Jeffrey N. McKnight

The Chromodomains of the Chd1 Chromatin Remodeler Regulate DNA Access to the ATPase Motor

Chromatin remodelers are ATP-driven machines that assemble, slide, and remove nucleosomes from DNA, but how the ATPase motors of remodelers are regulated is poorly understood. Here we show that the double chromodomain unit of the Chd1 remodeler blocks DNA binding and activation of the ATPase motor in the absence of nucleosome substrates. The Chd1 crystal structure reveals that an acidic helix joining the chromodomains can pack against a DNA-binding surface of the ATPase motor. Disruption of the chromodomain-ATPase interface prevents discrimination between nucleosomes and naked DNA and reduces the reliance on the histone H4 tail for nucleosome sliding. We propose that the chromodomains allow Chd1 to distinguish between nucleosomes and naked DNA by physically gating access to the ATPase motor, and we hypothesize that related ATPase motors may employ a similar strategy to discriminate among DNA-containing substrates.

Rapid DNA–protein cross-linking and strand scission by an abasic site in a nucleosome core particle

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ∼60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA–protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA–protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ∼4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA–protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.

Extranucleosomal DNA binding directs nucleosome sliding by Chd1.

ABSTRACT Chd1- and ISWI-type chromatin remodelers can sense extranucleosomal DNA and preferentially shift nucleosomes toward longer stretches of available DNA. The DNA-binding domains of these chromatin remodelers are believed to be responsible for sensing extranucleosomal DNA and are needed for robust sliding, but it is unclear how these domains contribute to directional movement of nucleosomes. Here, we show that the DNA-binding domain of Chd1 is not essential for nucleosome sliding but is critical for centering mononucleosomes on short DNA fragments. Remarkably, nucleosome centering was achieved by replacing the native DNA-binding domain of Chd1 with foreign DNA-binding domains of Escherichia coli AraC or Drosophila melanogaster engrailed. Introducing target DNA sequences recognized by the foreign domains enabled the remodelers to rapidly shift nucleosomes toward these binding sites, demonstrating that these foreign DNA-binding domains dictated the direction of sliding. Sequence-directed sliding occluded the target DNA sequences on the nucleosome enough to promote release of the remodeler. Target DNA sequences were highly stimulatory at multiple positions flanking the nucleosome and had the strongest influence when separated from the nucleosome by 23 or fewer base pairs. These results suggest that the DNA-binding domains affinity for extranucleosomal DNA is the key determinant for the direction that Chd1 shifts the nucleosome.

Dynamic regulation of transcription factors by nucleosome remodeling

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06249.001

Global Promoter Targeting of a Conserved Lysine Deacetylase for Transcriptional Shutoff during Quiescence Entry

Quiescence is a conserved cell-cycle state characterized by cell-cycle arrest, increased stress resistance, enhanced longevity, and decreased transcriptional, translational, and metabolic output. Although quiescence plays essential roles in cell survival and normal differentiation, the molecular mechanisms leading to this state are not well understood. Here, we determined changes in the transcriptome and chromatin structure of S. cerevisiae upon quiescence entry. Our analyses revealed transcriptional shutoff that is far more robust than previously believed and an unprecedented global chromatin transition, which are tightly correlated. These changes require Rpd3 lysine deacetylase targeting to at least half of gene promoters via quiescence-specific transcription factors including Xbp1 and Stb3. Deletion of RPD3 prevents cells from establishing transcriptional quiescence, leading to defects in quiescence entry and shortening of chronological lifespan. Our results define a molecular mechanism for global reprogramming of transcriptome and chromatin structure for quiescence driven by a highly conserved chromatin regulator.

Identification of Residues in Chromodomain Helicase DNA-Binding Protein 1 (Chd1) Required for Coupling ATP Hydrolysis to Nucleosome Sliding

Background: Chromatin remodelers slide nucleosomes in an ATP-dependent manner. Results: Residues between the ATPase motor and DNA-binding domain of chromodomain helicase DNA-binding protein 1 (Chd1) are required for sliding but not nucleosome-stimulated ATP hydrolysis. Conclusion: Residues outside the conserved ATPase core are required for efficiently utilizing the energy of ATP hydrolysis for sliding. Significance: Understanding the role of ATPase-coupling residues will be critical for revealing nucleosome sliding mechanisms. Chromatin remodelers are ATP-dependent machines responsible for directionally shifting nucleosomes along DNA. We are interested in defining which elements of the chromodomain helicase DNA-binding protein 1 (Chd1) remodeler are necessary and sufficient for sliding nucleosomes. This work focuses on the polypeptide segment that joins the ATPase motor to the C-terminal DNA-binding domain. We identify amino acid positions outside the ATPase motor that, when altered, dramatically reduce nucleosome sliding ability and yet have only ∼3-fold reduction in ATPase stimulation by nucleosomes. These residues therefore appear to play a role in functionally coupling ATP hydrolysis to nucleosome sliding, and suggest that the ATPase motor requires cooperation with external elements to slide DNA past the histone core.

