A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes.

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2ß. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.

Characterization of CSL (CBF-1, Su(H), Lag-1) mutants reveals differences in signaling mediated by Notch1 and Notch2.

Notch is a conserved signaling pathway that plays essential roles during embryonic development and postnatally in adult tissues; misregulated signaling results in human disease. Notch receptor-ligand interactions trigger cleavage of the Notch receptor and release of its intracellular domain (NICD) from the membrane. NICD localizes to the nucleus where it forms a transcriptionally active complex with the DNA-binding protein CSL and the coactivator Mastermind (MAM) to up-regulate transcription from Notch target genes. Previous studies have determined the structure of the CSL-NICD-MAM ternary complex and characterized mutations that affect complex assembly in functional assays. However, as CSL is expressed in all cell types, these studies have been limited to analyzing mutations in NICD and MAM. Here, we describe a novel set of cellular reagents to characterize how mutations in CSL affect its function as a transcriptional activator. Using retrovirally transduced embryonic fibroblasts from a CSL-null mouse, we generated cell lines that express either wild-type or mutant CSL molecules. We then analyzed these mutants for defects in Notch1- (NICD1) or Notch2 (NICD2)-mediated activation from two different transcriptional reporters (HES-1 or 4×CBS). Our results show that mutations targeted to the different domains of CSL display significant differences in their ability to adversely affect transcription from the two reporters. Additionally, a subset of CSL mutants is sensitive to whether NICD1 or NICD2 was used to activate the reporter. Taken together, these studies provide important molecular insights into how Notch transcription complexes assemble at different target genes and promoter arrangements in vivo.

Structure and function of the CSL-KyoT2 corepressor complex: a negative regulator of Notch signaling.

Notch refers to a highly conserved cell-to-cell signaling pathway with essential roles in embryonic development and tissue maintenance. Dysfunctional signaling causes human disease, highlighting the importance of pathway regulation. Notch signaling ultimately results in the activation of target genes, which is regulated by the nuclear effector CSL (CBF-1/RBP-J, Su(H), Lag-1). CSL dually functions as an activator and a repressor of transcription through differential interactions with coactivator or corepressor proteins, respectively. Although the structures of CSL-coactivator complexes have been determined, the structures of CSL-corepressor complexes are unknown. Here, using a combination of structural, biophysical, and cellular approaches, we characterize the structure and function of CSL in complex with the corepressor KyoT2. Collectively, our studies provide molecular insights into how KyoT2 binds CSL with high affinity and competes with coactivators, such as Notch, for binding CSL. These studies are important for understanding how CSL functions as both an activator and a repressor of transcription of Notch target genes.