ABSTRACT
The HES family of bHLH repressors plays a key role in regulating the differentiation of neural precursors in the vertebrate embryo. Members of the HES gene family are expressed in neural precursors as targets of the Notch signaling pathway, but how this occurs in the context of neurogenesis is not known. Here, we address this issue by identifying enhancers driving Notch-dependent gene expression of two Hes5-like genes expressed in Xenopus called Esr1 and Esr10. Using frog transgenesis, we identify enhancer elements driving expression of Esr1 and Esr10 in neural precursors or in response to ectopic expression of the proneural protein, Xngnr1. Using deletion and mutation analysis, we define motifs required for enhancer activity of both genes, namely Notch-responsive elements and, in the case of Esr10, E-box motifs. We find that Esr1 and Esr10 are differentially regulated both in terms of Notch input and its interaction with heterologous factors. These studies reveal inputs required for proneural expression of genes encoding bHLH repressors in the developing vertebrate nervous system.
Subject(s)
Gene Expression Regulation, Developmental , Membrane Proteins/metabolism , Neurons/physiology , Repressor Proteins/metabolism , Transcription Factors/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Animals , Animals, Genetically Modified , Base Sequence , Cell Differentiation/physiology , Enhancer Elements, Genetic , Genes, Reporter , In Situ Hybridization , Molecular Sequence Data , Mutation , Phylogeny , Promoter Regions, Genetic , Receptors, Notch , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Sequence Alignment , Signal Transduction/physiology , Transcription Factors/classification , Transcription Factors/genetics , Xenopus Proteins/classification , Xenopus Proteins/genetics , Xenopus laevis/anatomy & histologyABSTRACT
Signaling through the Notch pathway activates the proteolytic release of the Notch intracellular domain (ICD), a dedicated transcriptional coactivator of CSL enhancer-binding proteins. Here we show that chromatin-dependent transactivation by the recombinant Notch ICD-CBF1 enhancer complex in vitro requires an additional coactivator, Mastermind (MAM). MAM provides two activation domains necessary for Notch signaling in mammalian cells and in Xenopus embryos. We show that the central MAM activation domain (TAD1) recruits CBP/p300 to promote nucleosome acetylation at Notch enhancers and activate transcription in vitro. We also find that MAM expression induces phosphorylation and relocalization of endogenous CBP/p300 proteins to nuclear foci in vivo. Moreover, we show that coexpression with MAM and CBF1 strongly enhances phosphorylation and proteolytic turnover of the Notch ICD in vivo. Enhanced phosphorylation of the ICD and p300 requires a glutamine-rich region of MAM (TAD2) that is essential for Notch transcription in vivo. Thus MAM may function as a timer to couple transcription activation with disassembly of the Notch enhancer complex on chromatin.
