Chromosome segregation during mitosis requires assembly of the kinetochore complex at the centromere. Key to kinetochore assembly is the specific recognition of the histone variant CENP-A in the centromeric nucleosome by centromere protein C (CENP-C). We have defined the determinants of this recognition mechanism and discovered that CENP-C binds a hydrophobic region in the CENP-A tail and docks onto the acidic patch of histone H2A/H2B. We further find that the more broadly conserved CENP-C motif uses the same mechanism for CENP-A nucleosome recognition. Our findings reveal a conserved mechanism for protein recruitment to centromeres and a histone recognition mode whereby a disordered peptide binds the histone tail through nucleosome-docking-facilitated hydrophobic interactions.

Together with core histones, which make up the nucleosome, the linker histone (H1) is one of the five main histone protein families present in chromatin in eukaryotic cells. H1 binds to the nucleosome to form the next structural unit of metazoan chromatin, the chromatosome, which may help chromatin to fold into higher-order structures. Despite their important roles in regulating the structure and function of chromatin, linker histones have not been studied as extensively as core histones. Nevertheless, substantial progress has been made recently. The first near-atomic resolution crystal structure of a chromatosome core particle and an 11 Å resolution cryo-electron microscopy-derived structure of the 30 nm nucleosome array have been determined, revealing unprecedented details about how linker histones interact with the nucleosome and organize higher-order chromatin structures. Moreover, several new functions of linker histones have been discovered, including their roles in epigenetic regulation and the regulation of DNA replication, DNA repair and genome stability. Studies of the molecular mechanisms of H1 action in these processes suggest a new paradigm for linker histone function beyond its architectural roles in chromatin.

Summary Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses, previous cross-linking and fluorescence recovery after photobleach experiments, and helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures.

c Chlamydia trachomatis causes chronic inflammatory diseases of the eye and genital tract and has global medical importance. The chlamydial plasmid plays an important role in the pathophysiology of these diseases, as plasmid-deficient organisms are highly attenuated. The cryptic plasmid carries noncoding RNAs and eight conserved open reading frames (ORFs). To understand plasmid gene function, we generated plasmid shuttle vectors with deletions in each of the eight ORFs. The individual deletion mutants were used to transform chlamydiae and the transformants were characterized phenotypically and at the transcriptional level. We show that pgp1, -2, -6, and -8 are essential for plasmid maintenance, while the other ORFs can be deleted and the plasmid stably maintained. We further show that a pgp4 knockout mutant exhibits an in vitro phenotype similar to its isogenic plasmidless strain, in terms of abnormal inclusion morphology and lack of glycogen accumulation. Microarray and qRT-PCR analysis revealed that Pgp4 is a transcriptional regulator of plasmid-encoded pgp3 and multiple chromosomal genes, including the glycogen synthase gene glgA, that are likely important in chlamydial virulence. Our findings have major implications for understanding the plasmid's role in chlamydial pathogenesis at the molecular level.

Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

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Histone tails and their post-translational modifications (PTM) play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K16R17H18R19 in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1–15 in H4 can be detected, indicating that residues 16–22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch, K16R17H18R19.

BackgroundAmyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.Methodology/Principal FindingsAs revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.Conclusions/SignificanceWe suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.