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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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Chromatin Immunoprecipitation (ChIP).A. Confirmation of protein composition by Western Blot. The presence of (top row to bottom row) PARP-1, Ku70, Ku80, and integrin α2 in HEK293 cells, which express the ITGA2 (CA)12 allele, HeLa cells, which express the ITGA2 (CA)11 allele, or U937 cells, which do not express ITGA2, was confirmed by western blotting. The relative content of each protein in these three cell lines was comparable, except for the integrin α2, which is expressed at reduced levels in HeLa cells, and is absent in U937 cells. B. ChIP assays were performed using HEK293 cells (top row), HeLa cells (center row) and the control cell line U937 (bottom row). U937 bears the ITGA2 (CA)12 allele, but does not express any detectable ITGA2 mRNA, as determined by PCR (data not shown). Chromatin was sheared by sonication, and protein-DNA complexes were immunoprecipitated with antibodies against the PARP-1 or Ku80. The leftmost column represents 5% of total cross-linked chromatin before immunoprecipitation (5% input). Non-immune goat IgG served as a negative control (IgG control.). DNA retrieved after washing was amplified with primers specific for the test sequence encompassing the ITGA2 CA repeat region (beginning at nucleotide −708 and ending at nucleotide −552). Data from one experiment representative of three independent experiments are depicted. C. ChIP assays were performed exactly as in B, except that primers specific for a 3′-UTR negative control sequence were utilized. D. Semi-quantitation of PARP-1 and Ku80 bound to CA repeat sequences in vivo. The results of three ChIP assays such as that depicted in Figure 4B were analyzed semi-quantitatively. The relative binding of PARP-1 or Ku80 to the HEK 293 CA12 compared to HeLa CA11 sites is plotted on the ordinate as the fold-increase in density (HEK293/HeLa) of the photographic images corresponding to the amplified DNA sequences. Image densities were calculated using ImageJ software.
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pone-0008743-g004: Chromatin Immunoprecipitation (ChIP).A. Confirmation of protein composition by Western Blot. The presence of (top row to bottom row) PARP-1, Ku70, Ku80, and integrin α2 in HEK293 cells, which express the ITGA2 (CA)12 allele, HeLa cells, which express the ITGA2 (CA)11 allele, or U937 cells, which do not express ITGA2, was confirmed by western blotting. The relative content of each protein in these three cell lines was comparable, except for the integrin α2, which is expressed at reduced levels in HeLa cells, and is absent in U937 cells. B. ChIP assays were performed using HEK293 cells (top row), HeLa cells (center row) and the control cell line U937 (bottom row). U937 bears the ITGA2 (CA)12 allele, but does not express any detectable ITGA2 mRNA, as determined by PCR (data not shown). Chromatin was sheared by sonication, and protein-DNA complexes were immunoprecipitated with antibodies against the PARP-1 or Ku80. The leftmost column represents 5% of total cross-linked chromatin before immunoprecipitation (5% input). Non-immune goat IgG served as a negative control (IgG control.). DNA retrieved after washing was amplified with primers specific for the test sequence encompassing the ITGA2 CA repeat region (beginning at nucleotide −708 and ending at nucleotide −552). Data from one experiment representative of three independent experiments are depicted. C. ChIP assays were performed exactly as in B, except that primers specific for a 3′-UTR negative control sequence were utilized. D. Semi-quantitation of PARP-1 and Ku80 bound to CA repeat sequences in vivo. The results of three ChIP assays such as that depicted in Figure 4B were analyzed semi-quantitatively. The relative binding of PARP-1 or Ku80 to the HEK 293 CA12 compared to HeLa CA11 sites is plotted on the ordinate as the fold-increase in density (HEK293/HeLa) of the photographic images corresponding to the amplified DNA sequences. Image densities were calculated using ImageJ software.

Mentions: We used the ChIP assay to confirm that the co-activator protein complex is formed at the CA repeat sequence in vivo (Figure 4). We selected established cell lines that are homozygous for (CA)12 or (CA)11, but we have not yet identified a cell line that is homozygous for (CA)10. The HEK293 and U937 cell lines are both homozygous for the (CA)12 repeat, while HeLa cells are homozygous for the (CA)11 repeat. However, the U937 cell line serves as a negative control, because it is devoid of detectable α2β1 mRNA, even though it bears the ITGA2 (CA)12 repeat. Previous results indicate that the U937 ITGA2 promoter region is hypermethylated at CpG sites and transcriptionally silent [16].


