RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast

Abstract

RSC and SWI/SNF are related ATP-dependent chromatin remodeling machines that move nucleosomes, regulating access to DNA. We addressed their roles in nucleosome phasing relative to transcription start sites in yeast. SWI/SNF has no effect on phasing at the global level. In contrast, RSC depletion results in global nucleosome repositioning: Both upstream and downstream nucleosomal arrays shift toward the nucleosome-depleted region (NDR), with no change in spacing, resulting in a narrower and partly filled NDR. The global picture of RSC-depleted chromatin represents the average of a range of chromatin structures, with most genes showing a shift of the +1 or the -1 nucleosome into the NDR. Using RSC ChIP data reported by others, we show that RSC occupancy is highest on the coding regions of heavily transcribed genes, though not at their NDRs. We propose that RSC has a role in restoring chromatin structure after transcription. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighboring NDRs. These signals may be in phase, resulting in constructive interference and a regular array, or out of phase, resulting in destructive interference and fuzzy positioning. We propose a modified barrier model, in which a stable complex located at the NDR acts as a bidirectional phasing barrier. In RSC-depleted cells, this barrier has a smaller footprint, resulting in narrower NDRs. Thus, RSC plays a critical role in organizing yeast chromatin. [Supplemental material is available for this article.]The basic subunit of chromatin structure, the nucleosome, has inhibitory effects on transcription, DNA replication, and repair, which are accounted for by its compact structure and innate stability. The nucleosome core is composed of two molecules from each of the four core histones, H2A, H2B, H3, and H4, forming a histone octamer, around which is wrapped ;147 bp of DNA (Luger et al. 1997). Early nuclease digestion studies of chromatin in nuclei revealed a characteristic ladder of DNA bands in a gel, indicating that nucleosomes are regularly spaced along the DNA in vivo. The nucleosomes in the cells of higher eukaryotes are generally spaced farther apart (;195 bp per nucleosome) than in yeast (;165 bp) (van Holde 1988). Authentic nucleosomes can be reconstituted in vitro using purified histones and DNA, but they are not regularly spaced. Regular spacing requires the addition of ATP-dependent chromatin remodeling complexes, such as ISW1, ISW2, or ACF (Ito et al. 1997;Tsukiyama et al. 1999) and is affected by linker histone (Blank and Becker 1995). The remodeling complexes use the free energy of ATP hydrolysis to move nucleosomes along DNA (Clapier and Cairns 2009;Hota and Bartholomew 2011;Narlikar et al. 2013).Genome-wide studies of nucleosomal DNA isolated from cells by digestion with micrococcal nuclease (MNase) and analyzed using tiling microarrays or massively parallel sequencing have revealed that most yeast genes have a nucleosome-depleted reg...

Histone exchange, chromatin structure and the regulation of transcription

“…AT-rich sequences upstream of transcribed sequences exclude nucleosomes because they are less capable of bending around a histone octamer 90,91 . Chromatin remodellers such as the RSC complex maintain these nucleosome-depleted regions (NDRs) by sliding nucleosomes away from this region 92,93 (FIG. 2b).…”

Sequence-targeted nucleosome sliding in vivo by a hybrid Chd1 chromatin remodeler

“…Previous studies have reported that chromatin remodeling factors are responsible for the well positioned +1 and −1 nucleosomes at gene promoters and also for packing nucleosomes into organized arrays against these founding nucleosomes (Mavrich et al 2008; Gkikopoulos et al 2011;Zhang et al 2011;Ganguli et al 2014). However, the nature of the barriers and mechanism(s) through which these arrays are phased remain unclear (Hughes and Rando 2014).…”

Genomic Nucleosome Organization Reconstituted with Pure Proteins

“…This allows individual assembly stages to be dissected, defined, and the role of individual factors deciphered. Current approaches involving in vivo removal or inactivation of one or a few nucleosome-related activities, for example chromatin remodeling enzymes, via genetic manipulation (Badis et al, 2008; Ganguli et al, 2014; Gkikopoulos et al, 2011; Hartley and Madhani, 2009; Ocampo et al., 2016; Parnell et al, 2008; van Bakel et al, 2013; Whitehouse et al, 2007; Yen et al, 2012) have proven insightful for identifying factors involved, their contribution, and their genomic binding locations. Critically, however, direct versus indirect roles are not readily apparent due to the ever-present complex milieu of cellular proteins, and so cannot identify the minimal set of components and core mechanisms that directly establish the primary structure of chromatin.…”