Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

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 bp nucleosome bearing symmetric 25 bp 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.

Identification and Analysis of Six Phosphorylation Sites Within the Xenopus laevis Linker Histone H1.0 C-Terminal Domain Indicate Distinct Effects on Nucleosome Structure.

As a key structural component of the chromatin of higher eukaryotes, linker histones (H1s) are involved in stabilizing the folding of extended nucleosome arrays into higher-order chromatin structures and function as a gene-specific regulator of transcription in vivo. The H1 C-terminal domain (CTD) is essential for high-affinity binding of linker histones to chromatin and stabilization of higher-order chromatin structure. Importantly, the H1 CTD is an intrinsically disordered domain that undergoes a drastic condensation upon binding to nucleosomes. Moreover, although phosphorylation is a prevalent post-translational modification within the H1 CTD, exactly where this modification is installed and how phosphorylation influences the structure of the H1 CTD remains unclear for many H1s. Using novel mass spectrometry techniques, we identified six phosphorylation sites within the CTD of the archetypal linker histone Xenopus H1.0. We then analyzed nucleosome-dependent CTD condensation and H1-dependent linker DNA organization for H1.0 in which the phosphorylated serine residues were replaced by glutamic acid residues (phosphomimics) in six independent mutants. We find that phosphomimetics at residues S117E, S155E, S181E, S188E, and S192E resulted in a significant reduction in nucleosome-bound H1.0 CTD condensation compared with unphosphorylated H1.0, whereas S130E did not alter CTD structure. Furthermore, we found distinct effects among the phosphomimetics on H1-dependent linker DNA trajectory, indicating unique mechanisms by which this modification can influence H1 CTD condensation. These results bring to light a novel role for linker histone phosphorylation in directly altering the structure of nucleosome-bound H1 and a potential novel mechanism for its effects on chromatin structure and function.

Histone protein surface accessibility dictates direction of RSC-dependent nucleosome mobilization.

Chromatin remodeling enzymes use energy derived from ATP hydrolysis to mobilize nucleosomes and alter their structure to facilitate DNA access. The Remodels the Structure of Chromatin (RSC) complex has been extensively studied, yet aspects of how this complex functionally interacts with nucleosomes remain unclear. We introduce a steric mapping approach to determine how RSC activity depends on interaction with specific surfaces within the nucleosome. We find that blocking SHL + 4.5/-4.5 via streptavidin binding to the H2A N-terminal tail domains results in inhibition of RSC nucleosome mobilization. However, restriction enzyme assays indicate that remodeling-dependent exposure of an internal DNA site near the nucleosome dyad is not affected. In contrast, occlusion of both protein faces of the nucleosome by streptavidin attachment near the acidic patch completely blocks both remodeling-dependent nucleosome mobilization and internal DNA site exposure. However, we observed partial inhibition when only one protein surface is occluded, consistent with abrogation of one of two productive RSC binding orientations. Our results indicate that nucleosome mobilization requires RSC access to the trailing but not the leading protein surface, and reveals a mechanism by which RSC and related complexes may drive unidirectional movement of nucleosomes to regulate cis-acting DNA sequences in vivo.

Post-Translation Modifications and Mutations of Human Linker Histone Subtypes: Their Manifestation in Disease.

Linker histones (LH) are a critical component of chromatin in addition to the canonical histones (H2A, H2B, H3, and H4). In humans, 11 subtypes (7 somatic and 4 germinal) of linker histones have been identified, and their diverse cellular functions in chromatin structure, DNA replication, DNA repair, transcription, and apoptosis have been explored, especially for the somatic subtypes. Delineating the unique role of human linker histone (hLH) and their subtypes is highly tedious given their high homology and overlapping expression patterns. However, recent advancements in mass spectrometry combined with HPLC have helped in identifying the post-translational modifications (PTMs) found on the different LH subtypes. However, while a number of PTMs have been identified and their potential nuclear and non-nuclear functions explored in cellular processes, there are very few studies delineating the direct relevance of these PTMs in diseases. In addition, recent whole-genome sequencing of clinical samples from cancer patients and individuals afflicted with Rahman syndrome have identified high-frequency mutations and therefore broadened the perspective of the linker histone mutations in diseases. In this review, we compile the identified PTMs of hLH subtypes, current knowledge of the relevance of hLH PTMs in human diseases, and the correlation of PTMs coinciding with mutations mapped in diseases.

Multiple Genes of Candida albicans Influencing Echinocandin Susceptibility in Caspofungin-Adapted Mutants.