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)

Chromatin Immunoprecipitation (ChIP).A. Confirmation of protein composition by Western Blot. The presence of (top row to bottom row) PARP-1, Ku70, Ku80, and integrin α2 in HEK293 cells, which express the ITGA2 (CA)12 allele, HeLa cells, which express the ITGA2 (CA)11 allele, or U937 cells, which do not express ITGA2, was confirmed by western blotting. The relative content of each protein in these three cell lines was comparable, except for the integrin α2, which is expressed at reduced levels in HeLa cells, and is absent in U937 cells. B. ChIP assays were performed using HEK293 cells (top row), HeLa cells (center row) and the control cell line U937 (bottom row). U937 bears the ITGA2 (CA)12 allele, but does not express any detectable ITGA2 mRNA, as determined by PCR (data not shown). Chromatin was sheared by sonication, and protein-DNA complexes were immunoprecipitated with antibodies against the PARP-1 or Ku80. The leftmost column represents 5% of total cross-linked chromatin before immunoprecipitation (5% input). Non-immune goat IgG served as a negative control (IgG control.). DNA retrieved after washing was amplified with primers specific for the test sequence encompassing the ITGA2 CA repeat region (beginning at nucleotide −708 and ending at nucleotide −552). Data from one experiment representative of three independent experiments are depicted. C. ChIP assays were performed exactly as in B, except that primers specific for a 3′-UTR negative control sequence were utilized. D. Semi-quantitation of PARP-1 and Ku80 bound to CA repeat sequences in vivo. The results of three ChIP assays such as that depicted in Figure 4B were analyzed semi-quantitatively. The relative binding of PARP-1 or Ku80 to the HEK 293 CA12 compared to HeLa CA11 sites is plotted on the ordinate as the fold-increase in density (HEK293/HeLa) of the photographic images corresponding to the amplified DNA sequences. Image densities were calculated using ImageJ software.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806922&req=5

pone-0008743-g004: Chromatin Immunoprecipitation (ChIP).A. Confirmation of protein composition by Western Blot. The presence of (top row to bottom row) PARP-1, Ku70, Ku80, and integrin α2 in HEK293 cells, which express the ITGA2 (CA)12 allele, HeLa cells, which express the ITGA2 (CA)11 allele, or U937 cells, which do not express ITGA2, was confirmed by western blotting. The relative content of each protein in these three cell lines was comparable, except for the integrin α2, which is expressed at reduced levels in HeLa cells, and is absent in U937 cells. B. ChIP assays were performed using HEK293 cells (top row), HeLa cells (center row) and the control cell line U937 (bottom row). U937 bears the ITGA2 (CA)12 allele, but does not express any detectable ITGA2 mRNA, as determined by PCR (data not shown). Chromatin was sheared by sonication, and protein-DNA complexes were immunoprecipitated with antibodies against the PARP-1 or Ku80. The leftmost column represents 5% of total cross-linked chromatin before immunoprecipitation (5% input). Non-immune goat IgG served as a negative control (IgG control.). DNA retrieved after washing was amplified with primers specific for the test sequence encompassing the ITGA2 CA repeat region (beginning at nucleotide −708 and ending at nucleotide −552). Data from one experiment representative of three independent experiments are depicted. C. ChIP assays were performed exactly as in B, except that primers specific for a 3′-UTR negative control sequence were utilized. D. Semi-quantitation of PARP-1 and Ku80 bound to CA repeat sequences in vivo. The results of three ChIP assays such as that depicted in Figure 4B were analyzed semi-quantitatively. The relative binding of PARP-1 or Ku80 to the HEK 293 CA12 compared to HeLa CA11 sites is plotted on the ordinate as the fold-increase in density (HEK293/HeLa) of the photographic images corresponding to the amplified DNA sequences. Image densities were calculated using ImageJ software.
Mentions: We used the ChIP assay to confirm that the co-activator protein complex is formed at the CA repeat sequence in vivo (Figure 4). We selected established cell lines that are homozygous for (CA)12 or (CA)11, but we have not yet identified a cell line that is homozygous for (CA)10. The HEK293 and U937 cell lines are both homozygous for the (CA)12 repeat, while HeLa cells are homozygous for the (CA)11 repeat. However, the U937 cell line serves as a negative control, because it is devoid of detectable α2β1 mRNA, even though it bears the ITGA2 (CA)12 repeat. Previous results indicate that the U937 ITGA2 promoter region is hypermethylated at CpG sites and transcriptionally silent [16].

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