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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

Electrophoretic mobility shift assays with probe Sp1-1+2 (A) and probe Sp1-3 (B).Probe Sp1-1+2 − double-stranded oligonucleotide representing the -646/-611 sequence of the hFCGRT promoter, containing the potential transcriptional regulatory Sp1 motifs at positions -641 and -635; probe Sp1-3 − ds-oligonucleotide corresponding to the sequence -320/-293 of the hFCGRT promoter containing a putative Sp1 binding site at position -313. Probes were end-labeled with [γ32P]ATP] and incubated with nuclear extract (12 μg) in the absence of competitor (A and B, lanes 2) or in the presence of a 100-fold molar excess competitor: Sp1-1+2(WT)–unlabeled wild-type probe Sp1-1+2 (A, lane 3), Sp1-3(WT)–unlabeled wild-type probe Sp1-3 (B, lane 3), mSp1-1+2 – unlabeled probeSp1-1+2 containing mutation in the Sp1 sequence at positions -641 and -635 (A, lane 4), mSp1-2 – unlabeled probe Sp1-1+2 containing mutation in the Sp1 sequence at position -635 (A, lane 5), mSp1-3 – unlabeled probe Sp1-3 containing mutation in the Sp1 sequence at position -313 (B, lane 4). Labeled probe Sp1-1+2(WT) alone (A, lane 1), labeled probe Sp1-3(WT) alone (B, lane 1). DNA-protein complexes were resolved on 5% non-denaturing polyacrylamide gels and analyzed in a phosphor imager (Typhoon 8600) using ImageQuant software (Molecular Dynamics). Positions of specific DNA-protein complexes are indicated by arrows.
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pone.0135141.g002: Electrophoretic mobility shift assays with probe Sp1-1+2 (A) and probe Sp1-3 (B).Probe Sp1-1+2 − double-stranded oligonucleotide representing the -646/-611 sequence of the hFCGRT promoter, containing the potential transcriptional regulatory Sp1 motifs at positions -641 and -635; probe Sp1-3 − ds-oligonucleotide corresponding to the sequence -320/-293 of the hFCGRT promoter containing a putative Sp1 binding site at position -313. Probes were end-labeled with [γ32P]ATP] and incubated with nuclear extract (12 μg) in the absence of competitor (A and B, lanes 2) or in the presence of a 100-fold molar excess competitor: Sp1-1+2(WT)–unlabeled wild-type probe Sp1-1+2 (A, lane 3), Sp1-3(WT)–unlabeled wild-type probe Sp1-3 (B, lane 3), mSp1-1+2 – unlabeled probeSp1-1+2 containing mutation in the Sp1 sequence at positions -641 and -635 (A, lane 4), mSp1-2 – unlabeled probe Sp1-1+2 containing mutation in the Sp1 sequence at position -635 (A, lane 5), mSp1-3 – unlabeled probe Sp1-3 containing mutation in the Sp1 sequence at position -313 (B, lane 4). Labeled probe Sp1-1+2(WT) alone (A, lane 1), labeled probe Sp1-3(WT) alone (B, lane 1). DNA-protein complexes were resolved on 5% non-denaturing polyacrylamide gels and analyzed in a phosphor imager (Typhoon 8600) using ImageQuant software (Molecular Dynamics). Positions of specific DNA-protein complexes are indicated by arrows.

Mentions: Representative results from EMSA and supershift tests of the hFCGRT promoter are presented in Figs 2–5, Figs 6A and 7. They were obtained using the nuclear extract from PMA-differentiated THP-1 cells. A similar or identical banding pattern was observed with nuclear extracts from epithelial and endothelial cells (Fig 6B–6D, S1–S4 Figs).


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

Mikulska JE - PLoS ONE (2015)

Electrophoretic mobility shift assays with probe Sp1-1+2 (A) and probe Sp1-3 (B).Probe Sp1-1+2 − double-stranded oligonucleotide representing the -646/-611 sequence of the hFCGRT promoter, containing the potential transcriptional regulatory Sp1 motifs at positions -641 and -635; probe Sp1-3 − ds-oligonucleotide corresponding to the sequence -320/-293 of the hFCGRT promoter containing a putative Sp1 binding site at position -313. Probes were end-labeled with [γ32P]ATP] and incubated with nuclear extract (12 μg) in the absence of competitor (A and B, lanes 2) or in the presence of a 100-fold molar excess competitor: Sp1-1+2(WT)–unlabeled wild-type probe Sp1-1+2 (A, lane 3), Sp1-3(WT)–unlabeled wild-type probe Sp1-3 (B, lane 3), mSp1-1+2 – unlabeled probeSp1-1+2 containing mutation in the Sp1 sequence at positions -641 and -635 (A, lane 4), mSp1-2 – unlabeled probe Sp1-1+2 containing mutation in the Sp1 sequence at position -635 (A, lane 5), mSp1-3 – unlabeled probe Sp1-3 containing mutation in the Sp1 sequence at position -313 (B, lane 4). Labeled probe Sp1-1+2(WT) alone (A, lane 1), labeled probe Sp1-3(WT) alone (B, lane 1). DNA-protein complexes were resolved on 5% non-denaturing polyacrylamide gels and analyzed in a phosphor imager (Typhoon 8600) using ImageQuant software (Molecular Dynamics). Positions of specific DNA-protein complexes are indicated by arrows.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4529178&req=5

pone.0135141.g002: Electrophoretic mobility shift assays with probe Sp1-1+2 (A) and probe Sp1-3 (B).Probe Sp1-1+2 − double-stranded oligonucleotide representing the -646/-611 sequence of the hFCGRT promoter, containing the potential transcriptional regulatory Sp1 motifs at positions -641 and -635; probe Sp1-3 − ds-oligonucleotide corresponding to the sequence -320/-293 of the hFCGRT promoter containing a putative Sp1 binding site at position -313. Probes were end-labeled with [γ32P]ATP] and incubated with nuclear extract (12 μg) in the absence of competitor (A and B, lanes 2) or in the presence of a 100-fold molar excess competitor: Sp1-1+2(WT)–unlabeled wild-type probe Sp1-1+2 (A, lane 3), Sp1-3(WT)–unlabeled wild-type probe Sp1-3 (B, lane 3), mSp1-1+2 – unlabeled probeSp1-1+2 containing mutation in the Sp1 sequence at positions -641 and -635 (A, lane 4), mSp1-2 – unlabeled probe Sp1-1+2 containing mutation in the Sp1 sequence at position -635 (A, lane 5), mSp1-3 – unlabeled probe Sp1-3 containing mutation in the Sp1 sequence at position -313 (B, lane 4). Labeled probe Sp1-1+2(WT) alone (A, lane 1), labeled probe Sp1-3(WT) alone (B, lane 1). DNA-protein complexes were resolved on 5% non-denaturing polyacrylamide gels and analyzed in a phosphor imager (Typhoon 8600) using ImageQuant software (Molecular Dynamics). Positions of specific DNA-protein complexes are indicated by arrows.
Mentions: Representative results from EMSA and supershift tests of the hFCGRT promoter are presented in Figs 2–5, Figs 6A and 7. They were obtained using the nuclear extract from PMA-differentiated THP-1 cells. A similar or identical banding pattern was observed with nuclear extracts from epithelial and endothelial cells (Fig 6B–6D, S1–S4 Figs).

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