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


RESULTS
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
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.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
Cristina
C
Del Bianco
Anastasia
A
Vedenko
Sung Hee
SH
Choi
Michael F
MF
Berger
Leila
L
Shokri
Martha L
ML
Bulyk
Stephen C
SC
Blacklow
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism*; D046228:Microarray Analysis / Q000379:methods; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D051883:Receptor, Notch2 / Q000235:genetics / Q000378:metabolism; D000071656:Receptor, Notch3; D000074562:Receptor, Notch4; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C430872:MAML1 protein, human; C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; C499374:NOTCH3 protein, human; C106075:NOTCH4 protein, human; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; C497570:RBPJ protein, human; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000071656:Receptor, Notch3; D000074562:Receptor, Notch4; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D005982:Glutathione Transferase