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Enhanced binding of poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 promoter via an extended cytosine-adenosine repeat.

Cheli Y, Williams SA, Ballotti R, Nugent DJ, Kunicki TJ - PLoS ONE (2010)

Bottom Line: The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)(12) alleles of ITGA2 coincides with enhanced alpha2beta1 expression.The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2.These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin alpha2beta1 expression in human platelets and other cells.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT

Background: We have identified a cytosine-adenosine (CA) repeat length polymorphism in the 5'-regulatory region of the human integrin alpha2 gene ITGA2 that begins at -605. Our objective was to establish the contribution of this polymorphism to the regulation of integrin alpha2beta1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s).

Methodology/principal findings: In combination with the SNP C-52T, previously identified by us as a binding site for the transcription factor Sp1, four ITGA2 haplotypes can be distinguished, in the order in which they enhance ITGA2 transcription: (CA)(12)/-52C>(CA)(11)/-52C>(CA)(11)/-52T>(CA)(10)/-52T. By DNA affinity chromatography and chromatin immunoprecipitation (ChIP) assays, we show that poly (ADP-ribose)polymerase-1 (PARP-1) and Ku80/70 bind specifically and with enhanced affinity to the longer (CA)(12) repeat alleles.

Conclusions/significance: The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)(12) alleles of ITGA2 coincides with enhanced alpha2beta1 expression. The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2. These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin alpha2beta1 expression in human platelets and other cells.

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Related in: MedlinePlus

Identification of proteins that bind to the (CA)12 oligonucleotide sequence in vitro.A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA)12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in Figure S1. The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).
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pone-0008743-g003: Identification of proteins that bind to the (CA)12 oligonucleotide sequence in vitro.A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA)12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in Figure S1. The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).

Mentions: We used oligonucleotide affinity chromatography to capture proteins that bind to the (CA)12 repeat sequence in vitro (Figure 3). To eliminate non-specific binding, we performed the affinity chromatography in the presence of an excess of calf thymus DNA, as recommended by Kadonaga et al. [15]. Oligonucleotide/protein complexes were immobilized with streptavidin-Sepharose, and bound proteins were eluted from the oligonucleotide by addition of SDS and heating and then separated by SDS-PAGE (Figure 3A). The initial nuclear extract is depicted in lane 1. In the absence of calf thymus DNA, several proteins were complexed with biotin-CA12 (lane 2), including two prominent proteins with MWapp of 120 kDa (arrow a) and 80 kDa (arrow b). In the presence of calf thymus DNA, the same two proteins were still complexed with Biotin-CA12 (lane 3). The addition of a five-fold excess of control oligonucleotide together with Biotin-CA12 (lane 4) did not inhibit the binding of the 120 and 80 kDa proteins to Biotin-CA12. Finally, neither of these two proteins was complexed to the control oligonucleotide alone (lane 5).


Enhanced binding of poly(ADP-ribose)polymerase-1 and Ku80/70 to the ITGA2 promoter via an extended cytosine-adenosine repeat.

Cheli Y, Williams SA, Ballotti R, Nugent DJ, Kunicki TJ - PLoS ONE (2010)

Identification of proteins that bind to the (CA)12 oligonucleotide sequence in vitro.A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA)12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in Figure S1. The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806922&req=5