Candida albicans is an opportunistic human fungal pathogen that causes invasive infections in immunocompromised individuals. Despite the high anticandidal activity among the echinocandins (ECNs), a first-line therapy, resistance remains an issue. Furthermore, many clinical isolates display decreased ECN susceptibility, a physiological state which is thought to lead to resistance. Determining the factors that can decrease susceptibility is of high importance. We searched for such factors genome-wide by comparing the transcriptional profiles of five mutants that acquired decreased caspofungin susceptibility in vitro in the absence of canonical FKS1 resistance mutations. The mutants were derived from two genetic backgrounds and arose due to independent mutational events, some with monosomic chromosome 5 (Ch5). We found that the mutants exhibit common transcriptional changes. In particular, all mutants upregulate five genes from Ch2 in concert. Knockout experiments show that all five genes positively influence caspofungin and anidulafungin susceptibility and play a role in regulating the cell wall mannan and glucan contents. The functions of three of these genes, orf19.1766, orf19.6867, and orf19.5833, were previously unknown, and our work expands the known functions of LEU42 and PR26. Importantly, orf19.1766 and LEU42 have no human orthologues. Our results provide important clues as to basic mechanisms of survival in the presence of ECNs while identifying new genes controlling ECN susceptibility and revealing new targets for the development of novel antifungal drugs.

Whole-genome methods to define DNA and histone accessibility and long-range interactions in chromatin.

Defining the genome-wide chromatin landscape has been a goal of experimentalists for decades. Here we review highlights of these efforts, from seminal experiments showing discontinuities in chromatin structure related to gene activation to extensions of these methods elucidating general features of chromatin related to gene states by exploiting deep sequencing methods. We also review chromatin conformational capture methods to identify patterns in long-range interactions between genomic loci.

Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain.

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

A method for assessing histone surface accessibility genome-wide.

The assembly of DNA into nucleosomes and higher order chromatin structures serves not only as a means of compaction but also organizes the genome to facilitate crucial processes such as cell division and regulation of gene expression. Chromatin structure generally limits access to DNA, with the accessibility of DNA in chromatin being regulated through post translational modification of the histone proteins as well as the activity of chromatin remodeling proteins and architectural chromatin factors. There is great interest in assessing chromatin accessibility genome-wide to identify functional elements associated with enhancers, promoters, and other discontinuities in the compacted chromatin structure associated with gene expression. As the vast majority of techniques rely upon assessment of the exposure of the underlying DNA, we describe here a general method that can be used to assess exposure of internal and external histone protein surfaces. We demonstrate the feasibility of this method, in the organism S. cerevisiae. Our method relies on substitution of residues residing on selected histone protein surfaces with cysteine, and assessment of exposure by reaction with a thiol specific reagent, biotin-maleimide. We demonstrate that modified nucleosomes can be efficiently excised from nuclei treated with the reagent via a one-step purification process. After library preparation and deep sequencing, selected nucleosomes are typically ~25-fold enriched over background signals and exhibit phasing with respect to transcription start sites in yeast that is identical to an unselected population.

Creating superhydrophobic and antibacterial surfaces on gold by femtosecond laser pulses.

Femtosecond laser-induced surface structuring is a promising technique for the large-scale formation of nano- and microscale structures that can effectively modify materials' optical, electrical, mechanical, and tribological properties. Here we perform a systematic study on femtosecond laser-induced surface structuring on gold (Au) surface and their effect on both hydrophobicity and bacterial-adhesion properties. We created various structures including subwavelength femtosecond laser-induced periodic surface structures (fs-LIPSSs), fs-LIPSSs covered with nano/microstructures, conic and 1D-rod-like structures ( ≤ 6 µm), and spherical nanostructures with a diameter ≥ 10 nm, by raster scanning the laser beam, at different laser fluences. We show that femtosecond laser processing turns originally hydrophilic Au to a superhydrophobic surface. We determine the optimal conditions for the creation of the different surface structures and explain the mechanism behind the formed structures and show that the laser fluence is the main controlling parameter. We demonstrate the ability of all the formed surface structures to reduce the adhesion of Escherichia coli (E. coli) bacteria and show that fs-LIPSSs enjoys superior antibacterial adhesion properties due to its large-scale surface coverage. Approximately 99.03% of the fs-LIPSSs surface is free from bacterial adhesion. The demonstrated physical inhibition of bacterial colonies and biofilm formation without antibiotics is a crucial step towards reducing antimicrobial-resistant infections.

A nucleosome-free region locally abrogates histone H1-dependent restriction of linker DNA accessibility in chromatin.

Eukaryotic genomes are packaged into linker-oligonucleosome assemblies, providing compaction of genomic DNA and contributing to gene regulation and genome integrity. To define minimal requirements for initial steps in the transition of compact, closed chromatin to a transcriptionally active, open state, we developed a model in vitro system containing a single, unique, "target" nucleosome in the center of a 25-nucleosome array and evaluated the accessibility of the linker DNA adjacent to this target nucleosome. We found that condensation of H1-lacking chromatin results in ∼60-fold reduction in linker DNA accessibility and that mimics of acetylation within all four core histone tail domains of the target nucleosome synergize to increase accessibility ∼3-fold. Notably, stoichiometric binding of histone H1 caused >2 orders of magnitude reduction in accessibility that was marginally diminished by histone acetylation mimics. Remarkably, a nucleosome-free region (NFR) in place of the target nucleosome completely abrogated H1-dependent restriction of linker accessibility in the immediate vicinity of the NFR. Our results suggest that linker DNA is as inaccessible as DNA within the nucleosome core in fully condensed, H1-containing chromatin. They further imply that an unrecognized function of NFRs in gene promoter regions is to locally abrogate the severe restriction of linker DNA accessibility imposed by H1s.

