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SUMMARY Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 base-pair nucleosome bearing symmetric 25 base-pair linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

The nucleosomal subunit organization of chromatin provides a multitude of functions. Nucleosomes elicit an initial ~7-fold linear compaction of genomic DNA. They provide a critical mechanism for stable repression of genes and other DNA-dependent activities by restricting binding of trans-acting factors to cognate DNA sequences. Conversely they are engineered to be nearly meta-stable and disassembled (and reassembled) in a facile manner to allow rapid access to the underlying DNA during processes such as transcription, replication and DNA repair. Nucleosomes protect the genome from DNA damaging agents and provide a lattice onto which a myriad of epigenetic signals are deposited. Moreover, vast strings of nucleosomes provide a framework for assembly of the chromatin fiber and higher-order chromatin structures. Thus, in order to provide a foundation for understanding these functions, we present a review of the basic elements of nucleosome structure and stability, including the association of linker histones.

The structure of the nucleosome, the basic building block of the chromatin fiber, plays a key role in epigenetic regulatory processes that affect DNA-dependent processes in the context of chromatin. Members of the HMGN family of proteins bind specifically to nucleosomes and affect chromatin structure and function, including transcription and DNA repair. To better understand the mechanisms by which HMGN 1 and 2 alter chromatin, we analyzed their effect on the organization of histone tails and linker histone H1 in nucleosomes. We find that HMGNs counteract linker histone (H1)-dependent stabilization of higher order ‘tertiary’ chromatin structures but do not alter the intrinsic ability of nucleosome arrays to undergo salt-induced compaction and self-association. Surprisingly, HMGNs do not displace H1s from nucleosomes; rather these proteins bind nucleosomes simultaneously with H1s without disturbing specific contacts between the H1 globular domain and nucleosomal DNA. However, HMGNs do alter the nucleosome-dependent condensation of the linker histone C-terminal domain, which is critical for stabilizing higher-order chromatin structures. Moreover, HMGNs affect the interactions of the core histone tail domains with nucleosomal DNA, redirecting the tails to more interior positions within the nucleosome. Our studies provide new insights into the molecular mechanisms whereby HMGNs affect chromatin structure.

Background: Activation of GRK2 requires interaction with agonist-occupied GPCRs.Results: Residues on the GRK2 N terminus and kinase domain extension collaborate to create a GPCR docking site.Conclusion: Three GRK subfamilies use similar determinants to create the putative docking site, but subtle differences may dictate selectivity.Significance: Mapping the GRK-GPCR interface is required to understand the mechanism and specificity of GRK activation, and, therefore, the regulation of GPCRs.

Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures influencing nucleosome spacing on DNA and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, and focuses on recent advances that suggest modes of binding may influence chromatin organization while also highlighting remaining questions.

G protein‐coupled receptor (GPCR) kinases (GRKs) phosphorylate agonist‐activated GPCRs to initiate receptor desensitization, but the mechanism of kinase domain activation is unclear. Because agonist‐activated receptors dramatically stimulate the catalytic activity of GRKs, we sought to identify GRK2 residues located outside the active site that play a role in receptor interaction. Previous work suggested that active site tether (AST) residues of the kinase carboxyl‐tail extension are required for GRK activation, and that N‐terminal helix and AST interaction is important for receptor phosphorylation. We therefore carried out systematic mutagenesis of residues 3–18 and the AST of GRK2, and characterized these mutants using in vitro rhodopsin and peptide phosphorylation, GRK activation, intact cell β2‐adrenergic receptor phosphorylation, and cell based α2‐adrenergic receptor recruitment assays. Our results suggest that the N‐terminus and AST, both intrinsically disordered in the inactive kinase, play important roles in formation of the interaction site with activated receptors. Funding: NSF (RSM), NIH (JT), Canadian Institute for Health Research (MB), and FRSQ & Groupe de Recherche Universitaire sur le Médicament (AB).

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