Abstract
BackgroundCanonical 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 FindingsHere, 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/SignificanceThese 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.
Highlights
CSL is a DNA-binding transcription factor that orchestrates the transcriptional response to Notch receptor activation
For each 8-mer, we calculate its median signal intensity over the 32 spots at which it is present and a rank-based, protein-binding microarrays (PBMs) enrichment score (E-score), ranging from 20.5 to +0.5, that indicates the preference of a protein or protein complex for that 8-mer [20]
Our approach relied on the use of PBMs, a method that permits rapid, high-throughput characterization of the in vitro DNA binding specificities of proteins [16,17,18]
Summary
CSL (gene name RBPJ) is a DNA-binding transcription factor that orchestrates the transcriptional response to Notch receptor activation. After ligand binding induces proteolysis of Notch, ICN migrates to the nucleus, where it binds to CSL and recruits a protein of the Mastermind family to upregulate transcription of a typical target gene [1,2,3]. The preferred DNA binding sites for murine CSL and for the protein LAG-1, which is the homologue of CSL in C. elegans, have been analyzed by electrophoretic mobility shift assay, selection-based methods, and a bacterial onehybrid system. These methods led to the identification of an eight base-pair consensus binding sequence of CGTGGGAA [12]. 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
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