Summary The histone H2A-H2B heterodimer is an integral component of the nucleosome. The cellular localization and deposition of H2A-H2B into chromatin is regulated by numerous factors including histone chaperones such as Nucleosome Assembly Protein 1 (Nap1). We use hydrogen-deuterium exchange coupled to mass spectrometry to characterize H2A-H2B and Nap1. Unexpectedly, we find that at low ionic strength the α-helices in H2A-H2B are frequently sampling partially disordered conformations, and that binding to Nap1 reduces this conformational sampling. We identify the interaction surface between H2A-H2B and Nap1, and confirm its relevance both in vitro and in vivo. We show that two copies of H2A-H2B bound to a Nap1 homodimer form a tetramer with contacts between H2B chains similar to those in the four-helix bundle structural motif. The organization of the complex reveals that Nap1 competes with histone-DNA and inter-histone interactions observed in the nucleosome, thereby regulating the availability of histones for chromatin assembly.

No abstract

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 signalling 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.

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMGbox DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.During lymphocyte development, antigen receptor genes undergo a series of DNA rearrangements to generate functional exons encoding the antigen binding domains of these receptors (1). This rearrangement process, termed V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called recombination-activating gene (RAG) 1 -1 and RAG-2, bring two gene segments into close proximity and then introduce a DNA double-strand break (DSB) at the end of each coding segment.Adjacent to each coding segment lies a recombination signal sequence (RSS) that serves as the binding site for the RAG-1/2 protein complex (hereafter termed the "RAG complex") and directs the location of cleavage. The RSS contains a conserved heptamer and nonamer motif separated by either 12 or 23 bp of relatively nonconserved sequence (12-RSS and 23-RSS, respectively). Productive exon assembly is promoted by the 12/23 rule, a restriction that limits rearrangement to RSSs whose spacing between the heptamer and nonamer is different. RAGmediated cleavage of RSS pairs produces four DNA ends: two blunt, 5Ј-phosphorylated signal ends and two coding ends terminating in DNA hairpin structures (2-4). These reaction products originate from a two-step cleavage reaction in which the RSS is first nicked at its 5Ј-end, and then the resulting 3Ј-OH is covalently linked to the bottom strand by direct transesterification (5, 6). After DNA cleavage, signal ends are generally ligated together to form precise signal joints, but coding ends, being sealed as DNA hairpins, are first resolved and then processed and joined to create coding joints in which nucleotides are frequently gained or lost at the junction. DNA hairpin opening is most likely catalyzed by a complex containing Artemis and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) (7). Signal and coding joint formation is mediated by ubiquitously expr...

for the Low-Value Practices in Trauma Care Expert Consensus Group IMPORTANCE The use of quality indicators has been shown to improve injury care processes and outcomes. However, trauma quality indicators proposed to date exclusively target the underuse of recommended practices. Initiatives such as Choosing Wisely publish lists of practices to be questioned, but few apply to trauma care, and most have not successfully been translated to quality indicators. OBJECTIVE To develop a set of evidence and patient-informed, consensus-based quality indicators targeting reductions in low-value clinical practices in acute, in-hospital trauma care.DESIGN, SETTING, AND PARTICIPANTS This 2-round Research and Development/University of California at Los Angeles (RAND/UCLA) consensus study, conducted from April 20 to June 9, 2021, comprised an online questionnaire and a virtual workshop led by 2 independent moderators. Two panels of international experts from Canada, Australia, the US, and the UK, and local stakeholders from Québec, Canada, represented key clinical expertise involved in trauma care and included 3 patient partners. MAIN OUTCOMES AND MEASURESPanelists were asked to rate 50 practices on a 7-point Likert scale according to 4 quality indicator criteria: importance, supporting evidence, actionability, and measurability. RESULTSOf 49 eligible experts approached, 46 (94%; 18 experts [39%] aged Ն50 years; 37 men [80%]) completed at least 1 round and 36 (73%) completed both rounds. Eleven quality indicators were selected overall, 2 more were selected by the international panel and a further 3 by the local stakeholder panel. Selected indicators targeted low-value clinical practices in the following aspects of trauma care: (1) initial diagnostic imaging (head, cervical spine, ankle, and pelvis), (2) repeated diagnostic imaging (posttransfer computed tomography [CT] and repeated head CT), (3) consultation (neurosurgical and spine), ( 4) surgery (penetrating neck injury), (5) blood product administration, (6) medication (antibiotic prophylaxis and late seizure prophylaxis), (7) trauma service admission (blunt abdominal trauma), (8) intensive care unit admission (mild complicated traumatic brain injury), and ( 9) routine blood work (minor orthopedic surgery). CONCLUSIONS AND RELEVANCEIn this consensus study, a set of consensus-based quality indicators were developed that were informed by the best available evidence and patient priorities, targeting low-value trauma care. Selected indicators represented a trauma-specific list of practices, the use of which should be questioned. Trauma quality programs in high-income countries may use these study results as a basis to select context-specific quality indicators to measure and reduce low-value care.