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Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

ABSTRACT: The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the ?3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

SUBMITTER: Stephenson NL

PROVIDER: S-EPMC4537882 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

Stephenson Natalie L NL Avis Johanna M JM

FEBS open bio 20150730

The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic f ...[more]

PMID: 26288744

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Project description:BackgroundThe brain, spinal cord, and neural retina comprise the central nervous system (CNS) of vertebrates. Understanding the regulatory mechanisms that underlie the enormous cell-type diversity of the CNS is a significant challenge. Whole-genome mapping of DNase I-hypersensitive sites (DHSs) has been used to identify cis-regulatory elements in many tissues. We have applied this approach to the mouse CNS, including developing and mature neural retina, whole brain, and two well-characterized brain regions, the cerebellum and the cerebral cortex.ResultsFor the various regions and developmental stages of the CNS that we analyzed, there were approximately the same number of DHSs; however, there were many DHSs unique to each CNS region and developmental stage. Many of the DHSs are likely to mark enhancers that are specific to the specific CNS region and developmental stage. We validated the DNase I mapping approach for identification of CNS enhancers using the existing VISTA Browser database and with in vivo and in vitro electroporation of the retina. Analysis of transcription factor consensus sites within the DHSs shows distinct region-specific profiles of transcriptional regulators particular to each region. Clustering developmentally dynamic DHSs in the retina revealed enrichment of developmental stage-specific transcriptional regulators. Additionally, we found reporter gene activity in the retina driven from several previously uncharacterized regulatory elements surrounding the neurodevelopmental gene Otx2. Identification of DHSs shared between mouse and human showed region-specific differences in the evolution of cis-regulatory elements.ConclusionsOverall, our results demonstrate the potential of genome-wide DNase I mapping to cis-regulatory questions regarding the regional diversity within the CNS. These data represent an extensive catalogue of potential cis-regulatory elements within the CNS that display region and temporal specificity, as well as a set of DHSs common to CNS tissues. Further examination of evolutionary conservation of DHSs between CNS regions and different species may reveal important cis-regulatory elements in the evolution of the mammalian CNS.

Dataset Information

Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

Publications

Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

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