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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.


RT-PCR analysis of human FCGRT mRNA in cell lines: (A) THP-1, HUVEC, Caco-2, and Lu 106, (B) HSkMEC.Total RNA from these cell lines was subjected toRT-PCR using primers specific for human FCGRT and GAPDH. For control samples, reverse transcription was omitted. The amplified PCR products with (+) or without (-) reverse transcription (RT) were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Arrows indicate the location of the amplification products of expected sizes for human FCGRT and GAPDH. The size marker-GeneRuler-100 bp DNA ladder (M). Total RNA from the HL-60 cell line was subjected to RT-PCR analysis (as negative control, FCGRT mRNA is not expressed in this cell line).
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pone.0135141.g001: RT-PCR analysis of human FCGRT mRNA in cell lines: (A) THP-1, HUVEC, Caco-2, and Lu 106, (B) HSkMEC.Total RNA from these cell lines was subjected toRT-PCR using primers specific for human FCGRT and GAPDH. For control samples, reverse transcription was omitted. The amplified PCR products with (+) or without (-) reverse transcription (RT) were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Arrows indicate the location of the amplification products of expected sizes for human FCGRT and GAPDH. The size marker-GeneRuler-100 bp DNA ladder (M). Total RNA from the HL-60 cell line was subjected to RT-PCR analysis (as negative control, FCGRT mRNA is not expressed in this cell line).

Mentions: For studies on the regulation of transcription of human FCGRT, human epithelial and endothelial cell lines and PMA-differentiated THP-1 cells were used. These cell lines were chosen, because they represent cell types that have been found to express hFcRn in vivo. Although hFcRn was previously reported to be expressed in THP-1 cells [14] and the human intestinal Caco-2 cell line [31], the presence of the human FCGRT mRNA was verified in the selected Caco-2, Lu 106, HUVEC, HSkMEC, and THP-1 cell lines by RT-PCR to make sure that these cells are a suitable model systems for studying transcriptional regulation of human FCGRT. The results of RT-PCR screening are shown in Fig 1, where the PCR product of the expected size of 457 bp is clearly visible. Sequencing of this PCR product demonstrated 100% identity with the previously reported sequence of the human FCGRT gene [26].


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

Mikulska JE - PLoS ONE (2015)

RT-PCR analysis of human FCGRT mRNA in cell lines: (A) THP-1, HUVEC, Caco-2, and Lu 106, (B) HSkMEC.Total RNA from these cell lines was subjected toRT-PCR using primers specific for human FCGRT and GAPDH. For control samples, reverse transcription was omitted. The amplified PCR products with (+) or without (-) reverse transcription (RT) were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Arrows indicate the location of the amplification products of expected sizes for human FCGRT and GAPDH. The size marker-GeneRuler-100 bp DNA ladder (M). Total RNA from the HL-60 cell line was subjected to RT-PCR analysis (as negative control, FCGRT mRNA is not expressed in this cell line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4529178&req=5

pone.0135141.g001: RT-PCR analysis of human FCGRT mRNA in cell lines: (A) THP-1, HUVEC, Caco-2, and Lu 106, (B) HSkMEC.Total RNA from these cell lines was subjected toRT-PCR using primers specific for human FCGRT and GAPDH. For control samples, reverse transcription was omitted. The amplified PCR products with (+) or without (-) reverse transcription (RT) were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium bromide. Arrows indicate the location of the amplification products of expected sizes for human FCGRT and GAPDH. The size marker-GeneRuler-100 bp DNA ladder (M). Total RNA from the HL-60 cell line was subjected to RT-PCR analysis (as negative control, FCGRT mRNA is not expressed in this cell line).
Mentions: For studies on the regulation of transcription of human FCGRT, human epithelial and endothelial cell lines and PMA-differentiated THP-1 cells were used. These cell lines were chosen, because they represent cell types that have been found to express hFcRn in vivo. Although hFcRn was previously reported to be expressed in THP-1 cells [14] and the human intestinal Caco-2 cell line [31], the presence of the human FCGRT mRNA was verified in the selected Caco-2, Lu 106, HUVEC, HSkMEC, and THP-1 cell lines by RT-PCR to make sure that these cells are a suitable model systems for studying transcriptional regulation of human FCGRT. The results of RT-PCR screening are shown in Fig 1, where the PCR product of the expected size of 457 bp is clearly visible. Sequencing of this PCR product demonstrated 100% identity with the previously reported sequence of the human FCGRT gene [26].

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.