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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 the AP-1 binding motif on the transcriptional activity of the hFCGRT promoter.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B) Summarized results of luciferase activity tests. The pGL3-711(mAP1) mutant construct was prepared as described in Materials and Methods. This construct is a derivative of the pGl3-711(WT) wild-type construct containing a mutation in the AP-1 binding site at position -276. Transfection and normalization were performed as described in the legend to Fig 8. Values are expressed as means ± SD of three to six independent experiments performed in duplicate. The promoter activity of mutant constructs and pGL3-basic is represented as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. Significant repression of transcription compared with the wild-type construct, *P < 0.01 versus pGL3-711(WT).
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pone.0135141.g009: Effect of the AP-1 binding motif on the transcriptional activity of the hFCGRT promoter.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B) Summarized results of luciferase activity tests. The pGL3-711(mAP1) mutant construct was prepared as described in Materials and Methods. This construct is a derivative of the pGl3-711(WT) wild-type construct containing a mutation in the AP-1 binding site at position -276. Transfection and normalization were performed as described in the legend to Fig 8. Values are expressed as means ± SD of three to six independent experiments performed in duplicate. The promoter activity of mutant constructs and pGL3-basic is represented as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. Significant repression of transcription compared with the wild-type construct, *P < 0.01 versus pGL3-711(WT).

Mentions: The pGL3-711(mAP1) construct carrying mutation in the AP-1 motif demonstrated drastic reduction in the ability to drive the reporter activity (Fig 9B), suggesting that the AP-1 site plays an essential role in the activation of the hFCGRT promoter in Caco-2, Lu 106, HSkMEC and THP-1 cells. Mutation of the AP-1 binding site at position -276 caused a decrease in the activity of the hFCGRT promoter in Caco-2 and HSkMEC cells to a level that was not significantly different from that of the promoterless luciferase plasmid pGL3-basic, while in Lu106 and THP-1 cells the activity of the promoter was decreased to 23% and 29%, respectively, compared to the wild-type promoter activity.


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

Mikulska JE - PLoS ONE (2015)

Effect of the AP-1 binding motif on the transcriptional activity of the hFCGRT promoter.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B) Summarized results of luciferase activity tests. The pGL3-711(mAP1) mutant construct was prepared as described in Materials and Methods. This construct is a derivative of the pGl3-711(WT) wild-type construct containing a mutation in the AP-1 binding site at position -276. Transfection and normalization were performed as described in the legend to Fig 8. Values are expressed as means ± SD of three to six independent experiments performed in duplicate. The promoter activity of mutant constructs and pGL3-basic is represented as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. Significant repression of transcription compared with the wild-type construct, *P < 0.01 versus pGL3-711(WT).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4529178&req=5

pone.0135141.g009: Effect of the AP-1 binding motif on the transcriptional activity of the hFCGRT promoter.(A) Schematic diagram representing wild-type and mutant promoter constructs. Mutations are marked with crosses. (B) Summarized results of luciferase activity tests. The pGL3-711(mAP1) mutant construct was prepared as described in Materials and Methods. This construct is a derivative of the pGl3-711(WT) wild-type construct containing a mutation in the AP-1 binding site at position -276. Transfection and normalization were performed as described in the legend to Fig 8. Values are expressed as means ± SD of three to six independent experiments performed in duplicate. The promoter activity of mutant constructs and pGL3-basic is represented as a percentage of the normalized activity of the wild-type pGL3-711(WT) construct, which is defined as 100%. Significant repression of transcription compared with the wild-type construct, *P < 0.01 versus pGL3-711(WT).
Mentions: The pGL3-711(mAP1) construct carrying mutation in the AP-1 motif demonstrated drastic reduction in the ability to drive the reporter activity (Fig 9B), suggesting that the AP-1 site plays an essential role in the activation of the hFCGRT promoter in Caco-2, Lu 106, HSkMEC and THP-1 cells. Mutation of the AP-1 binding site at position -276 caused a decrease in the activity of the hFCGRT promoter in Caco-2 and HSkMEC cells to a level that was not significantly different from that of the promoterless luciferase plasmid pGL3-basic, while in Lu106 and THP-1 cells the activity of the promoter was decreased to 23% and 29%, respectively, compared to the wild-type promoter activity.

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