HMGN1 and 2 remodel core and linker histone tail domains within chromatin.

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.

When push comes to shove: SWI/SNF uses a nucleosome to get rid of a nucleosome.

A report in this issue of Molecular Cell (Dechassa et al., 2010) provides evidence that a translocating SWI/SNF-nucleosome complex efficiently displaces neighboring nucleosomes in vitro and may account for SWI/SNF-dependent nucleosome eviction in vivo.

Chromatin structure-dependent conformations of the H1 CTD.

Linker histones are an integral component of chromatin but how these proteins promote assembly of chromatin fibers and higher order structures and regulate gene expression remains an open question. Using Förster resonance energy transfer (FRET) approaches we find that association of a linker histone with oligonucleosomal arrays induces condensation of the intrinsically disordered H1 CTD in a manner consistent with adoption of a defined fold or ensemble of folds in the bound state. However, H1 CTD structure when bound to nucleosomes in arrays is distinct from that induced upon H1 association with mononucleosomes or bare double stranded DNA. Moreover, the H1 CTD becomes more condensed upon condensation of extended nucleosome arrays to the contacting zig-zag form found in moderate salts, but does not detectably change during folding to fully compacted chromatin fibers. We provide evidence that linker DNA conformation is a key determinant of H1 CTD structure and that constraints imposed by neighboring nucleosomes cause linker DNAs to adopt distinct trajectories in oligonucleosomes compared to H1-bound mononucleosomes. Finally, inter-molecular FRET between H1s within fully condensed nucleosome arrays suggests a regular spatial arrangement for the H1 CTD within the 30 nm chromatin fiber.

HMGB1 Stimulates Activity of Polymerase ß on Nucleosome Substrates.

The process of base excision repair (BER) recognizes and repairs small lesions or inappropriate bases on DNA through either a short-patch or long-patch pathway. The enzymes involved in BER have been well-characterized on DNA substrates, and, somewhat surprisingly, many of these enzymes, including several DNA glycosylases, AP endonuclease (APE), FEN1 endonuclease, and DNA ligases, have been shown to have activity on DNA substrates within nucleosomes. DNA polymerase ß (Pol ß), however, exhibits drastically reduced or no activity on nucleosomal DNA. Interestingly, acetylation of Pol ß, by the acetyltransferase p300, inhibits its 5' dRP-lyase activity and presumably pushes repair of DNA substrates through the long-patch base excision repair (LP-BER) pathway. In addition to the major enzymes involved in BER, a chromatin architectural factor, HMGB1, was found to directly interact with and enhance the activity of APE1 and FEN1, and thus may aid in altering the structure of the nucleosome to be more accessible to BER factors. In this work, we investigated whether acetylation of Pol ß, either alone or in conjunction with HMGB1, facilitates its activity on nucleosome substrates. We find acetylated Pol ß exhibits enhanced strand displacement synthesis activity on DNA substrates, but, similar to the unmodified enzyme, has little or no activity on nucleosomes. Preincubation of DNA templates with HMGB1 has little or no stimulatory effect on Pol ß and even is inhibitory at higher concentrations. In contrast, preincubation of nucleosomes with HMGB1 rescues Pol ß gap-filling activity in nucleosomes, suggesting that this factor may help overcome the repressive effects of chromatin.

Linker histones: novel insights into structure-specific recognition of the nucleosome.

Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing the 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, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while highlighting the remaining questions.

Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

Radioresistance of GGG sequences to prompt strand break formation from direct-type radiation damage.

As humans, we are constantly exposed to ionizing radiation from natural, man-made and cosmic sources which can damage DNA, leading to deleterious effects including cancer incidence. In this work, we introduce a method to monitor strand breaks resulting from damage due to the direct effect of ionizing radiation and provide evidence for sequence-dependent effects leading to strand breaks. To analyze only DNA strand breaks caused by radiation damage due to the direct effect of ionizing radiation, we combined an established technique to generate dehydrated DNA samples with a technique to analyze single-strand breaks on short oligonucleotide sequences via denaturing gel electrophoresis. We find that direct damage primarily results in a reduced number of strand breaks in guanine triplet regions (GGG) when compared to isolated guanine (G) bases with identical flanking base context. In addition, we observe strand break behavior possibly indicative of protection of guanine bases when flanked by pyrimidines and sensitization of guanine to strand break when flanked by adenine (A) bases in both isolated G and GGG cases. These observations provide insight into the strand break behavior in GGG regions damaged via the direct effect of ionizing radiation. In addition, this could be indicative of DNA sequences that are naturally more susceptible to strand break due to the direct effect of ionizing radiation.

A distinct switch in interactions of the histone H4 tail domain upon salt-dependent folding of nucleosome arrays.

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes. Likewise, H4 tail-H2A contacts stabilize array folding. However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.

Intra- and inter-nucleosome interactions of the core histone tail domains in higher-order chromatin structure.

Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array-array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, effect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as inter-nucleosomal and inter-array interactions of the H3 and H4 N-terminal tails.