Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.

Meiotic double-strand breaks (DSBs) are formed by Spo11 in conjunction with at least nine other proteins whose roles are not well understood. We find that two of these proteins, Rec102 and Rec104, interact physically, are mutually dependent for proper subcellular localization, and share a requirement for Spo11 and Ski8 for their recruitment to meiotic chromosomes, suggesting that they work together as a functional unit. Rec102 associated extensively with chromatin loops during leptotene and zygotene and showed preferential binding in the vicinity at least of most DSB sites, consistent with a direct role in DSB formation. However, Rec102 was associated with both DSB-hot and DSB-cold regions, ruling out a simple model in which sites of DSB formation are dictated by where Rec102/104 complexes load. Both proteins persisted on chromatin until pachytene before abruptly disappearing, indicating that they remain on chromosomes well after DSB formation. These studies reveal unexpected behaviors for Rec102 and Rec104, and point to distinct roles and subcomplexes among the DSB proteins.

The N-terminal tail domains of the core histones play important roles in gene regulation, but the exact mechanisms through which they act are not known. Recent studies suggest that the tail domains may influence the ability of RNA polymerase to elongate through the nucleosomal DNA and, thus, that posttranslational modification of the tail domains may provide a control point for gene regulation through effects on the elongation rate. We take advantage of an experimental system that uses bacteriophage T7 RNA polymerase as a probe for aspects of nucleosome transcription that are dominated by the properties of nucleosomes themselves. With this system, experiments can analyze the synchronous, real-time, single-passage transcription on the nucleosomal template. Here, we use this system to directly test the hypothesis that the tail domains may influence the "elongatability" of nucleosomal DNA and to identify which of the tail domains may contribute to this. The results show that the tail domains strongly influence the rate of elongation and suggest that the effect is dominated by the N-terminal domains of the (H3-H4) 2 tetramer. They further imply that tail-mediated octamer transfer is not essential for elongation through the nucleosome. Acetylation of the tail domains leads to effects on elongation that are similar to those arising from complete removal of the tail domains.Each of the four core histones of the nucleosome has a ϳ15-to 45-amino-acid highly positively charged N-terminal tail domain. These tail domains are of particular significance because they are the sites for posttranslational modifications that are linked to chromosome function. In particular, histone acetylation has been the subject of recent interest because it establishes a link between tail domain function and gene regulation. Each of the core histone proteins can be acetylated in vivo on multiple lysines within the N-terminal domains. Many generegulatory proteins have been found to encode histone acetylases or deacetylases, or to act in combination with other proteins that themselves are histone acetylases or deacetylases (14,18,39,40,45,52,54,55).Remarkably, despite their evolutionary conservation, individual tail domains can be deleted with little effect on the viability or even the growth rate of yeast (17,36,50). In addition, nucleosomes from which the N-terminal tails have been entirely removed are essentially unchanged in overall structure and stability (2,8,9,24). Subtle phenotypes that are observed in yeast mutants lacking one or another of these conserved tail domains are mimicked by point mutations that simulate lysine acetylation (e.g., lysine to glutamine). This suggests that at least some aspects of tail domain function in gene activation (or derepression) can be achieved, equivalently, either by eliminating the distinctive positive charge of lysine residues (by natural acetylation or by lysine to glutamine mutation) or by deleting the tail domains altogether.The exact mechanisms through which the tail domains contribute to gene reg...

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