DNA Sequence Plays a Major Role in Determining Nucleosome Positions in Yeast CUP1 Chromatin

Abstract

The role of DNA sequence in determining nucleosome positions in vivo was investigated by comparing the positions adopted by nucleosomes reconstituted on a yeast plasmid in vitro using purified core histones with those in native chromatin containing the same DNA, described previously. Nucleosomes were reconstituted on a 2.5 kilobase pair DNA sequence containing the yeast TRP1ARS1 plasmid with CUP1 as an insert (TAC-DNA). Multiple, alternative, overlapping nucleosome positions were mapped on TAC-DNA. For the 58 positioned nucleosomes identified, the relative positioning strengths and the stabilities to salt and temperature were determined. These positions were, with a few exceptions, identical to those observed in native, remodeled TAC chromatin containing an activated CUP1 gene. Only some of these positions are utilized in native, unremodeled chromatin. These observations suggest that DNA sequence is likely to play a very important role in positioning nucleosomes in vivo. We suggest that events occurring in yeast CUP1 chromatin determine which positions are occupied in vivo and when they are occupied.Eukaryotic DNA is packaged into chromatin, the basic structural repeat unit of which is the nucleosome. The nucleosome core contains 147 bp 1 of DNA wrapped in about 1.75 superhelical turns around a central octamer composed of two each of the four core histones H2A, H2B, H3, and H4. Its structure has been solved at high resolution by x-ray crystallography (1). In recent years, it has become clear that the nucleosome is not just a means of packaging large amounts of DNA into the nucleus but is also involved in regulation of gene expression (2, 3). The latter role stems from the fact that sequence-specific DNAbinding proteins often cannot recognize their sites when they are tightly wrapped in a nucleosome. The solution to this problem apparently resides in the activities of the many different chromatin remodeling and histone acetylase complexes recently identified. These facilitate the disruption or displacement of nucleosomes, allowing factors to bind, transcription complexes to form, and RNA polymerase II to initiate transcription.One of the most interesting aspects of nucleosome structure is that the histone octamer does not bind to DNA randomly but exhibits preferences for some sequences over others, a phenomenon referred to as nucleosome positioning (4, 5). Positioning is actually determined by the central H3-H4 tetramer (6). The translational position of a nucleosome is defined by the 147-bp DNA sequence it occupies (see Ref. 7 for a detailed analysis). The affinity of the histone octamer for a particular 147-bp DNA sequence defines its positioning strength. Strongly positioned nucleosomes are those that contain sequences that bind the octamer much more tightly than neighboring sequences. There are many examples of these, the most well known being the 5 S RNA gene (8). There is a great deal of evidence for the importance of positioned nucleosomes in gene regulation (4). In budding yeast, the best studied example ...

A SWI/SNF- and INO80-dependent nucleosome movement at the INO1 promoter

“…The INO1 ORF and its promoter are organized into a set of well-positioned overlapping nucleosomes during repressing conditions. Such overlapping chromatin structures were also observed in other genes such as CUP1 (28,35) and HIS3 (36). It is interesting to observe overlapping positions under repressing conditions.…”

“…However, there are many more than seven bands in repressed chromatin and the neighboring bands were significantly less than 147 bp apart. Based on our previous work, these bands probably indicate alternative overlapping nucleosome positions (28,29,35). Thus, our map revealed the presence of overlapping nucleosome positions on INO1.…”

Activation of Saccharomyces cerevisiae HIS3 Results in Gcn4p-Dependent, SWI/SNF-Dependent Mobilization of Nucleosomes over the Entire Gene

“…An important issue is whether the chromatin structure of HIS3 is typical of yeast genes. We have studied nucleosome positions on only one other gene, CUP1 (28)(29)(30), with similar results. However, since we were unable to identify the remodeling complexes acting at CUP1, we turned to HIS3 for further studies, because it was known to be affected by the SWI/SNF complex.…”

Nucleosomal signatures impose nucleosome positioning in coding and noncoding sequences in the genome

“…In some cases, the nucleosomal pattern in vitro coincides with that of native chromatin in discrete genomic regions (Shen and Clark 2001;Allan et al 2013;Beh et al 2015). However, unlike the situation in vitro, the 601 and 603 artificial sequences do not preferentially form nucleosomes when integrated into the genome of S. cerevisiae (Gaykalova et al 2011;Perales et al 2011).…”