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Notch and MAML-1 complexation do not detectably alter the DNA binding specificity of the transcription factor CSL.

Del Bianco C, Vedenko A, Choi SH, Berger MF, Shokri L, Bulyk ML, Blacklow SC - PLoS ONE (2010)

Bottom Line: Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors.Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined.

Principal findings: Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.

Conclusions/significance: These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes.

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Related in: MedlinePlus

Detection of CSL-Notch1 complexes on DNA by monitoring the capture of GST-Notch-1.Columns from left to right show: experimental design, strategy for detection of immobilized complexes, PBM scans, and a DNA sequence logo representing the bound 8-mers. The protein mixtures incubated in separate chambers of the same microarray chip were as follows: A) GST-Notch1/CSL-His6 complexes, B) GST-Notch1/CSL-His6/MAML complexes, and C) GST-Notch1 alone (see methods). C: CSL; N: Notch; M: MAML. Cartoons representing CSL-His6, Notch, and MAML proteins are colored green, blue, and red, respectively.
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pone-0015034-g004: Detection of CSL-Notch1 complexes on DNA by monitoring the capture of GST-Notch-1.Columns from left to right show: experimental design, strategy for detection of immobilized complexes, PBM scans, and a DNA sequence logo representing the bound 8-mers. The protein mixtures incubated in separate chambers of the same microarray chip were as follows: A) GST-Notch1/CSL-His6 complexes, B) GST-Notch1/CSL-His6/MAML complexes, and C) GST-Notch1 alone (see methods). C: CSL; N: Notch; M: MAML. Cartoons representing CSL-His6, Notch, and MAML proteins are colored green, blue, and red, respectively.

Mentions: To confirm that our approach detected multiprotein complexes bound to the DNA, and not merely GST-CSL that was no longer in complex with Notch1 (or MAML-1), we also assembled complexes using hexahistidine-tagged CSL, and prepared a GST-Notch1 fusion protein for use in place of unlabeled Notch1. This scheme allowed for indirect detection of CSL binding to DNA by monitoring the subsequent capture of GST-Notch1 by CSL-DNA complexes [21]. Again, the observed DNA binding site preferences closely resembled those seen upon binding of GST-CSL alone, with GST-Notch1 alone serving as the negative control (Figure 4). This experiment provides an unambiguous demonstration that it is possible to monitor the loading of multiple protein components onto DNA using PBMs.


Notch and MAML-1 complexation do not detectably alter the DNA binding specificity of the transcription factor CSL.

Del Bianco C, Vedenko A, Choi SH, Berger MF, Shokri L, Bulyk ML, Blacklow SC - PLoS ONE (2010)

Detection of CSL-Notch1 complexes on DNA by monitoring the capture of GST-Notch-1.Columns from left to right show: experimental design, strategy for detection of immobilized complexes, PBM scans, and a DNA sequence logo representing the bound 8-mers. The protein mixtures incubated in separate chambers of the same microarray chip were as follows: A) GST-Notch1/CSL-His6 complexes, B) GST-Notch1/CSL-His6/MAML complexes, and C) GST-Notch1 alone (see methods). C: CSL; N: Notch; M: MAML. Cartoons representing CSL-His6, Notch, and MAML proteins are colored green, blue, and red, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991368&req=5

pone-0015034-g004: Detection of CSL-Notch1 complexes on DNA by monitoring the capture of GST-Notch-1.Columns from left to right show: experimental design, strategy for detection of immobilized complexes, PBM scans, and a DNA sequence logo representing the bound 8-mers. The protein mixtures incubated in separate chambers of the same microarray chip were as follows: A) GST-Notch1/CSL-His6 complexes, B) GST-Notch1/CSL-His6/MAML complexes, and C) GST-Notch1 alone (see methods). C: CSL; N: Notch; M: MAML. Cartoons representing CSL-His6, Notch, and MAML proteins are colored green, blue, and red, respectively.
Mentions: To confirm that our approach detected multiprotein complexes bound to the DNA, and not merely GST-CSL that was no longer in complex with Notch1 (or MAML-1), we also assembled complexes using hexahistidine-tagged CSL, and prepared a GST-Notch1 fusion protein for use in place of unlabeled Notch1. This scheme allowed for indirect detection of CSL binding to DNA by monitoring the subsequent capture of GST-Notch1 by CSL-DNA complexes [21]. Again, the observed DNA binding site preferences closely resembled those seen upon binding of GST-CSL alone, with GST-Notch1 alone serving as the negative control (Figure 4). This experiment provides an unambiguous demonstration that it is possible to monitor the loading of multiple protein components onto DNA using PBMs.

Bottom Line: Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors.Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Background: Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined.

Principal findings: Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins.

Conclusions/significance: These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes.

Show MeSH
Related in: MedlinePlus