Project description:γ-secretase is an intra-membrane-cleaving aspartyl protease implicated in the processing of a wide range of type I membrane proteins including the Notch receptor and the amyloid-β precursor protein (APP). It thus regulates a diverse array of cellular and biological processes including the differentiation of neuronal embryonic stem cells, or intestinal stem cells, with the latter controlling the self-renewing of the intestinal epithelium. Indeed, proteolysis of these proteins by γ-secretase triggers signaling cascades by releasing intracellular domains (ICDs) which, following association with adaptor proteins and nuclear translocation, modulate the transcription of different genes by binding directly to their promoters. The pronounced proliferative and regenerative effects of Notch signaling and its implication in the generation of the Aβ-peptides, makes γ-secretase a therapeutic target for several types of cancer and for Alzheimer’s disease. To investigate the broad effects of γ-secretase activity onto the cellular transcriptome, Chinese hamster ovary (CHO) cells with enhanced γ-secretase were compared to cells with abolished γ-secretase activity via a microarray designed for a genetically close species, mouse. Our findings will potentially help to decipher the biology of γ-secretase, including a better understanding of the roles of this enzyme in gene transcription.
Project description:γ-secretase is an intra-membrane-cleaving aspartyl protease implicated in the processing of a wide range of type I membrane proteins including the Notch receptor and the amyloid-β precursor protein (APP). It thus regulates a diverse array of cellular and biological processes including the differentiation of neuronal embryonic stem cells, or intestinal stem cells, with the latter controlling the self-renewing of the intestinal epithelium. Indeed, proteolysis of these proteins by γ-secretase triggers signaling cascades by releasing intracellular domains (ICDs) which, following association with adaptor proteins and nuclear translocation, modulate the transcription of different genes by binding directly to their promoters. The pronounced proliferative and regenerative effects of Notch signaling and its implication in the generation of the Aβ-peptides, makes γ-secretase a therapeutic target for several types of cancer and for Alzheimerâs disease. To investigate the broad effects of γ-secretase activity onto the cellular transcriptome, Chinese hamster ovary (CHO) cells with enhanced γ-secretase were compared to cells with abolished γ-secretase activity via a microarray designed for a genetically close species, mouse. Our findings will potentially help to decipher the biology of γ-secretase, including a better understanding of the roles of this enzyme in gene transcription. We compared the transcriptomes of two CHO cell lines displaying extreme differences in γ-secretase activity. The S-1 cell line overexpressed the four components of γ-secretase (NCT, APH1aL, PS1, and PEN2) and was characterized by a marked increase in the level of PS1 heterodimers associated with 8-fold increased γ-secretase activity compared to untransfected controls. The other cell line consisted of wild type CHO cells incubated with DAPT, a well-known γ-secretase inhibitor. The two cell lines were used in combination with a mouse microarray to analyze gene transcription under enhanced γ-secretase. Two samples, S1 and DAPT treated CHO- were used in biological triplicates each.
Project description:RNA-sequencing analysis of a wildtype CHO cell line (CHO-K1), a mutant CHO cell line (pgsA745) that lacks xylosyltransferase (XylT) activity, and pgsA745 stably transfected with XylT1 (pgsA745-XylT1).