Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters

Abstract

Transcriptional activation is often associated with chromatin remodeling. However, little is known about the dynamics of remodeling of nucleosome arrays in vivo. Upon induction of Saccharomyces cerevisiae PHO5, a novel kinetic assay of DNA methyltransferase accessibility showed that nucleosomes adjacent to the histone-free upstream activating sequence (UASp1) are disrupted earlier and at higher frequency in the cell population than are those more distal. Individually cloned molecules, each representing the chromatin state of a full promoter from a single cell, revealed multiple promoter classes with either no remodeling or variable numbers of disrupted nucleosomes. Individual promoters in the remodeled fraction were highly enriched for contiguous blocks of disrupted nucleosomes, the majority of which overlapped the UAS region. These results support a probabilistic model in which chromatin remodeling at PHO5 spreads from sites of transactivator association with DNA and attenuates with distance.

Single-cell nucleosome mapping reveals the molecular basis of gene expression heterogeneity

“…31 and Fig. S4), and the nucleosomes of this gene were shown to undergo significant rearrangement upon phosphate starvation, as determined by electron microscopy (30) and methyltransferase accessibility (34). In agreement with previous studies (35,43,44), PCR-based nucleosome-scanning assay showed marked shifts in chromatin structure (Fig.…”

“…1B) and genome-wide micrococcal nuclease (MNaseseq) were performed (Fig. 1C), confirming previously reported nucleosome positioning on the PHO5 promoter for positions of the +1 through −3 nucleosomes of the PHO5 promoter under phosphate-rich conditions, which is nonpermissive for gene expression (30,(32)(33)(34)(35).…”

“…1C). The majority of these cells had a −2 nucleosome occluding the high-affinity UAS, as previously shown by other methods (30,(32)(33)(34)(35). However, the single-cell analysis showed that about 10% of cells do not have a nucleosome over the highaffinity UAS (Fig.…”

“…3A). Indeed, previous predictions and experimental work suggested that the −2 nucleosome needed to be removed in order for the −1 nucleosome to remodel (30,34). Single-cell nucleosome mapping showed that upon phosphate starvation and activation of the PHO5 gene, 76% of cells now had a nucleosome-depleted architecture, compared with only 10% when cells were grown in phosphate-rich media (Fig.…”

“…To map nucleosomes in individual cells, we modified a method developed in the Kladde and Jones laboratories based upon the ability of nucleosomes to block the GpC-specific DNA methyltransferase M.CviPI from modifying DNA wrapped around a histone octamer (34,36,37). In comparison with other nucleosome-mapping methods, this method is less destructive than MNase for mapping the DNA at the ends of the nucleosome or chemical mapping, which cleaves the DNA at the center of the nucleosome (19).…”