CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methylCpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKLdependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.The transition from inert seed to growing seedling marks the culmination of a remarkable transformation in developmental identity and transcriptional programs. During seed formation, numerous genes are expressed that contribute to establishment of the body plan, laying down of storage reserves, and desiccation tolerance (1, 2). Many of these genes are widely expressed in the developing seed only to be silenced by the time the seed germinates, often for the remainder of the life cycle of the plant (3). Although several key regulators have been identified that activate various seed transcriptional programs, such as the LEAFY COTY-LEDON (LEC) genes and ABSCISIC ACID-INSENSITIVE3 (ABI3) (4, 5), relatively little is known about the mechanisms that ensure that these programs are restricted to developing seeds. This repression is critical for subsequent stages of plant development. In particular, continued expression of LEC genes leads to continued expression of seed-specific programs and substantial alteration of seedling development (6 -8).PICKLE (PKL) has previously been shown to play a significant role in repression of seed-associated genes. In pkl plants, many seed-associated traits continue to be expressed after germination, including accumulation of seed storage reserves and the ability to undergo somatic embryogenesis (9, 10). Primary roots of pkl seedlings that continue to express seed-associated developmental programs stop elongating and adopt a green tuberous phenotype referred to as the "pickle root" phenotype. PKL codes for a predicted ATP-dependent chromatin remodeling factor in the CHD3 family (11,12). In animal systems, CHD3 proteins are a component of the Mi-2/NuRD complex (13-16). This complex includes several other proteins, in...

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain–containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch.

The Notch signaling pathway controls patterning and cell fate decisions during development in metazoans, and is associated with human diseases such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and certain cancers. Studies over the last several years have revealed sophisticated regulation of both the membrane-bound Notch receptor and its ligands by vesicle trafficking. This is perhaps most evident in neural progenitor cells in Drosophila, which divide asymmetrically to segregate Numb, an endocytic adaptor protein that acts as a Notch pathway inhibitor, to one daughter cell. Here, we discuss recent findings addressing how receptor and ligand trafficking to specific membrane compartments control activation of the Notch pathway in asymmetrically dividing cells and other tissues.

Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky-Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes.

Background: Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The Drosophila homolog of auxilin (dAux), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood.