A combination of computational and experimental approaches identifies DNA sequence constraints associated with target site binding specificity of the transcription factor CSL

Abstract

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.

The Canonical Notch Signaling Pathway: Structural and Biochemical Insights into Shape, Sugar, and Force

“…For example, while the affinity of CSL to DNA is relatively modest (~60 – 1,000 nM; (Friedmann and Kovall, 2010; Torella et al, 2014)) and does not change in affinity or specificity when in complex with NICD in vitro (Del Bianco et al, 2010; Friedmann et al, 2008), the in vivo NICD/CSL complex appears to bind better to DNA than CSL alone. Indeed, genome-wide analyses identified enrichment of CSL at enhancers in both Drosophila (Skalska et al, 2015) and T-cells (Wang et al, 2014) after a short pulse of Notch stimulation.…”

Cofunctional Subpathways Were Regulated by Transcription Factor with Common Motif, Common Family, or Common Tissue

“…Some studies have indicated that motif sequence is associated with TF function. TFs with similar motifs often recognize target genes with similar expression patterns [ 10 ], and TFs with different motifs may directly influence different TF function [ 11 ], and TFs influence pathways initiated by specific DNA-binding motifs [ 34 ]. To explore whether the common-motif TFs tended to regulate cofunctional subpathways, we extracted 1061 human DNA motifs from the Transfac database.…”

“…Dissecting the characteristics of TFs is helpful for understanding their regulatory function in complex biological systems. An increasing number of studies have demonstrated that TFs with similar motifs often (which were defined as common-motif TFs in the paper) recognize target genes with similar expression patterns [ 10 ], and in a similar way, TFs with different motifs may directly show different functions [ 11 ]. Several researchers have revealed that TFs in the common family (were defined as common-family TFs in the paper) have strong homology and the TFs are functionally related [ 12 ]; TFs in the transcription factor family are functional redundancies [ 13 ]; and TF family members share a relatively high degree of similarity in genomic structure and gene arrangement [ 12 – 14 ].…”

Structural and functional studies of the RBPJ-SHARP complex reveal conserved corepressor binding site

“…Classical models of transcriptional regulation in the Notch pathway posit that CSL is constitutively bound to DNA, and that corepressors are replaced by NICD when signaling is activated 43,44 . Given the comparable high affinities of both SHARP and NICD for RBPJ 27,30 , as well as the more modest affinity of CSL for DNA (~0.5-1.0 μM) 45,46 , it is difficult to reconcile how cofactor exchange on DNA, i.e . NICD replacing SHARP on RBPJ, might work at the molecular level.…”