ABSTRACT
Methylmercury (MeHg) is a persistent environmental toxin that has targeted effects on fetal neural development. Although a number of cytotoxic mechanisms of MeHg have been characterized in cultured cells, its mode of action in the developing nervous system in vivo is less clear. Studies of MeHg-affected rodent and human brains show disrupted cortical and cerebellar architecture suggestive of mechanisms that augment cell signaling pathways potentially affecting cell migration and proliferation. We previously identified the Notch receptor pathway, a highly conserved signaling mechanism fundamental for neural development, as a target for MeHg-induced signaling in Drosophila neural cell lines. Here we have expanded our use of the Drosophila model to resolve a broader spectrum of transcriptional changes resulting from MeHg exposure in vivo and in vitro. Several Notch target genes within the Enhancer-of-split (E(spl)C) and Bearded (BrdC) complexes are upregulated with MeHg exposure in the embryo and in cultured neural cells. However, the profile of MeHg-induced E(spl)C and BrdC gene expression differs significantly from that seen with activation of the Notch receptor. Targeted knockdown of Notch and of the downstream coactivator Suppressor of Hairless (Su(H)), shows no effect on MeHg-induced transcription, indicating a novel Notch-independent mechanism of action for MeHg. MeHg transcriptional activation is partially mimicked by iodoacetamide but not by N-ethylmaleimide, two thiol-specific electrophiles, revealing a degree of specificity of cellular thiol targets in MeHg-induced transcriptional events.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila/drug effects , Embryo, Nonmammalian/drug effects , Methylmercury Compounds/toxicity , Receptors, Notch/genetics , Repressor Proteins/genetics , Animals , Cell Line , Central Nervous System/abnormalities , Central Nervous System/drug effects , Drosophila/embryology , Drosophila/genetics , Embryo, Nonmammalian/abnormalities , Female , Gene Expression Profiling , Methylmercury Compounds/pharmacokinetics , RNA, Messenger/metabolismABSTRACT
Yeast Taf1p is an integral component of the multiprotein transcription factor TFIID. By using coimmunoprecipitation assays, coupled with a comprehensive set of deletion mutants encompassing the entire open reading frame of TAF1, we have discovered an essential role of a small portion of yeast Taf1p. This domain of Taf1p, termed region 4, consisting of amino acids 200 to 303, contributes critically to the assembly and stability of the 15-subunit TFIID holocomplex. Region 4 of Taf1p is mutationally sensitive, can assemble several Tafps into a partial TFIID complex, and interacts directly with Taf4p and Taf6p. Mutations in Taf1p-region 4 induce temperature-conditional growth of yeast cells. At the nonpermissive temperature these mutations have drastic effects on both TFIID integrity and mRNA synthesis. These data are consistent with the hypothesis that Taf1p subserves a critical scaffold function within the TFIID complex. The significance of these data with regard to TFIID structure and function is discussed.
Subject(s)
Protein Structure, Tertiary/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , TATA-Binding Protein Associated Factors/metabolism , Transcription Factor TFIID/biosynthesis , Transcription Factor TFIID/metabolism , Mutation , Protein Structure, Tertiary/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , TATA-Binding Protein Associated Factors/genetics , Temperature , Transcription Factor TFIID/chemistry , Transcription Factor TFIID/geneticsABSTRACT
The Delta protein is a single-pass transmembrane ligand for the Notch family of receptors. Delta binding to Notch invokes regulated intramembrane proteolysis and nuclear translocation of the Notch intracellular domain. Delta is proteolytically processed at two sites, Ala(581) and Ala(593) in the juxtamembrane and transmembrane domains, respectively (Mishra-Gorur, K., Rand, M. D., Perez-Villamil, B., and Artavanis-Tsakonas, S. (2002) J. Cell Biol. 159, 313-324). Controversy over the role of Delta processing in propagating Notch signals has stemmed from conflicting reports on the activity or inactivity of soluble extracellular domain products of Delta. We have examined Delta proteolysis in greater detail and report that Delta undergoes three proteolytic cleavages in the region of the juxtamembrane and transmembrane domains. Only one of these cleavages, analogous to cleavage at Ala(581), is dependent on the Kuzbanian ADAM metalloprotease. The two additional cleavages correspond to the previously described cleavage at Ala(593) and a novel unidentified site within or close to the transmembrane domain. Delta processing is up-regulated in co-cultures with Notch-expressing cells and is similarly induced by p-aminophenylmercuric acetate, a well documented activator of metalloproteases. Furthermore, expression of a truncated intracellular isoform of Delta shows prominent nuclear localization. Altogether, these data demonstrate a role for Notch in inducing Delta proteolysis and implicate a nuclear function for Delta, consistent with a model of bi-directional signaling through Notch-Delta interactions.
