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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: 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.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

Role of Sp1 binding sites in the promoter activity of the human FCGRT gene.(A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using TransIT-2020 transfection reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P < 0.01 vs. pGL3-711(WT) construct; **P < 0.01 compared with pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2) and pGL3-711(mSp1-1+2+3).
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pone.0135141.g008: Role of Sp1 binding sites in the promoter activity of the human FCGRT gene.(A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using TransIT-2020 transfection reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P < 0.01 vs. pGL3-711(WT) construct; **P < 0.01 compared with pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2) and pGL3-711(mSp1-1+2+3).

Mentions: To investigate the function of individual Sp1 sites, each Sp1 site alone or in several combinations was mutated in the pGl3-711(WT) promoter construct. The activities of mutant promoters were compared to that of the wild-type promoter construct. As shown in Fig 8B, the pGL3-711(WT) wild-type construct increased the luciferase activity 10-fold in Caco-2, THP-1, HSkMEC cell lines, and 20-fold in Lu 106 cells compared with the promoterless pGL3-basic vector. Mutation of the Sp1 binding site at -641 [pGl3-711(mSp1-1) construct] and -635 [pGl3-711(mSp1-2) construct] as well as the double Sp1 mutant [pGL3-711(mSp1-1+2)] reduced the hFCGRT promoter activity to about 50% of the wild-type promoter activity in Caco-2, HSkMEC and THP-1 cell lines, and 56–66% in Lu 106 cells, suggesting that the two sites are involved in transcription of the human FCGRT gene in an interdependent manner. Mutation of the Sp1 site at -313 [pGl3-711(mSp1-3) construct] had a dramatic effect on the promoter activity in all the selected cells, as there was an 85–89% decrease observed in the luciferase activity. When the Sp1 sites at positions -641, -635, and -313 were mutated simultaneously [pGL3-711(mSp1-1+2+3) construct], luciferase expression driven by this mutant construct was comparable to that of the wild-type pGL3-711 (WT) in Lu 106 cells, while in Caco-2, THP-1 and HSkMEC cells it was reduced only to 55–60% of the wild-type promoter activity, essentially abating the positive effect of the Sp1 site at -313. These results indicated that the Sp1 sites located at positions -641, -635, and -313 acted as positive regulators of the hFCGRT promoter activity. The Sp1 motif at -313 may be the most important regulatory sequence for transcription of the human FCGRT gene in all model cells, because mutation of this site had a stronger effect on promoter activity than did mutations in the Sp1 binding sites at -641 and -635. The data also suggested that the partly overlapping Sp1 sites at positions -641 and -635 might suppress the positive effect of the Sp1 sequence at-313 on the human FCGRT gene transcription.


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

Mikulska JE - PLoS ONE (2015)

Role of Sp1 binding sites in the promoter activity of the human FCGRT gene.(A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using TransIT-2020 transfection reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P < 0.01 vs. pGL3-711(WT) construct; **P < 0.01 compared with pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2) and pGL3-711(mSp1-1+2+3).
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pone.0135141.g008: Role of Sp1 binding sites in the promoter activity of the human FCGRT gene.(A) Schematic diagram representing the wild-type promoter construct and the mutant promoter constructs. Mutations are marked with crosses. (B) Summary of the results of luciferase activity. Mutant promoter constructs were generated from the wild-type pGL3-711(WT) subjected to site-directed mutagenesis by the QuikChange Lightning Site-directed Mutagenesis Kit, as described in Materials and Methods. Mutant promoter constructs pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2), pGL3-711(mSp1-3) and pGL3-711(mSp1-1+2+3) contain mutations in the Sp1sequence at positions -641, -635, -641 plus -635, -313, -641 plus -635 plus -313, respectively. Using TransIT-2020 transfection reagent, pGL3-basic vector, wild-type pGL3-711(WT) construct and site-directed mutant constructs were transiently cotransfected with the phRG-B plasmid (internal control) into THP-1, Lu 106, Caco-2, and HSkMEC cell lines. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. The transfected THP-1 cells were exposed to PMA (100 ng/ml) for 4 hours prior to harvesting. Transfection efficiency was normalized to the Renilla luciferase activity derived from the phRG-B plasmid and expressed as relative luciferase activity. The data shown are means ± SD of three to six independent experiments performed in duplicate for each construct. The transcriptional activity of the mutant constructs and pGL3-basic is plotted as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. The differences between the wild-type construct and mutant constructs were statistically significant; *P < 0.01 vs. pGL3-711(WT) construct; **P < 0.01 compared with pGL3-711(mSp1-1), pGL3-711(mSp1-2), pGL3-711(mSp1-1+2) and pGL3-711(mSp1-1+2+3).
Mentions: To investigate the function of individual Sp1 sites, each Sp1 site alone or in several combinations was mutated in the pGl3-711(WT) promoter construct. The activities of mutant promoters were compared to that of the wild-type promoter construct. As shown in Fig 8B, the pGL3-711(WT) wild-type construct increased the luciferase activity 10-fold in Caco-2, THP-1, HSkMEC cell lines, and 20-fold in Lu 106 cells compared with the promoterless pGL3-basic vector. Mutation of the Sp1 binding site at -641 [pGl3-711(mSp1-1) construct] and -635 [pGl3-711(mSp1-2) construct] as well as the double Sp1 mutant [pGL3-711(mSp1-1+2)] reduced the hFCGRT promoter activity to about 50% of the wild-type promoter activity in Caco-2, HSkMEC and THP-1 cell lines, and 56–66% in Lu 106 cells, suggesting that the two sites are involved in transcription of the human FCGRT gene in an interdependent manner. Mutation of the Sp1 site at -313 [pGl3-711(mSp1-3) construct] had a dramatic effect on the promoter activity in all the selected cells, as there was an 85–89% decrease observed in the luciferase activity. When the Sp1 sites at positions -641, -635, and -313 were mutated simultaneously [pGL3-711(mSp1-1+2+3) construct], luciferase expression driven by this mutant construct was comparable to that of the wild-type pGL3-711 (WT) in Lu 106 cells, while in Caco-2, THP-1 and HSkMEC cells it was reduced only to 55–60% of the wild-type promoter activity, essentially abating the positive effect of the Sp1 site at -313. These results indicated that the Sp1 sites located at positions -641, -635, and -313 acted as positive regulators of the hFCGRT promoter activity. The Sp1 motif at -313 may be the most important regulatory sequence for transcription of the human FCGRT gene in all model cells, because mutation of this site had a stronger effect on promoter activity than did mutations in the Sp1 binding sites at -641 and -635. The data also suggested that the partly overlapping Sp1 sites at positions -641 and -635 might suppress the positive effect of the Sp1 sequence at-313 on the human FCGRT gene transcription.

Bottom Line: 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.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