No abstract

We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing pairedend DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes. To characterize DNA-binding features of transcription factors, we plotted the length of each fragment versus its position in the genome, which defined the minimal protected region of each factor. This process led to the precise mapping of protected and exposed regions at and around binding sites, and also determination of the degree to which they are flanked by phased nucleosomes and subnucleosome-sized particles. Our protocol and mapping method provide a general strategy for epigenome characterization, including nucleosome phasing and dynamics, ATP-dependent nucleosome remodelers, and transcription factors, from a singlesequenced sample.Saccharomyces cerevisiae | V-plot | transcription factor binding sites S hort-read deep-sequencing technologies have the potential of revolutionizing epigenomic profiling by making it possible to map DNA fragments with single base-pair resolution at reasonable cost. This ideal has been achieved for nucleosomes, which can be mapped at high resolution by treatment with micrococcal nuclease (MNase). MNase is a single strand-specific secreted glycoprotein that cleaves one strand when DNA breathes, then cleaves the other strand, resulting in a double-strand break. MNase then "nibbles" on the exposed DNA ends until it encounters an obstruction, such as a nucleosome, where the histone cores protect the DNA from further encroachment. Although MNase has long been used for studying nucleosomes (1), its mechanism of action on DNA suggests that it will stop "nibbling" at any obstruction, such as a DNA-binding protein. We had previously shown that MNase digestion of Drosophila nuclei followed by low-salt native chromatin extraction can be used to map both nucleosomes and paused RNA Polymerase II using paired-end sequencing (2). Similarly, Kent et al. used MNase digestion of uncrosslinked yeast nuclei to map binding sites for both nucleosomes and smaller particles identified as sequence-specific transcription factors (3). These studies showed that MNaseprotected DNA fragments as small as ∼50 bp could be recovered and mapped.A limitation of using paired-end sequencing as a read-out method for MNase mapping is that standard sequencing library preparation methods are optimized for DNA fragments of a few hundred base pairs, whereas the fragments protected by DNAbinding proteins are an order-of-magnitude smaller. Here we introduce a rapid Solexa library preparation protocol that efficiently recovers...

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we ''footprinted'' nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).