Models for Golgi traffic: a critical assessment

A variety of secretory cargoes move through the Golgi, but the pathways and mechanisms of this traffic are still being debated. Here, we evaluate the strengths and weaknesses of five current models for Golgi traffic: (1) anterograde vesicular transport between stable compartments, (2) cisternal progression/maturation, (3) cisternal progression/maturation with heterotypic tubular transport, (4) rapid partitioning in a mixed Golgi, and (5) stable compartments as cisternal progenitors. Each model is assessed for its ability to explain a set of key observations encompassing multiple cell types. No single model can easily explain all of the observations from diverse organisms. However, we propose that cisternal progression/maturation is the best candidate for a conserved core mechanism of Golgi traffic, and that some cells elaborate this core mechanism by means of heterotypic tubular transport between cisternae.     

Methods for chromatin assembly and remodeling

Packaging of the DNA in nucleosomes restricts its accessibility to regulatory factors and enzymatic complexes, making a local remodeling of the nucleosome structure a prerequisite to the establishment of protein-DNA interactions. The use of an experimental system in which one nucleosome is reconstituted on a short linear DNA fragment allows gel fractionation of nucleosomes according to their translational positions, whose locations are dependent on the underlying DNA sequence. Nucleosome mobilization by chromatin remodeling factors is easily detected by observing band disappearance in gel, which in turn provides evidence for histone octamer displacement. Here, we provide methods for chromatin assembly that we have been using in our analysis for nucleosome mobilization by chromatin remodeling factors. These methods are straightforward and easy to follow. Thus, they may provide a good starting assay system for analysis of nucleosome movements by other chromatin remodeling machines.     

Phosphoinositides as ligands of PHD-fingers: implications for chromatin remodeling

Phosphatidylinositol phosphates (PtdInsPs) play critical roles in signal transduction pathways, cytoskele-tal regulation, and vesicular trafficking and are implicated in multiple disease pathways. In the nucleus, PtdlnsPs have been found associated with chromatin, but their specific nuclear-binding proteins have not been identified. In a biochemical screen for PtdInsP-binding proteins, we identified a protein (ING2) containing a Plant Homeodomain (PHD) finger, a motif present in many chromatin- regulatory proteins. We show that the PHD domains of ING2 and of other diverse nuclear proteins bind to the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we provide evidence for a functional interaction     

Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin.     

Cohesin: a critical chromatin organizer in mammalian gene regulation

Cohesins are evolutionarily conserved essential multi-protein complexes that are important for higher-order chromatin organization. They play pivotal roles in the maintenance of genome integrity through mitotic chromosome regulation, DNA repair and replication, as well as gene regulation critical for proper development and cellular differentiation. In this review, we will discuss the multifaceted functions of mammalian cohesins and their apparent functional hierarchy in the cell, with particular focus on their actions in gene regulation and their relevance to human developmental disorders.     

Rad51 polymerization reveals a new chromatin remodeling mechanism

Rad51 protein is a well known protagonist of homologous recombination in eukaryotic cells. Rad51 polymerization on single-stranded DNA and its role in presynaptic filament formation have been extensively documented. Rad51 polymerizes also on double-stranded DNA but the significance of this filament formation remains unclear. We explored the behavior of Saccharomyces cerevisiae Rad51 on dsDNA and the influence of nucleosomes on Rad51 polymerization mechanism to investigate its putative role in chromatin accessibility to recombination machinery. We combined biochemical approaches, transmission electron microscopy (TEM) and atomic force microscopy (AFM) for analysis of the effects of the Rad51 filament on chromatinized templates. Quantitative analyses clearly demonstrated the occurrence of chromatin remodeling during nucleoprotein filament formation. During Rad51 polymerization, recombinase proteins moved all the nucleosomal arrays in front of the progressing filament. This polymerization process had a powerful remodeling effect, as Rad51 destabilized the nucleosomes along considerable stretches of DNA. Similar behavior was observed with RecA. Thus, recombinase polymerization is a powerful mechanism of chromatin remodeling. These remarkable features open up new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics.