Human SRCAP and Drosophila melanogaster DOM Are Homologs That Function in the Notch Signaling Pathway

Abstract

The putative ATPase chromatin-remodeling machine SRCAP was identified in a yeast two-hybrid protein screen by interaction with the histone acetylase CBP. SRCAP is implicated in the transcriptional coactivation of cyclic AMP-and steroid-dependent promoters, but no natural chromosomal targets for SRCAP regulation have been identified. DOM is the unique SRCAP homolog in Drosophila melanogaster. The goal of this study was to test whether SRCAP is a functional homolog of DOM and to identify potential activities and targets of SRCAP in vivo. We show that human SRCAP complements recessive domino mutant phenotypes. This rescue depends on an intact ATPase homology domain. SRCAP colocalizes extensively with DOM on Drosophila polytene chromosomes and is recruited to sites of active transcription, such as steroid-regulated loci, but not to activated heat shock loci. We show that SRCAP recruits Drosophila CBP to ectopic chromosomal sites, providing the first evidence to suggest that SRCAP and CBP interact directly or indirectly on chromosomes. We show that DOM is a Notch pathway activator in Drosophila and that wild-type SRCAP-but not an ATPase domain mutant-can substitute for DOM in Notch-dependent wing development. We show that SRCAP potentiates Notch-dependent gene activation in HeLa cells. Taken together, these data implicate SRCAP and DOM in developmental gene activation.Most regulated gene expression in eukaryotes is the result of transcriptional regulation. Current models of transcriptional regulation invoke the targeted recruitment of enzymes that make covalent changes in histone and/or DNA structure, in ATPases that remodel histone-DNA interactions, and, in cases of gene activation, in RNA polymerase itself. A major challenge is to identify the diverse roles of chromatin modifiers in transcriptional regulation.SRCAP (SNF2-related CBP-activating protein) shares homology with members of the SNF2/SWI2 class of chromatinremodeling enzymes (20). SRCAP was originally identified in a yeast two-hybrid protein screen for proteins that interact with the histone acetyltransferase CBP (CREB-binding protein). Subsequent studies using transiently transfected reporters have implicated SRCAP as a coactivator for CREB-and nuclear hormone receptor-mediated transcriptional activation (30, 31). The mechanism by which SRCAP activates transcription has not been completely elucidated. Results from a number of studies, including mammalian two-hybrid and coimmunoprecipitation experiments, indicate SRCAP may function in part by recruitment of CBP or other coactivators, such as the arginine methyltransferase CARM-1 and the nuclear hormone receptor coactivator GRIP-1 (20, 31). A role for the conserved ATPase/helicase homology domain in regulation of transcription has not been established, since this activity is dispensable for SRCAP activity in transient transfection assays, nor have the natural chromosomal promoters that utilize SRCAP to regulate transcription been identified.In mammals, the protein most homologous to SRCAP is p400. p400 was ...

Nipped-A, the Tra1/TRRAP Subunit of the Drosophila SAGA and Tip60 Complexes, Has Multiple Roles in Notch Signaling during Wing Development

“…Domino-A is more restricted in expression than domino-B, and domino-B is likely the major domino product expressed in wing disks (47). Previous studies indicated that a domino mutation increased the severity of the N nd-1 mutant phenotype, suggesting that domino-B participates in Notch signaling at the wing margin, and cultured cell transfection experiments with mammalian SRCAP suggest that it supports transcriptional activation by Notch (16). Figure 8 shows that, similar to the previously reported effect of the dom 7 mutation, the dom 1 mutation increased the severity of the N nd-1 mutant phenotype.…”

Tip60 Histone Acetyltransferase Acts as a Negative Regulator of Notch1 Signaling by Means of Acetylation

“…Recent reports have shown that using genetic studies in flies, the Nipped-A/Tra1/TRRAP proteins and the SRCAP/ DOM proteins are required for the activity of Notch through its coactivator protein, mastermind, during wing development and are key component of the Tip60 histone acetylase complex (20,26,44). The Notch intracellular domain was not identified among the proteins associated with Nipped-A/Tra1/TRRAP and SRCAP/DOM in the Tip60 complexes (9).…”

“…Genetic studies using the fly system have shown that TRRAP and domino have functions in Notch signaling. However, findings do not rule out the possibility that TRRAP and domino can also act independently of Tip60 (20,26). Cellular responses to DNA damage also involve the HAT Tip60, as the overexpression of a dominant negative HAT-defective Tip60 mutant decreases both DNA repair and apoptosis upon the induction of DNA double-stranded breaks (12,33,69).…”

Drosophila Reptin and Other TIP60 Complex Components Promote Generation of Silent Chromatin

“…That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests to us that Reptin functions through the fly TIP60 complex. Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast (Eissenberg et al 2005). Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes (Krogan et al 2003;Kobor et al 2004;Mizuguchi et al 2004).…”