL3-711(WT)PMA construct) or LPS (pGL3-711(WT)LPS construct), which is defined as 100%. Data shown are means ± SD of three to six independent experiments performed in duplicate. Significant reduction in the promoter activity of the mutant construct compared to wild-type construct, *P < 0.01 vs. pGL3-711(WT)PMA and pGL3-711(WT)LPS. **–denotes a significant difference (**P < 0.01) between the promoter activity of the wild-type construct in stimulated (PMA or LPS) and unstimulated cells.
Mentions: The activity of the hFCGRT promoter was increased 2-fold in Caco-2, Lu 106 and HSkMEC cell lines stimulated by PMA (Fig 11B) and LPS (Fig 11C) in comparison with unstimulated cells. When the C/EBPβ site at -497 was mutated [pGL3-711(mC/EBPβ-1) construct], promoter activity was reduced by almost 50%, reaching a level comparable to that driven by the wild-type pGL3-711(WT) construct in untreated cells.

Bottom Line: Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter.In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation.EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.

ABSTRACT
Human epithelial, endothelial and PMA-differentiated THP-1 cell lines were used as model systems to study the transcriptional regulation of the human FCGRT gene encoding the alpha chain of hFcRn. The data obtained from site-directed mutagenesis in transient transfection experiments indicate that the Sp1 sites at positions -641, -635, and -313, CF1/YY1 elements at positions -586 and -357, and the AP-1 motif at -276 within the-660/-233 fragment of the human FCGRT promoter (hFCGRT) participate in the regulation of human FCGRT in all selected cell lines. However, their individual contribution to promoter activity is not equivalent. The Sp1 binding site at -313 and the AP-1 site at -276 are critical for the activity of the hFCGRT promoter in epithelial and endothelial cells. Moreover, the CF1/YY1 site at -586 in differentiated THP-1 cells, plays an essential role in the transcriptional activity of the promoter. In addition, the C/EBPbeta binding site at -497 of the hFCGRT promoter in epithelial and endothelial cells, and the C/EBPbeta motif located at -497 and -233 within the hFCGRT promoter in differentiated THP-1 cells may function as positive regulatory sequences in response to LPS or PMA stimulation. EMSA and supershift analyses showed that the functionally identified binding motifs in the hFCGRT promoter were able to specifically interact with their corresponding (Sp1, Sp2, Sp3, c-Fos, c-Jun, YY1, and C/EBPbeta or C/EBPdelta) transcription factors (TFs), suggesting their possible involvement in the regulation of the human FCGRT gene expression.

No MeSH data available.


Related in: MedlinePlus