Transcriptional Repression in the Notch Pathway

Abstract

The Notch pathway is a conserved cell-to-cell signaling mechanism that mediates cell fate decisions in metazoans. Canonical signaling results in changes in gene expression, which is regulated by the nuclear effector of the pathway CSL (CBF1/RBP-J, Su(H), Lag-1). CSL is a DNA binding protein that functions as either a repressor or an activator of transcription, depending upon whether it is complexed by transcriptional corepressor or coactivator proteins, respectively. In stark contrast to CSL-coactivator complexes, e.g. the transcriptionally active CSLNotch-Mastermind ternary complex, the structure and function of CSL-corepressor complexes are poorly understood. The corepressor MINT (Msx2-interacting nuclear target protein) has been shown in vivo to antagonize Notch signaling and shown in vitro to biochemically interact with CSL; however, the molecular details of this interaction are only partially defined. Here, we provide a quantitative thermodynamic binding analysis of CSL-MINT complexes. Using isothermal titration calorimetry, we demonstrate that MINT forms a high affinity complex with CSL, and we also delineate the domains of MINT and CSL that are necessary and sufficient for complex formation. Moreover, we show in cultured cells that this region of MINT can inhibit Notch signaling in transcriptional reporter assays. Taken together, our results provide functional insights into how CSL is converted from a repressor to an activator of transcription.

Structure and Function of the CSL-KyoT2 Corepressor Complex: A Negative Regulator of Notch Signaling

“…This analysis shows that the overall free energy of binding for CSL-KyoT2 complexes is independent of temperature and the ΔC p of binding is −0.57 kcal/mol·K. Previously, we found the ΔC p of binding for CSL-RAM complexes to be −0.62 kcal/mol·K (VanderWielen et al, 2011), which is very similar to the value determined here for CSL-KyoT2 interactions.…”

Conformational Locking upon Cooperative Assembly of Notch Transcription Complexes

“…Our working interpretation is that MAML1 binding retards exchange in ANK repeats 2 and 3 by stabilizing the folded conformation of these repeats when bound, because MAML1 binding does not greatly further retard exchange in ANK repeats 4–6, which are intrinsically more protected when unbound. These findings nicely complement previous reports that the affinity of ANK for CSL (> 20 µM based on fluorescence measurements) is low by comparison to RAM (Del Bianco et al, 2008), and that the inclusion of ANK in the same polypeptide chain does not enhance the binding enthalpy of RAM for CSL (Vanderwielen et al, 2011). …”

“…Recent studies investigating the interaction of the MINT co-repressor with CSL indicate that residues 2776–2833 of MINT bind to CSL with low nanomolar affinity (Vanderwielen et al, 2011). This work implicates both the BTD and CTD of CSL as contributing to MINT binding, but the binding interface remains largely undetermined.…”

“…Second, it is not clear whether RAM-mediated recruitment of mammalian NICD to CSL leads to stable docking of the ANK domain onto the CTD and NTD of CSL, or whether this step merely increases the effective concentration of the ANK domain for its binding site by constraining it to remain nearby (Bertagna et al, 2008). Whereas FRET assays have shown that transfer from a donor site on the ANK domain to an acceptor on CSL-bound DNA occurs upon association of RAMANK proteins with CSL (Del Bianco et al, 2008), calorimetry studies indicate that the binding enthalpy associated with association of RAMANK and CSL is not distinguishable from binding of RAM to CSL, suggesting that the interface between the ANK domain and CSL is either incompletely formed or in dynamic equilibrium with an open conformation in the absence of MAML1 (Vanderwielen et al, 2011). Lastly, cooperative assembly of dimeric Notch transcription complexes requires the presence of MAML1, even though the only visible contacts between the individual NTCs in the crystal structure of the dimeric complex lie in the second and third ANK repeats of the Notch ANK domain (Arnett et al, 2010; Nam et al, 2007), raising the question of how this cooperativity is orchestrated biochemically.…”

Dissecting and Circumventing the Requirement for RAM in CSL-Dependent Notch Signaling

“…the observations that the ANK domain only binds at very high concentrations and requires stabilization by MAML [15], [20], [22], and iii.) in the absence of MAML, RAM binds to CSL with similar affinity as RAMANK [23], differing by two-fold at most [24]. It has been proposed that the covalent linkage of RAM and ANK by an intrinsically disordered segment increases the effective concentration of ANK near CSL, thereby promoting MAML association and downstream activation [16], [25].…”