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GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

He X, Syed KS, Tillo D, Mann I, Weirauch MT, Vinson C - G3 (Bethesda) (2015)

Bottom Line: The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes.CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding.We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.

No MeSH data available.


Related in: MedlinePlus

CREB1 enhances GABPα binding to several SNPs in the ETS↔CRE motif. (A) GABPα-GST (8 ng) binding to 1960 features containing the ETS↔CRE 16-mer CCGGAAGTGACGTCAC and 48 SNPs on the ETS-CRE array. The first column of the figure contains 40 black spots representing GABPα-GST binding to the 40 features containing the consensus ETS↔CRE motif CCGGAAGTGACGTCAC. The rest of the columns represent 40 features for each of the 48 SNPs, as indicated. (B) The normalized GABPα-GST binding in the presence of equal concentration (8 ng:8 ng) of the CREB1 plasmid on the ETS-CRE array. (C) Histogram of the ratio of GABPα-GST array intensities ± CREB1 to the consensus and SNP probes. Horizontal dashed line indicates the ratio of GABPα+CREB1/GABPα binding to the consensus motif. (D–F) Same as in (A–C), but for GABPα-GST (± CREB1) binding to 1960 features containing the weaker ETS↔CRE 16-mer GCGGAAGTGACGTCAC motif and 48 SNPs on the ETS-CRE array.
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fig3: CREB1 enhances GABPα binding to several SNPs in the ETS↔CRE motif. (A) GABPα-GST (8 ng) binding to 1960 features containing the ETS↔CRE 16-mer CCGGAAGTGACGTCAC and 48 SNPs on the ETS-CRE array. The first column of the figure contains 40 black spots representing GABPα-GST binding to the 40 features containing the consensus ETS↔CRE motif CCGGAAGTGACGTCAC. The rest of the columns represent 40 features for each of the 48 SNPs, as indicated. (B) The normalized GABPα-GST binding in the presence of equal concentration (8 ng:8 ng) of the CREB1 plasmid on the ETS-CRE array. (C) Histogram of the ratio of GABPα-GST array intensities ± CREB1 to the consensus and SNP probes. Horizontal dashed line indicates the ratio of GABPα+CREB1/GABPα binding to the consensus motif. (D–F) Same as in (A–C), but for GABPα-GST (± CREB1) binding to 1960 features containing the weaker ETS↔CRE 16-mer GCGGAAGTGACGTCAC motif and 48 SNPs on the ETS-CRE array.

Mentions: CREB1 increased binding of GABPα-GST to the canonical motif (C/) approximately two-fold (P < 0.0001, t-test). CREB1 also increases binding to all SNPs, although none is more strongly bound than the canonical motif. In particular, CREB1 increased GABPα-GST binding to the weakly bound SNPs in the core GGA trinucleotide three-fold (Figure 3, A–C). CREB1 has variable effects on increased GABPα-GST binding for two groups of SNPs in bold (CCGGAAGT) at the beginning and end of the ETS motif, with increases of up to 20-fold for four SNPs, TCGGAAGT, CGGGAAGT, CCGGACGT, and CCGGAACT. Importantly, these are the same SNPs that strongly reduced GABPα-GST binding in the absence of CREB1. There is a nonlinear relationship between GABPα array intensities and cooperativity (ratio of GABPα+CREB/GABPα; Figure S4), suggesting that the increased cooperativity by CREB1 is not simply due to a decrease in affinity of the GABPα monomer site. SNPs localized within the CREB1 motif caused only a slight decrease in CREB1-dependent GABPα-GST binding, suggesting that these SNPs instead act by decreasing CREB1 binding, as expected. Similar results were obtained at two other GABPα-GST and CREB1 concentrations (Figure S5 and Figure S6), but in the concentration of 2.5 ng of GABPα-GST and GABPα-GST+CREB1 there is a clear decrease in binding to SNPs localized within the CREB1 motif (Figure S5, C and F), which indicate the binding ability of GABPα depends less on the changes of CREB1 binding when GABPα concentrations are high.


GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

He X, Syed KS, Tillo D, Mann I, Weirauch MT, Vinson C - G3 (Bethesda) (2015)

CREB1 enhances GABPα binding to several SNPs in the ETS↔CRE motif. (A) GABPα-GST (8 ng) binding to 1960 features containing the ETS↔CRE 16-mer CCGGAAGTGACGTCAC and 48 SNPs on the ETS-CRE array. The first column of the figure contains 40 black spots representing GABPα-GST binding to the 40 features containing the consensus ETS↔CRE motif CCGGAAGTGACGTCAC. The rest of the columns represent 40 features for each of the 48 SNPs, as indicated. (B) The normalized GABPα-GST binding in the presence of equal concentration (8 ng:8 ng) of the CREB1 plasmid on the ETS-CRE array. (C) Histogram of the ratio of GABPα-GST array intensities ± CREB1 to the consensus and SNP probes. Horizontal dashed line indicates the ratio of GABPα+CREB1/GABPα binding to the consensus motif. (D–F) Same as in (A–C), but for GABPα-GST (± CREB1) binding to 1960 features containing the weaker ETS↔CRE 16-mer GCGGAAGTGACGTCAC motif and 48 SNPs on the ETS-CRE array.
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fig3: CREB1 enhances GABPα binding to several SNPs in the ETS↔CRE motif. (A) GABPα-GST (8 ng) binding to 1960 features containing the ETS↔CRE 16-mer CCGGAAGTGACGTCAC and 48 SNPs on the ETS-CRE array. The first column of the figure contains 40 black spots representing GABPα-GST binding to the 40 features containing the consensus ETS↔CRE motif CCGGAAGTGACGTCAC. The rest of the columns represent 40 features for each of the 48 SNPs, as indicated. (B) The normalized GABPα-GST binding in the presence of equal concentration (8 ng:8 ng) of the CREB1 plasmid on the ETS-CRE array. (C) Histogram of the ratio of GABPα-GST array intensities ± CREB1 to the consensus and SNP probes. Horizontal dashed line indicates the ratio of GABPα+CREB1/GABPα binding to the consensus motif. (D–F) Same as in (A–C), but for GABPα-GST (± CREB1) binding to 1960 features containing the weaker ETS↔CRE 16-mer GCGGAAGTGACGTCAC motif and 48 SNPs on the ETS-CRE array.
Mentions: CREB1 increased binding of GABPα-GST to the canonical motif (C/) approximately two-fold (P < 0.0001, t-test). CREB1 also increases binding to all SNPs, although none is more strongly bound than the canonical motif. In particular, CREB1 increased GABPα-GST binding to the weakly bound SNPs in the core GGA trinucleotide three-fold (Figure 3, A–C). CREB1 has variable effects on increased GABPα-GST binding for two groups of SNPs in bold (CCGGAAGT) at the beginning and end of the ETS motif, with increases of up to 20-fold for four SNPs, TCGGAAGT, CGGGAAGT, CCGGACGT, and CCGGAACT. Importantly, these are the same SNPs that strongly reduced GABPα-GST binding in the absence of CREB1. There is a nonlinear relationship between GABPα array intensities and cooperativity (ratio of GABPα+CREB/GABPα; Figure S4), suggesting that the increased cooperativity by CREB1 is not simply due to a decrease in affinity of the GABPα monomer site. SNPs localized within the CREB1 motif caused only a slight decrease in CREB1-dependent GABPα-GST binding, suggesting that these SNPs instead act by decreasing CREB1 binding, as expected. Similar results were obtained at two other GABPα-GST and CREB1 concentrations (Figure S5 and Figure S6), but in the concentration of 2.5 ng of GABPα-GST and GABPα-GST+CREB1 there is a clear decrease in binding to SNPs localized within the CREB1 motif (Figure S5, C and F), which indicate the binding ability of GABPα depends less on the changes of CREB1 binding when GABPα concentrations are high.

Bottom Line: The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes.CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding.We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.

No MeSH data available.


Related in: MedlinePlus