Molecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood. Positive and negative regulatory elements that function only in specific cell types or in response to extracellular inducers have been identified. A number of cases of inducible and tissue-specific gene expression involve the activation of preexisting transcription factors, rather than the synthesis of new proteins. This activation may involve covalent modification of the protein or an allosteric change in its structure. The modification of regulatory proteins may play a central role in the mechanisms of eukaryotic gene regulation.

GAL4 is a yeast regulatory protein that binds to specific sites within a DNA sequence called UASG (galactose upstream activating sequence) and activates transcription of linked genes. This activation requires two functions of the protein: a DNA binding domain located near the amino terminus, and one or more 'activating regions'. The 'activating regions' are highly acidic (see also ref. 12) and can be replaced, for example, by a short peptide designed to form a negatively charged, amphipathic alpha-helix. GAL4, as well as deletion derivatives bearing one or more 'activating regions' attached to the DNA binding domain, activates transcription in cultured mammalian cells from mammalian promoters linked to a UASG (refs 14, 15). Here we show that GAL4, when expressed in particular tissues of Drosophila larvae, stimulates tissue-specific transcription of a Drosophila promoter linked to GAL4 binding sites.

Endocytosis modulates the Notch signaling pathway in both the signaling and receiving cells. One recent hypothesis is that endocytosis of the ligand Delta by the signaling cells is essential for Notch activation in the receiving cells. Here, we present evidence in strong support of this model. We show that in the developing Drosophila eye Fat facets (Faf), a deubiquitinating enzyme, and its substrate Liquid facets (Lqf), an endocytic epsin, promote Delta internalization and Delta signaling in the signaling cells. We demonstrate that while Lqf is necessary for three different Notch/Delta signaling events at the morphogenetic furrow, Faf is essential only for one:Delta signaling by photoreceptor precluster cells, which prevents recruitment of ectopic neurons. In addition, we show that the ubiquitin-ligase Neuralized(Neur), which ubiquitinates Delta, functions in the signaling cells with Faf and Lqf. The results presented bolster one model for Neur function in which Neur enhances Delta signaling by stimulating Delta internalization in the signaling cells. We propose that Faf plays a role similar to that of Neur in the Delta signaling cells. By deubiquitinating Lqf, which enhances the efficiency of Delta internalization, Faf stimulates Delta signaling.

Photoreceptor nuclei in the Drosophila eye undergo developmentally regulated migrations. Nuclear migration is known to require the perinuclear protein Klarsicht, but the function of Klarsicht has been obscure. Here, we show that Klarsicht is required for connecting the microtubule organizing center (MTOC) to the nucleus. In addition, in a genetic screen for klarsicht-interacting genes, we identified Lam Dm(0), which encodes nuclear lamin. We find that, like Klarsicht, lamin is required for photoreceptor nuclear migration and for nuclear attachment to the MTOC. Moreover, perinuclear localization of Klarsicht requires lamin. We propose that nuclear migration requires linkage of the MTOC to the nucleus through an interaction between microtubules, Klarsicht, and lamin. The Klarsicht/lamin interaction provides a framework for understanding the mechanistic basis of human laminopathies.

A diverse family of proteins has been discovered with a small C-terminal KASH domain in common. KASH domain proteins are localized uniquely to the outer nuclear envelope, enabling their cytoplasmic extensions to tether the nucleus to actin filaments or microtubules. KASH domains are targeted to the outer nuclear envelope by SUN domains of inner nuclear envelope proteins. Several KASH protein genes were discovered as mutant alleles in model organisms with defects in developmentally regulated nuclear positioning. Recently, KASH-less isoforms have been found that connect the cytoskeleton to organelles other than the nucleus. A widened view of these proteins is now emerging, where KASH proteins and their KASH-less counterparts are cargo-specific adaptors that not only link organelles to the cytoskeleton but also regulate developmentally specific organelle movements.