pone-0008743-g003: Identification of proteins that bind to the (CA)12 oligonucleotide sequence in vitro.A. One mg of Dami nuclear extract protein was incubated with one of four biotinylated (CA)12 oligonucleotide probes in binding buffer. Oligonucleotide/protein complexes were adsorbed to streptavidin-agarose beads, and the bound proteins were eluted, separated by SDS-PAGE and visualized using silver stain. The following protein samples are depicted: (lane1) Starting nuclear extract; (lane 2) Proteins bound to CA12 in the absence of calf thymus DNA; (lane 3) Proteins bound to CA12 in the presence of calf thymus DNA; (lane 4); Proteins bound to CA12 in the presence of calf thymus DNA+ 5-fold molar excess of control oligonucleotide; and (lane 5) Proteins bound to control oligonucleotide in the presence of calf thymus DNA. The two prominent protein bands with MWApp of 120 kDa and 80 kDa (positions indicated by arrows a and b, respectively, to the right of the panel) were excised and processed by MS/MS. Peptides recovered and sequenced by MS/MS are depicted in Figure S1. The positions held by molecular weight standards (Amersham Biosciences, Pittsburgh, PA) are indicated to the left of the panel. B. Dami nuclear extract proteins were incubated in vitro with the biotin-conjugated oligonucleotide probes: CA12, CA11, CA10 or control oligonucleotide (C). The resultant oligonucleotide/protein complexes were pulled down with streptavidin agarose. Nuclear proteins present in the complexes were separated by SDS-PAGE and identified by western blotting using the specific antibodies indicated to the left of the figure. The presence of PARP-1, Ku80 and Ku70 was confirmed in this manner. Antibodies specific for Sp1 served as a negative control, since the oligonucleotides used in these assays does not contain the Sp1 binding site. To confirm comparable oligonucleotide loading, the same samples were electrophoresed in a 2% agarose gel and stained with ethidium bromide (probes; negative image).
Mentions: We used oligonucleotide affinity chromatography to capture proteins that bind to the (CA)12 repeat sequence in vitro (Figure 3). To eliminate non-specific binding, we performed the affinity chromatography in the presence of an excess of calf thymus DNA, as recommended by Kadonaga et al. [15]. Oligonucleotide/protein complexes were immobilized with streptavidin-Sepharose, and bound proteins were eluted from the oligonucleotide by addition of SDS and heating and then separated by SDS-PAGE (Figure 3A). The initial nuclear extract is depicted in lane 1. In the absence of calf thymus DNA, several proteins were complexed with biotin-CA12 (lane 2), including two prominent proteins with MWapp of 120 kDa (arrow a) and 80 kDa (arrow b). In the presence of calf thymus DNA, the same two proteins were still complexed with Biotin-CA12 (lane 3). The addition of a five-fold excess of control oligonucleotide together with Biotin-CA12 (lane 4) did not inhibit the binding of the 120 and 80 kDa proteins to Biotin-CA12. Finally, neither of these two proteins was complexed to the control oligonucleotide alone (lane 5).

Bottom Line: The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)(12) alleles of ITGA2 coincides with enhanced alpha2beta1 expression.The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2.These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin alpha2beta1 expression in human platelets and other cells.

View Article: PubMed Central - PubMed

Affiliation: The Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, United States of America.

ABSTRACT

Background: We have identified a cytosine-adenosine (CA) repeat length polymorphism in the 5'-regulatory region of the human integrin alpha2 gene ITGA2 that begins at -605. Our objective was to establish the contribution of this polymorphism to the regulation of integrin alpha2beta1 expression, which is known to vary several-fold among normal individuals, and to investigate the underlying mechanism(s).

Methodology/principal findings: In combination with the SNP C-52T, previously identified by us as a binding site for the transcription factor Sp1, four ITGA2 haplotypes can be distinguished, in the order in which they enhance ITGA2 transcription: (CA)(12)/-52C>(CA)(11)/-52C>(CA)(11)/-52T>(CA)(10)/-52T. By DNA affinity chromatography and chromatin immunoprecipitation (ChIP) assays, we show that poly (ADP-ribose)polymerase-1 (PARP-1) and Ku80/70 bind specifically and with enhanced affinity to the longer (CA)(12) repeat alleles.

Conclusions/significance: The increased binding of PARP-1 and Ku80/70, known components of transcription co-activator complexes, to the longer (CA)(12) alleles of ITGA2 coincides with enhanced alpha2beta1 expression. The most likely explanation for these findings is that PARP-1 and Ku80/70 contribute to the transcriptional regulation of ITGA2. These observations provide new insight into the mechanisms(s) underlying haplotype-dependent variability in integrin alpha2beta1 expression in human platelets and other cells.

Show MeSH
Related in: MedlinePlus