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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

Notch receptors 1–3 do not detectably alter the DNA binding-site preferences of CSL-His6.DNA binding specificity motifs and 8-mer PBM enrichment scores were calculated for complexes comprised of A) CSL-His6 and GST-Notch1, B) CSL-His6 and GST-Notch2 and C) CSL-His6 and GST-Notch3. Motifs were derived using the Seed-and-Wobble algorithm as previously described [20], [31].
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pone-0015034-g005: Notch receptors 1–3 do not detectably alter the DNA binding-site preferences of CSL-His6.DNA binding specificity motifs and 8-mer PBM enrichment scores were calculated for complexes comprised of A) CSL-His6 and GST-Notch1, B) CSL-His6 and GST-Notch2 and C) CSL-His6 and GST-Notch3. Motifs were derived using the Seed-and-Wobble algorithm as previously described [20], [31].

Mentions: Finally, we investigated how incorporating the RAMANK domain from the four different Notch homologues (Notch1-4) into CSL-containing complexes influenced the DNA-binding properties of CSL. This experiment was performed with preassembled complexes of unlabeled CSL, GST-Notch, and MAML-1 to ensure that all detected complexes included both Notch and CSL, with the presence of MAML-1 inferred based on previous studies [7], [8], [22]. We inspected the PBM-derived motifs for potential differences among the difference complexes. We also performed an analysis of the comprehensive 8-mer PBM data to look for reproducible trends (here, 6-mers) that may be consistently favored or disfavored for binding by any of the protein complexes as compared to GST-CSl alone. The data do not reveal any detectable differences – either by examination of the PBM-derived motifs or by a comprehensive search for potential preferred 6-mers (see Methods) – in the binding specificities of CSL for DNA when it is assembled in complexes with the four different Notch receptors, nor for the different Notch complexes when compared with one another (Figure 5 and Supplementary Figure S1), leading to the conclusion that CSL DNA binding site preferences are essentially unaffected by complexation with any of the human Notch proteins (see also Supplementary Figure S2).


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)

Notch receptors 1–3 do not detectably alter the DNA binding-site preferences of CSL-His6.DNA binding specificity motifs and 8-mer PBM enrichment scores were calculated for complexes comprised of A) CSL-His6 and GST-Notch1, B) CSL-His6 and GST-Notch2 and C) CSL-His6 and GST-Notch3. Motifs were derived using the Seed-and-Wobble algorithm as previously described [20], [31].
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015034-g005: Notch receptors 1–3 do not detectably alter the DNA binding-site preferences of CSL-His6.DNA binding specificity motifs and 8-mer PBM enrichment scores were calculated for complexes comprised of A) CSL-His6 and GST-Notch1, B) CSL-His6 and GST-Notch2 and C) CSL-His6 and GST-Notch3. Motifs were derived using the Seed-and-Wobble algorithm as previously described [20], [31].
Mentions: Finally, we investigated how incorporating the RAMANK domain from the four different Notch homologues (Notch1-4) into CSL-containing complexes influenced the DNA-binding properties of CSL. This experiment was performed with preassembled complexes of unlabeled CSL, GST-Notch, and MAML-1 to ensure that all detected complexes included both Notch and CSL, with the presence of MAML-1 inferred based on previous studies [7], [8], [22]. We inspected the PBM-derived motifs for potential differences among the difference complexes. We also performed an analysis of the comprehensive 8-mer PBM data to look for reproducible trends (here, 6-mers) that may be consistently favored or disfavored for binding by any of the protein complexes as compared to GST-CSl alone. The data do not reveal any detectable differences – either by examination of the PBM-derived motifs or by a comprehensive search for potential preferred 6-mers (see Methods) – in the binding specificities of CSL for DNA when it is assembled in complexes with the four different Notch receptors, nor for the different Notch complexes when compared with one another (Figure 5 and Supplementary Figure S1), leading to the conclusion that CSL DNA binding site preferences are essentially unaffected by complexation with any of the human Notch proteins (see also Supplementary Figure S2).

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