Global nucleosome distribution and the regulation of transcription in yeast
© BioMed Central Ltd 2004
Published: 30 September 2004
Recent studies show that active regulatory regions of the yeast genome have a lower density of nucleosomes than other regions, and that there is an inverse correlation between nucleosome density and the transcription rate of a gene. This may be the result of transcription factors displacing nucleosomes.
It has been known for nearly three decades that there is a relationship between the chromatin structure of a gene and its transcriptional status. This relationship was first identified when nuclease-hypersensitive sites were observed to appear at the 5' end of genes upon activation of transcription [1, 2]. Later, the transcription-dependent changes in the chromatin of a gene came to be understood better through examination of the chromatin structures of individual genes - such as PHO5, GAL1 and GAL10 - under active and inactive conditions [3–7]. These studies found that the nucleosomes - the basic repeating units of chromatin, each consisting of a histone octamer encircled by about 146 base-pairs of DNA - are modified, unfolded or lost at the promoters of genes upon activation of transcription. It remains unclear, however, whether such remodeling or loss of nucleosomes is a general feature of eukaryotic gene regulation. Recently, two papers have analyzed the nucleosome distribution throughout the yeast genome. The authors of the new studies [8, 9] propose that the genome-wide distribution of nucleosomes is heterogeneous and that this pattern may be involved in, or result from, the regulation of gene expression.
Nucleosomes are depleted from regulatory regions of the yeast genome
A recent study by Lee et al.  analyzes nucleosome distribution over the entire yeast genome, and another by Bernstein et al.  investigates nucleosome occupancy specifically at yeast promoters . Both groups used a combination of chromatin immunoprecipitation and microarray analysis: after cross-linking proteins to DNA, different sets of antibodies were used to immunoprecipitate histones from yeast cell extracts, thereby enriching for DNA that is bound to histones. Lee et al.  performed comparative hybridization of the enriched DNA and total genomic DNA on microarrays in order to determine the relative histone occupancy at the entire genome, whereas Bernstein et al.  used the same approach with only intergenic DNA in order to determine histone occupancy in intergenic regions. Although it should be kept in mind that the degree of histone cross-linking may not always accurately reflect the presence or absence of histones, these studies do make a compelling argument for differences in nucleosome density across the genome.
One of the recent studies  found that the distribution of histones is heterogeneous over the genome, such that intergenic regions appear to have a sparser distribution of nucleosomes than the open reading frames (ORFs). Furthermore, the regulatory regions, such as promoters, have even fewer nucleosomes than other intergenic regions. Importantly, there is an inverse correlation between nucleosome occupancy at a promoter region and the transcription rate of the gene downstream of the promoter: the upstream regions of active genes have a lower density of nucleosomes than those of less-transcribed genes. Interestingly, the transcription rate also affects nucleosome occupancy within the ORFs. ORFs that are transcribed at rates of more than about 30 mRNAs per hour have a lower density of nucleosomes than ORFs overall. This is an important observation because it suggests not only that nucleosomes are transiently dissociated from DNA during the elongation phase of transcription but also that they are not fully replaced within at the coding regions of heavily transcribed genes after each RNA polymerase passes along [10–12].
The heterogeneous distribution of nucleosomes over the genome is consistent with an earlier study showing that one can physically fractionate regions transcribed by RNA polymerase II from other regions in the genome . This fractionation is done by cross-linking chromatin to DNA in vivo and then separating the aqueous phase from the organic phase in phenol:chloroform extractions. Free DNA segregates into the aqueous phase and DNA bound to proteins remains in the organic phase. This results in differential segregation of intergenic regions of the genome into the aqueous phase. Nagy et al.  proposed that this may be a result of different efficiency of chromatin cross-linking along the genome and that these differences in efficiency might be mediated through differentially modified histone tails. The heterogeneous distribution of nucleosomes suggests, however, that the regulatory regions are simply depleted of nucleosomes, and other proteins bound at these regions may not cross-link to DNA as efficiently as histones. In either case, the physical fractionation of yeast chromatin suggests that the chromatin is organized differently between coding and noncoding regions of the genome, and the heterogeneous distribution of nucleosomes may be part of this organization.
Nucleosome occupancy at the promoters of individual genes is inversely proportional to their transcription rate
In order to understand further the relationship between nucleosome occupancy and the transcriptional status of a gene, Lee et al.  analyzed nucleosome occupancy over the entire genome after heat shock, a treatment that changes the transcription profile of the yeast genome considerably. When yeast cells are growing rapidly at an optimal temperature, some of the most active genes are those encoding ribosomal proteins, and these genes are also most repressed upon heat shock. Both studies [8, 9] observed that the promoters of ribosomal protein genes are the most depleted of nucleosomes when cells are rapidly growing. When the cells are heat shocked, these genes are rapidly repressed and their nucleosome occupancy increases . This suggests that nucleosome occupancy is either the cause or the result of the transcriptional status of a gene.
This raises an important question: what are the determinants of nucleosome occupancy, and how do they relate to transcription? An attractive answer to this question might be that transcription factors replace nucleosomes at the promoters. One transcription factor that is known to target the promoters of ribosomal protein genes is Rap1p . An unbiased search for sequence motifs at the promoters that are most depleted of nucleosomes during rapid growth also identified Rap1p-binding sites in ribosomal protein promoters . What role, then, does Rap1p play in nucleosome occupancy after heat shock? It was previously shown that Rap1p can move or displace nucleosomes at the promoters of ribosomal protein genes , so one prediction is that the loss of nucleosomes may be a result of Rap1p binding at the promoters. This idea is supported by the results of an experiment showing that when the Rap1p-binding site is deleted at a number of ribosomal protein promoters, nucleosome occupancy increases at these promoters . In contrast, after heat shock, although nucleosome occupancy at the promoters increases, Rap1p remains bound . This observation is consistent with the result of another experiment: when the transcription of ribosomal proteins is repressed by rapamycin treatment and nucleosomes return to their promoters, Rap1p remains bound . Together, these observations suggest that Rap1p binding alone is insufficient to keep nucleosomes off promoters, and it probably requires additional cofactors and/or chromatin-remodeling factors.
The determinants of global nucleosome distribution
As well as suggesting the general model shown in Figure 1, the recent studies identify a heterogeneous distribution of nucleosomes in the yeast genome [8, 9]. These findings offer an important factor that should be taken into account when interpreting genome-wide experiments involving post-translationally modified nucleosomes. When examining the distribution of modified histones in the genome, one should keep in mind that the histones are organized heterogeneously in the genome such that the regulatory regions possess fewer nucleosomes. Thus, the apparent loss of a histone modification may in fact represent the absence of histones . Overall, recent studies suggest a genome-wide depletion of nucleosomes over regulatory regions that might be a common feature of eukaryotic genomes.
This work was supported by postdoctoral fellowship grant PF-02-012-01-GMC from the American Cancer Society to M.J.C., and NIGMS, National Institutes of Health grant GM047867 to J.L.W.
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