We have mapped the dynamic remodeling of most nucleosomes in the yeast genome during a transcriptional perturbation using a combination of micrococcal nuclease digestion, isolation of mononucleosome associated DNA and Solexa sequencing. Using a Parzen window–based approach, which is a generally applicable method to analyze all similar datasets derived from ultra-high–throughput sequencing, we defined the dynamic remodeling of approximately 50,000 nucleosomes at single-nucleotide resolution in normally growing cells and in cells that were transcriptionally perturbed by heat shock for 15 min. Our study independently confirms expectations about nucleosomal positioning based on previous smaller scale and lower resolution studies, but also reveals novel features about chromatin structure and transcriptional activity, especially given that previous studies have not examined the dynamic repositioning of nucleosomes in response to genome-wide transcriptional reprogramming.
Our results showed that in addition to a positioned nucleosome at the TSS, genes in general tend to also contain a well-positioned nucleosome at the 3′ end of the coding region. Yeast genes are thus demarcated by a well-positioned nucleosome at each end of their transcribed regions, with a nucleosome-free gap just beyond. This could potentially reflect chromatin organization that facilitates RNA polymerase initiation as well as termination. Most coding regions also showed strongly and regularly positioned nucleosomes, although the strength of the nucleosome positioning was weaker in genes transcribed at high rates. Interestingly, the first well-positioned boundary nucleosome downstream of the TSS, which is likely to be an H2A.Z variant–containing nucleosome based on previous studies [9
], showed similar stability in genes transcribed at high and low rates (B), suggesting that this chromatin landmark is important for demarcating promoters.
Upon transcriptional perturbation, the majority of nucleosomes did not change positions, either at promoters or within coding sequences ( and ). Gene-specific remodeling was restricted to the discrete eviction, appearance, or repositioning of one or two nucleosomes localized to promoters. Remodeling events at genes that were activated or repressed upon heat shock could be classified into distinct patterns, indicating that there is no simple rule for nucleosome remodeling at promoters to activate and repress genes. Thus, although activation was generally and quantitatively associated with nucleosome eviction and transcriptional repression with nucleosome appearance (C), there were cases in which strongly positioned nucleosomes appeared at activated promoters ( and S5
). Translational repositioning of nucleosomes would seem like eviction and appearance at different spots in the same promoter. These observations suggest that nucleosome remodeling at promoters is not a trivial consequence of transcriptional activity appearing as overall openness of chromatin at activated promoters and obstruction at repressed promoters, but rather, that the precise placement of individual nucleosomes at promoters mechanistically regulates transcription by modulating access of trans
-acting factors to specific sites.
In addition to chromatin remodeling specifically at regulated promoters, many promoters however showed dynamic single-nucleosome remodeling during the physiological perturbation even in the absence of any resulting transcriptional change (E), indicating that selective, activity-specific remodeling was accompanied by a certain number of background, nonspecific remodeling events. We speculate that these background single-nucleosome remodeling events poise promoters for rapid future transcriptional activity, by either assembling partial preinitiation complexes [31
], or by exchanging core histones with one or more histone variants [32
]. A recent study showed that nucleosomes are globally positioned by Isw2 acting at the boundary between genes and intergenic regions, and that some of the Isw2-dependent remodeling occurs independent of transcription [11
]. Therefore, the background remodeling seen in the absence of transcriptional changes in our study could potentially reflect nonspecific remodeling by ISW-like complexes.
We classified transcription factors into three classes based on change in accessibility of their binding sites upon transcriptional perturbation. All the prominent stress-related transcription factors belonged to the category showing a strong increase in accessibility upon transcriptional perturbation. In addition, we found that Rap1 and Fhl1 binding sites showed an increase in accessibility even though the majority of their target genes, namely the ribosomal protein genes, showed a decrease in transcription upon heat-shock stress. When transcription of the ribosomal protein genes is repressed by heat shock, osmotic shock, or inhibition of the TOR pathway by rapamycin, it is known that Ifh1 leaves the promoter, but Rap1 and Fhl1 remain bound [30
]. It is possible that Rap1 and Fhl1 play a role in recruiting chromatin remodelers to bring about a repressive chromatin structure at the ribosomal protein genes. Previous studies have indicated that the primary discriminant between a functional and a nonfunctional transcription factor binding site in vivo is the presence of stably positioned nucleosomes covering the latter [4
]. Our results above indicate that superimposed on this, there is a second mode of regulation at functional binding sites of stress-related transcription factors brought about by a stimulus-dependent remodeling of one or two nucleosomes, making the site more accessible for stable binding of transcription factors. Alternatively, binding of the transcription factor(s) could result in the remodeling of nucleosomes via the help of chromatin remodelers.
The work described here is the first study of genome-wide dynamic nucleosome remodeling events at single-base resolution. More such studies in yeast and higher eukaryotes will shed light on the relationship between epigenetic changes at high resolution and the global regulation of gene expression.