Decoupling nucleosome recognition from DNA binding dramatically alters the properties of the Chd1 chromatin remodeler

Chromatin remodelers can either organize or disrupt nucleosomal arrays, yet the mechanisms specifying these opposing actions are not clear. Here, we show that the outcome of nucleosome sliding by Chd1 changes dramatically depending on how the chromatin remodeler is targeted to nucleosomes. Using a Chd1–streptavidin fusion remodeler, we found that targeting via biotinylated DNA resulted in directional sliding towards the recruitment site, whereas targeting via biotinylated histones produced a distribution of nucleosome positions. Remarkably, the fusion remodeler shifted nucleosomes with biotinylated histones up to 50 bp off the ends of DNA and was capable of reducing negative supercoiling of plasmids containing biotinylated chromatin, similar to remodelling characteristics observed for SWI/SNF-type remodelers. These data suggest that forming a stable attachment to nucleosomes via histones, and thus lacking sensitivity to extranucleosomal DNA, seems to be sufficient for allowing a chromatin remodeler to possess SWI/SNF-like disruptive properties.

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

ATP-dependent chromatin remodelers regulate chromatin dynamics by modifying nucleosome positions and occupancy. DNA-dependent processes such as replication and transcription rely on chromatin to faithfully regulate DNA accessibility, yet how chromatin remodelers achieve well-defined nucleosome positioning in vivo is poorly understood. Here, we report a simple method for site-specifically altering nucleosome positions in live cells. By fusing the Chd1 remodeler to the DNA binding domain of the Saccharomyces cerevisiae Ume6 repressor, we have engineered a fusion remodeler that selectively positions nucleosomes on top of adjacent Ume6 binding motifs in a highly predictable and reproducible manner. Positioning of nucleosomes by the fusion remodeler recapitulates closed chromatin structure at Ume6-sensitive genes analogous to the endogenous Isw2 remodeler. Strikingly, highly precise positioning of single founder nucleosomes by either chimeric Chd1-Ume6 or endogenous Isw2 shifts phased chromatin arrays in cooperation with endogenous chromatin remodelers. Our results demonstrate feasibility of engineering precise nucleosome rearrangements through sequence-targeted chromatin remodeling and provide insight into targeted action and cooperation of endogenous chromatin remodelers in vivo.

Genome‐Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High‐Resolution Histone ChIP, and NCAM

Because histones bind DNA very tightly, the location on DNA and the level of occupancy of a given DNA sequence by nucleosomes can profoundly affect accessibility of non‐histone proteins to chromatin, affecting virtually all DNA‐dependent processes, such as transcription, DNA repair, DNA replication and recombination. Therefore, it is often necessary to determine positions and occupancy of nucleosomes to understand how DNA‐dependent processes are regulated. Recent technological advances made such analyses feasible on a genome‐wide scale at high resolution. In addition, we have recently developed a method to measure nuclease accessibility of nucleosomes on a global scale. This unit describes methods to map nucleosome positions, to determine nucleosome density, and to determine nuclease accessibility of nucleosomes using deep sequencing. Curr. Protoc. Mol. Biol. 108:21.28.1‐21.28.16.

The conserved HDAC Rpd3 drives transcriptional quiescence in S. cerevisiae

Quiescence is a ubiquitous cell cycle stage conserved from microbes through humans and is essential to normal cellular function and response to changing environmental conditions. We recently reported a massive repressive event associated with quiescence in Saccharomyces cerevisiae, where Rpd3 establishes repressive chromatin structure that drives transcriptional shutoff [6]. Here, we describe in detail the experimental procedures, data collection, and data analysis related to our characterization of transcriptional quiescence in budding yeast (GEO: GSE67151). Our results provide a bona fide molecular event driven by widespread changes in chromatin structure through action of Rpd3 that distinguishes quiescence as a unique cell cycle stage in S. cerevisiae.