Limits...
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

Identification of transcription factors binding to the Sp1 sequences within the -660/-233 fragment of the hFCGRT promoter.Supershift experiments were performed by preincubating the nuclear extract from differentiated THP-1 cells on ice for 1 h with 2 μg of rabbit polyclonal antibodies specific for the Sp family transcription factors or normal rabbit IgG prior to the addition of 32P-labeled wild-type probes: Sp1-1+2(WT), (A); Sp1-3(WT), (B). Labeled probe alone (A and B, lanes 1); labeled probe incubated with nuclear extract in the absence of antibodies (A and B, lanes 2); in the presence of rabbit polyclonal antibodies: anti-Sp1 (A and B, lanes 4), anti-Sp2 (A and B, lanes 5), anti-Sp3 (A and B, lanes 6), anti-AP-2 (A, lane 7), normal rabbit IgG (A and B, lanes 3). Shifted bands are imarked with an asterisk. Results were analyzed by a phosphor imager.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4529178&req=5

pone.0135141.g003: Identification of transcription factors binding to the Sp1 sequences within the -660/-233 fragment of the hFCGRT promoter.Supershift experiments were performed by preincubating the nuclear extract from differentiated THP-1 cells on ice for 1 h with 2 μg of rabbit polyclonal antibodies specific for the Sp family transcription factors or normal rabbit IgG prior to the addition of 32P-labeled wild-type probes: Sp1-1+2(WT), (A); Sp1-3(WT), (B). Labeled probe alone (A and B, lanes 1); labeled probe incubated with nuclear extract in the absence of antibodies (A and B, lanes 2); in the presence of rabbit polyclonal antibodies: anti-Sp1 (A and B, lanes 4), anti-Sp2 (A and B, lanes 5), anti-Sp3 (A and B, lanes 6), anti-AP-2 (A, lane 7), normal rabbit IgG (A and B, lanes 3). Shifted bands are imarked with an asterisk. Results were analyzed by a phosphor imager.

Mentions: Incubation of nuclear extracts with radiolabeled ds-oligonucleotide Sp1-1+2, containing putative partly overlapping Sp1 binding motifs located at -641 and -635, produced two DNA-protein complexes (Fig 2A, lane 2). A single band was revealed in reaction with ds-oligonucleotide Sp1-3 containing a putative binding site at position -313 (Fig 2B, lane 2). These complexes were abolished when nuclear extracts were preincubated with a 100-fold molar excess of unlabeled wild-type oligonucleotides (Fig 2A and 2B, lanes 3), but not the mutant oligonucleotides (Fig 2A, lanes 4,5 and 2B, lane 4). The competition results indicated that nuclear proteins present in the selected cell lines interacted specifically with GC box at position -641, -635 and -313. To determine the nature of the transcription factor interacting with the identified regulatory Sp1 elements within the hFCGRT promoter, supershift assays were performed with antibodies against the Sp family transcription factors. The upper complex 1 supershifted with antibodies specific for Sp1 (Fig 3A, lane 4) and Sp2 (Fig 3A, lane 5). Antibody against Sp3 caused a loss of the faster migrating complex 2 (Fig 3A, lane 6). Both Sp1 and Sp2 specific antibodies reduced the complex formation with the Sp1-3 probe (Fig 3B, lanes 4 and 5), but a small amount of supershifted complex was also observed. In contrast, the control normal rabbit IgG (Fig 3A and 3B, lanes 3) had no effect. Taken together, supershift experiments showed that members of the Sp transcription factor family (Sp1, Sp2, Sp3) are involved in specific interactions with Sp1 binding sites at positions -641 and -635 within the hFCGRT promoter in the selected cell lines, whereas the Sp1 and Sp2 nuclear proteins specifically bind to the Sp1 sequence located at -313 in the hFCGRT promoter.


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

Mikulska JE - PLoS ONE (2015)

Identification of transcription factors binding to the Sp1 sequences within the -660/-233 fragment of the hFCGRT promoter.Supershift experiments were performed by preincubating the nuclear extract from differentiated THP-1 cells on ice for 1 h with 2 μg of rabbit polyclonal antibodies specific for the Sp family transcription factors or normal rabbit IgG prior to the addition of 32P-labeled wild-type probes: Sp1-1+2(WT), (A); Sp1-3(WT), (B). Labeled probe alone (A and B, lanes 1); labeled probe incubated with nuclear extract in the absence of antibodies (A and B, lanes 2); in the presence of rabbit polyclonal antibodies: anti-Sp1 (A and B, lanes 4), anti-Sp2 (A and B, lanes 5), anti-Sp3 (A and B, lanes 6), anti-AP-2 (A, lane 7), normal rabbit IgG (A and B, lanes 3). Shifted bands are imarked with an asterisk. Results were analyzed by a phosphor imager.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0135141.g003: Identification of transcription factors binding to the Sp1 sequences within the -660/-233 fragment of the hFCGRT promoter.Supershift experiments were performed by preincubating the nuclear extract from differentiated THP-1 cells on ice for 1 h with 2 μg of rabbit polyclonal antibodies specific for the Sp family transcription factors or normal rabbit IgG prior to the addition of 32P-labeled wild-type probes: Sp1-1+2(WT), (A); Sp1-3(WT), (B). Labeled probe alone (A and B, lanes 1); labeled probe incubated with nuclear extract in the absence of antibodies (A and B, lanes 2); in the presence of rabbit polyclonal antibodies: anti-Sp1 (A and B, lanes 4), anti-Sp2 (A and B, lanes 5), anti-Sp3 (A and B, lanes 6), anti-AP-2 (A, lane 7), normal rabbit IgG (A and B, lanes 3). Shifted bands are imarked with an asterisk. Results were analyzed by a phosphor imager.
Mentions: Incubation of nuclear extracts with radiolabeled ds-oligonucleotide Sp1-1+2, containing putative partly overlapping Sp1 binding motifs located at -641 and -635, produced two DNA-protein complexes (Fig 2A, lane 2). A single band was revealed in reaction with ds-oligonucleotide Sp1-3 containing a putative binding site at position -313 (Fig 2B, lane 2). These complexes were abolished when nuclear extracts were preincubated with a 100-fold molar excess of unlabeled wild-type oligonucleotides (Fig 2A and 2B, lanes 3), but not the mutant oligonucleotides (Fig 2A, lanes 4,5 and 2B, lane 4). The competition results indicated that nuclear proteins present in the selected cell lines interacted specifically with GC box at position -641, -635 and -313. To determine the nature of the transcription factor interacting with the identified regulatory Sp1 elements within the hFCGRT promoter, supershift assays were performed with antibodies against the Sp family transcription factors. The upper complex 1 supershifted with antibodies specific for Sp1 (Fig 3A, lane 4) and Sp2 (Fig 3A, lane 5). Antibody against Sp3 caused a loss of the faster migrating complex 2 (Fig 3A, lane 6). Both Sp1 and Sp2 specific antibodies reduced the complex formation with the Sp1-3 probe (Fig 3B, lanes 4 and 5), but a small amount of supershifted complex was also observed. In contrast, the control normal rabbit IgG (Fig 3A and 3B, lanes 3) had no effect. Taken together, supershift experiments showed that members of the Sp transcription factor family (Sp1, Sp2, Sp3) are involved in specific interactions with Sp1 binding sites at positions -641 and -635 within the hFCGRT promoter in the selected cell lines, whereas the Sp1 and Sp2 nuclear proteins specifically bind to the Sp1 sequence located at -313 in the hFCGRT promoter.

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