A map of nucleosome positions in yeast at base-pair resolution

Abstract

The exact positions of nucleosomes along genomic DNA can influence many aspects of chromosome function, yet existing methods for mapping nucleosomes do not provide the necessary single base pair accuracy to determine these positions. Here we develop and apply a new approach for direct mapping of nucleosome centers based on chemical modification of engineered histones. The resulting map locates nucleosome positions genome-wide in unprecedented detail and accuracy. It reveals novel aspects of the in vivo nucleosome organization that are linked to transcription factor binding, RNA polymerase pausing, and the higher order structure of the chromatin fiber.

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

“…By comparing the MAC and MIC results, we dissected relative contributions of cisdeterminants and trans-determinants to nucleosome distribution (manuscript under review). Tetrahymena is one of the few model systems in which histone mutagenesis can be readily performed (Liu et al, 2004), allowing complete replacement of endogenous histones with mutated ones needed for Cu + chelation, hydroxyl radical production, and subsequent cleavage of nucleosomal DNA around the dyad (Brogaard et al, 2012a). DNA fragments corresponding to ~200 bp NRL was generated by chemical cleavage, establishing it as a valid alternative for mapping nucleosome distribution in Tetrahymena ( Figure 6).…”

“…While MNase digestion progressively removes the linker DNA, leaving mono-nucleosomes as the key product, hydroxyl radical cleavage occurs at two symmetric sites 6 bp away from the nucleosome dyad, allowing direct measurement of the exact position of a nucleosome and nucleosome repeat length (NRL) at base-pair resolution ( Figure 6A). Using an established histone mutagenesis procedure (Liu et al, 2004), H4 S47C mutation was introduced into Tetrahymena, allowing a chelated Cu + to be positioned near the nucleosome dyad, as well as localized production of hydroxyl radical and cleavage of nucleosomal DNA (Brogaard et al, 2012a;Flaus et al, 1996). Similarly, H3 C110A mutation was introduced to limit spurious reactions.…”

“…It has been adapted for mapping of in vivo nucleosome distribution in Saccharomyces cerevisiae at base-pair resolution (Brogaard et al, 2012a). Briefly, after serine 47 of histone H4 protein is mutated to a cysteine (H4 S47C), nucleosomal DNA can be precisely cleaved at sites adjacent to the nucleosomal dyad by hydroxyl radicals generated by hydrogen peroxide and chelated Cu + (Flaus et al, 1996).…”

“…Briefly, after serine 47 of histone H4 protein is mutated to a cysteine (H4 S47C), nucleosomal DNA can be precisely cleaved at sites adjacent to the nucleosomal dyad by hydroxyl radicals generated by hydrogen peroxide and chelated Cu + (Flaus et al, 1996). Its precise cleavage and lack of sequence bias provide a valuable alternative to MNase digestion for high resolution mapping of nucleosome distribution (Brogaard et al, 2012a).…”

Contributions of Sequence to the Higher-Order Structures of DNA

“…Moreover, whether or not the regular chromatin fibers characterized in vitro and in silico are relevant to the higher-order organization of the genome in vivo remains an open question (21). The chromatin fibers characterized at the mesoscale level tend to be regular structures with evenly spaced nucleosomes, as opposed to the unevenly and sometimes more widely spaced arrays of nucleosomes detected at basepair resolution on whole genomes (22,23). The latter features have a dramatic effect on the mesoscale properties of simulated chromatin fibers, with the depletion of nucleosomes enhancing the predicted looping propensities to levels consistent with genomic data (24).…”