ABSTRACT
In vertebrate retinal progenitor cells, the proneural factor Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and -independent pathways.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/genetics , Immunoglobulin J Recombination Signal Sequence-Binding Protein/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Retina/embryology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Gene Expression Regulation , Mice , Nerve Tissue Proteins/metabolism , Receptors, Notch/metabolism , Regulatory Elements, Transcriptional , Retina/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
Notch is an intercellular signaling pathway that is highly conserved in metazoans and is essential for proper cellular specification during development and in the adult organism. Misregulated Notch signaling underlies or contributes to the pathogenesis of many human diseases, most notably cancer. Signaling through the Notch pathway ultimately results in changes in gene expression, which is regulated by the transcription factor CSL. Upon pathway activation, CSL forms a ternary complex with the intracellular domain of the Notch receptor (NICD) and the transcriptional coactivator Mastermind (MAM) that activates transcription from Notch target genes. While detailed in vitro studies have been conducted with mammalian and worm orthologous proteins, less is known regarding the molecular details of the Notch ternary complex in Drosophila. Here we thermodynamically characterize the assembly of the fly ternary complex using isothermal titration calorimetry. Our data reveal striking differences in the way the RAM (RBP-J associated molecule) and ANK (ankyrin) domains of NICD interact with CSL that is specific to the fly. Additional analysis using cross-species experiments suggest that these differences are primarily due to fly CSL, while experiments using point mutants show that the interface between fly CSL and ANK is likely similar to the mammalian and worm interface. Finally, we show that the binding of the fly RAM domain to CSL does not affect interactions of the corepressor Hairless with CSL. Taken together, our data suggest species-specific differences in ternary complex assembly that may be significant in understanding how CSL regulates transcription in different organisms.
Subject(s)
DNA-Binding Proteins/chemistry , Drosophila Proteins/chemistry , Immunoglobulin J Recombination Signal Sequence-Binding Protein/chemistry , Protein Structure, Tertiary , Receptors, Notch/chemistry , Transcriptional Activation/genetics , Amino Acid Sequence/genetics , Animals , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein/genetics , Models, Molecular , Receptors, Notch/genetics , Signal Transduction , ThermodynamicsABSTRACT
Regulation of transcription is fundamental to development and physiology, and occurs through binding of transcription factors to specific DNA sequences in the genome. CSL (CBF1/Suppressor of Hairless/LAG-1), a core component of the Notch signaling pathway, is one such transcription factor that acts in concert with co-activators or co-repressors to control the activity of associated target genes. One fundamental question is how CSL can recognize and select among different DNA sequences available in vivo and whether variations between selected sequences can influence its function. We have therefore investigated CSL-DNA recognition using computational approaches to analyze the energetics of CSL bound to different DNAs and tested the in silico predictions with in vitro and in vivo assays. Our results reveal novel aspects of CSL binding that may help explain the range of binding observed in vivo. In addition, using molecular dynamics simulations, we show that domain-domain correlations within CSL differ significantly depending on the DNA sequence bound, suggesting that different DNA sequences may directly influence CSL function. Taken together, our results, based on computational chemistry approaches, provide valuable insights into transcription factor-DNA binding, in this particular case increasing our understanding of CSL-DNA interactions and how these may impact on its transcriptional control.
