Notch signaling : From receptor cleavage to chromatin remodeling

Sammanfattning: One of the principal signaling mechanisms that control development of multicellular organisms is the Notch signaling pathway. The transmembrane Notch receptor, presented at the cell surface, interacts with ligand on neighbouring cells. Ligand interaction is followed by proteolytic processing, where cleavage by the gamma-secretase complex releases the intracellular (IC) part of the receptor. The Notch IC translocates to the nucleus, where it displaces corepressors from the DNA-binding protein CSL. Notch IC in complex with CSL, mastermind, p300, PCAF and other associated proteins activate transcription of target genes, including genes of the HES and HEY families. This thesis work is focused on two specific aspects of Notch signaling: i) processing of the receptor by the Á-secretase complex and ii) the mechanisms by which the IC functions as a transcriptional activator. The gamma-secretase complex, consisting of presenilin (PS), nicastrin, Aph-1 and Pen-2 proteins, processes not only the Notch receptor, but a number of other transmembrane proteins, including the amyloid precursor protein (APP). Aberrant processing of APP is implicated as a cause of Alzheimer Ls disease. Two mammalian PS homologs exist, but little is known about the degree of functional specialization of the two homologs. The incentive to address this question stems in the findings that knockout animals for the two homologs exhibit significantly different phenotypes, implicating a role for PS1, but not for PS2, in Notch signaling. In Paper I we characterize the functional similarity between complexes of various PS composition. We show that endoproteolysis, NTF-CTF interactions and the assembly and activity of gamma-secretase complexes are very conserved between PS1 and PS2. The Aph-1 protein was found in genetic screens in C. elegans and has been linked to Notch signaling because of the phenotype of the loss-of-function mutant. In paper II, we analyze whether the mammalian homologs of the protein are actually part of the complex processing the Notch receptor, and whether they have a role only in complex assembly or are important also for cleavage activity. We present evidence that Aph-1 is present at the cell surface in an active gamma-secretase complex, and interacts with the Notch receptor, both before and after ligand activation. Four Notch receptors are found in mammals, Notch1-4. Notch1 and 3 exhibit somewhat different properties, i.e. Notch1 is a potent activator of typical Notch regulated genes, while Notch3 is a very poor activator in the same context. Notch3 is even capable of repressing Notch1 mediated transactivation. In Paper III we explore underpinning mechanisms behind these differences. We find that Notch3 IC, like Notch1 IC, can displace the co-repressor SMRT from CSL and interacts with the SKIP and PCAF proteins. We also identify two distinct regions in the Notch IC that are critical for the difference between the Notch1 and Notch3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch1 derived ankyrin repeat region are potent activators. Second, a novel region in the Notch IC, the RE/AC region (for repression/activation), is required for Notch1 IC's ability to activate and for Notch3 IC's ability to repress a HES promoter. In Paper IV we use a cell free system, with purified components of the complex and naked or chromatin reconstituted DNA to establish the importance of the individual components for activation of transcription and in what step they exert their function. Mastermind, CSL, and Notch IC are all required for optimal transcription from DNA, whereas transcription from chromatin in addition requires p300. The transcriptional activity of p300 is dependent on acetyl coenzyme A, indicating that it functions as a histone acetyltransferase when mediating Notch IC function. PCAF is unable to promote transcription on its own but enhances Notch IC-mediated transcription from chromatin in conjunction with p300. These data define a critical role for p300 in the potentiation of Notch IC function by mastermind and indicate direct effects of CSL, Notch IC, and mastermind on the general transcription machinery.