Răzvan V. Chereji

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

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.

Genome-wide profiling of nucleosome sensitivity and chromatin accessibility in Drosophila melanogaster

Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to ‘phasing’ off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.

The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo

Abstract Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo. We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure.

Heavy transcription of yeast genes correlates with differential loss of histone H2B relative to H4 and queued RNA polymerases

Eukaryotic chromatin is composed of nucleosomes, which contain nearly two coils of DNA wrapped around a central histone octamer. The octamer contains an H3-H4 tetramer and two H2A-H2B dimers. Gene activation is associated with chromatin disruption: a wider nucleosome-depleted region (NDR) at the promoter and reduced nucleosome occupancy over the coding region. Here, we examine the nature of disrupted chromatin after induction, using MNase-seq to map nucleosomes and subnucleosomes, and a refined high-resolution ChIP-seq method to map H4, H2B and RNA polymerase II (Pol II) genome-wide. Over coding regions, induced genes show a differential loss of H2B relative to H4, which correlates with Pol II density and the appearance of subnucleosomes. After induction, Pol II is surprisingly low at the promoter, but accumulates on the gene and downstream of the termination site, implying that dissociation is very slow. Thus, induction-dependent chromatin disruption reflects both eviction of H2A-H2B dimers and the presence of queued Pol II elongation complexes. We propose that slow Pol II dissociation after transcription is a major factor in chromatin disruption and that it may be of critical importance in gene regulation.

Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation

Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.

Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo

Abstract Glucocorticoid hormone plays a major role in metabolism and disease. The hormone-bound glucocorticoid receptor (GR) binds to a specific set of enhancers in different cell types, resulting in unique patterns of gene expression. We have addressed the role of chromatin structure in GR binding by mapping nucleosome positions in mouse adenocarcinoma cells. Before hormone treatment, GR-enhancers exist in one of three chromatin states: (i) Nucleosome-depleted enhancers that are DNase I-hypersensitive, associated with the Brg1 chromatin remodeler and flanked by nucleosomes incorporating histone H2A.Z. (ii) Nucleosomal enhancers that are DNase I-hypersensitive, marked by H2A.Z and associated with Brg1. (iii) Nucleosomal enhancers that are inaccessible to DNase I, incorporate little or no H2A.Z and lack Brg1. Hormone-induced GR binding results in nucleosome shifts at all types of GR-enhancer, coinciding with increased recruitment of Brg1. We propose that nucleosome-depleted GR-enhancers are formed and maintained by other transcription factors which recruit Brg1 whereas, at nucleosomal enhancers, GR behaves like a pioneer factor, interacting with nucleosomal sites and recruiting Brg1 to remodel the chromatin.

Precise genome-wide mapping of single nucleosomes and linkers in vivo

We developed a chemical cleavage method that releases single nucleosome dyad-containing fragments, allowing us to precisely map both single nucleosomes and linkers with high accuracy genome-wide in yeast. Our single nucleosome positioning data reveal that nucleosomes occupy preferred positions that differ by integral multiples of the DNA helical repeat. By comparing nucleosome dyad positioning maps to existing genomic and transcriptomic data, we evaluated the contributions of sequence, transcription, and histones H1 and H2A.Z in defining the chromatin landscape. We present a biophysical model that neglects DNA sequence and shows that steric occlusion suffices to explain the salient features of nucleosome positioning.

SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast

The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.

SPT6 interacts with NSD2 and facilitates interferon‐induced transcription

The role of the histone chaperone SPT6 in mammalian cells is not fully understood. Here, we investigated the involvement of SPT6 in type I interferon (IFN)‐induced transcription in murine fibroblasts. In RNA‐seq analysis, Spt6 siRNA attenuates about half of ~ 200 IFN‐stimulated genes (ISGs), while not affecting housekeeping genes. ISGs with high mRNA induction are more susceptible to Spt6 siRNA than those with lower levels of induction. ChIP analysis shows that SPT6 is recruited to highly inducible, Spt6 siRNA‐sensitive ISGs, but not to other siRNA‐insensitive ISGs. Furthermore, SPT6 recruitment is abrogated in cells lacking the histone methyltransferase NSD2. In co‐IP experiments, SPT6 interacts with NSD2. In summary, SPT6 facilitates IFN‐induced transcription, highlighting its critical role in gene activation.

Major Determinants of Nucleosome Positioning

The compact structure of the nucleosome limits DNA accessibility and inhibits the binding of most sequence-specific proteins. Nucleosomes are not randomly located on the DNA but positioned with respect to the DNA sequence, suggesting models in which critical binding sites are either exposed in the linker, resulting in activation, or buried inside a nucleosome, resulting in repression. The mechanisms determining nucleosome positioning are therefore of paramount importance for understanding gene regulation and other events that occur in chromatin, such as transcription, replication, and repair. Here, we review our current understanding of the major determinants of nucleosome positioning: DNA sequence, nonhistone DNA-binding proteins, chromatin-remodeling enzymes, and transcription. We outline the major challenges for the future: elucidating the precise mechanisms of chromatin opening and promoter activation, identifying the complexes that occupy promoters, and understanding the multiscale problem of chromatin fiber organization.