Pamela N. Dyer

Reconstitution of Nucleosome Core Particles from Recombinant Histones and DNA

Publisher Summary The ability to prepare nucleosome core particles (NCPs), or nucleosomal arrays, from recombinant histone proteins and defined-sequence DNA has become a requirement in many projects that address the role of histone modifications, histone variants, or histone mutations in nucleosome and chromatin structure. The cloning strategies for the construction of plasmids containing multiple repeats of defined DNA sequences, and the subsequent large-scale isolation of defined sequence DNA for nucleosome reconstitution are described. This chapter also describes adapted procedures to prepare nucleosomes with histones from other species, and for the refolding and reconstitution of (H2A– H2B) dimers and (H3–H4) 2 tetramers. Methods to reconstitute nucleosomes from different histone subcomplexes are described. A flow chart for all procedures involved in the preparation of synthetic nucleosomes is also presented.

Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA

H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A. The major sequence differences are in the docking domain that tethers the H2A–H2B dimer to the (H3–H4)2 tetramer; in addition, the C‐terminal tail is absent in H2A.Bbd. We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd‐NCP), and found that Bbd‐NCP had a more relaxed structure in which only 118±2 bp of DNA is protected against digestion with micrococcal nuclease. The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd‐NCP indicates that the distance between the DNA ends is increased significantly. The Bbd docking domain is largely responsible for this behavior, as shown by domain‐swap experiments. Bbd‐containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB. The structural properties of Bbd‐NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin.

Crystal structures of histone Sin mutant nucleosomes reveal altered protein–DNA interactions

Here we describe 11 crystal structures of nucleosome core particles containing individual point mutations in the structured regions of histones H3 and H4. The mutated residues are located at the two protein–DNA interfaces flanking the nucleosomal dyad. Five of the mutations partially restore the in vivo effects of SWI/SNF inactivation in yeast. We find that even nonconservative mutations of these residues (which exhibit a distinct phenotype in vivo) have only moderate effects on global nucleosome structure. Rather, local protein–DNA interactions are disrupted and weakened in a subtle and complex manner. The number of lost protein–DNA interactions correlates directly with an increased propensity of the histone octamer to reposition with respect to the DNA, and with an overall destabilization of the nucleosome. Thus, the disruption of only two to six of the ∼120 direct histone–DNA interactions within the nucleosome has a pronounced effect on nucleosome mobility and stability. This has implications for our understanding of how these structures are made accessible to the transcription and replication machinery in vivo.

Histone chaperone FACT action during transcription through chromatin by RNA polymerase II

FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)–nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT–H2A/H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.

Structure of histone-based chromatin in Archaea

Origin of DNA compaction As a repeating unit in eukaryotic chromatin, a nucleosome wraps DNA in superhelical turns around a histone octamer. Mattiroli et al. present the crystal structure of an archaeal histone-DNA complex in which the histone-mediated DNA geometry is exactly the same as that in the nucleosome. Comparing features of archaeal and eukaryotic chromatin structures offers important insights into the evolution of eukaryotic nucleosomes. Science, this issue p. 609 Archaeal histone homodimers form a complex with DNA that is similar to the eukaryotic nucleosome. Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

In Vitro Chromatin Assembly: Strategies and Quality Control

Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signaling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter, we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

In Vitro Chromatin Assembly

Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signaling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter, we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

Investigating the Dynamics of Destabilized Nucleosomes Using Methyl-TROSY NMR

The nucleosome core particle (NCP), comprised of histone proteins wrapped with ∼146 base pairs of DNA, provides both protection and controlled access to DNA so as to regulate vital cellular processes. High-resolution structures of nucleosomes and nucleosome complexes have afforded a clear understanding of the structural role of NCPs, but a detailed description of the dynamical properties that facilitate DNA-templated processes is only beginning to emerge. Using methyl-TROSY NMR approaches we evaluate the effect of point mutations designed to perturb key histone interfaces that become destabilized during nucleosome remodeling in an effort to probe NCP plasticity. Notably the NCP retains its overall structural integrity, yet relaxation experiments of mutant nucleosomes reveal significant dynamics within a central histone interface associated with alternative NCP conformations populated to as much as 15% under low salt conditions. This work highlights the inherent plasticity of NCPs and establishes methyl-TROSY NMR as a valuable compliment to current single molecule methods in quantifying NCP dynamic properties.