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The highly positively charged and intrinsically disordered H1 C-terminal domain (CTD) undergoes extensive condensation upon binding to nucleosomes, and stabilizes nucleosomes and higher-order chromatin structures but its interactions in chromatin are not well defined. Using single-molecule FRET we found that about half of the H1 CTDs in H1-nucleosome complexes exhibit well-defined FRET values indicative of distinct, static conformations, while the remainder of the population exhibits exchange between multiple defined FRET structures. Moreover, crosslinking studies indicate that the first 30 residues of the H1 CTD participate in relatively localized contacts with the first â¼25 bp of linker DNA, and that two separate regions in the CTD contribute to H1-dependent organization of linker DNA. Finally, we show that acetylation mimetics within the histone H3 tail markedly reduce the overall extent of H1 CTD condensation and significantly increase the fraction of H1 CTDs undergoing dynamic exchange between FRET states. Our results indicate the nucleosome-bound H1 CTD adopts loosely defined structures that exhibit significantly enhanced dynamics and decondensation upon epigenetic acetylation within the H3 tail.
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Histonas , Nucleosomas , Histonas/genética , Código de Histonas , Procesamiento Proteico-Postraduccional , CromatinaRESUMEN
Despite their importance, how linker histone H1s interact in chromatin and especially how the highly positively charged and intrinsically disordered H1 C-terminal domain (CTD) binds and stabilizes nucleosomes and higher-order chromatin structures remains unclear. Using single-molecule FRET we found that about half of the H1 CTDs in H1-nucleosome complexes exhibit well-defined FRET values indicative of distinct, static conformations, while the remainder of the population exhibits dynamically changing values, similar to that observed for H1 in the absence of nucleosomes. We also find that the first 30 residues of the CTD participate in relatively localized interactions with the first â¼20 bp of linker DNA, and that two separate regions in the CTD contribute to H1-dependent organization of linker DNA, consistent with some non-random CTD-linker DNA interactions. Finally, our data show that acetylation mimetics within the histone H3 tail induce decondensation and enhanced dynamics of the nucleosome-bound H1 CTD. (148 words).
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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.
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Cromatina , Histonas , Humanos , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Espectrometría de Masas , MutaciónRESUMEN
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.
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Candida albicans , Equinocandinas , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Caspofungina/farmacología , Farmacorresistencia Fúngica/genética , Equinocandinas/farmacología , Proteínas Fúngicas/genética , Lipopéptidos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
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.
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Ensamble y Desensamble de Cromatina , Histonas/química , Nucleosomas/química , Adenosina Trifosfato/metabolismo , ADN/genética , ADN/metabolismo , Histonas/metabolismo , Estreptavidina/metabolismoRESUMEN
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.
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Histonas , Nucleosomas , Animales , Cromatina , ADN/química , Histonas/metabolismo , Fosforilación , Xenopus laevis/metabolismoRESUMEN
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.
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Cromatina , Histonas , Cromosomas , ADN/genética , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Análisis de Secuencia de ADNRESUMEN
Candida albicans is a prevalent fungal pathogen of humans. Understanding the development of decreased susceptibility to ECN drugs of this microbe is of substantial interest, as it is viewed as an intermediate step allowing the formation of FKS1 resistance mutations. We used six previously characterized mutants that decreased caspofungin susceptibility either by acquiring aneuploidy of chromosome 5 (Ch5) or by aneuploidy-independent mechanisms. When we exposed these caspofungin-adapted mutants to caspofungin again, we obtained 60 evolved mutants with further decreases in caspofungin susceptibility, as determined with CLSI method. We show that the initial adaptation to caspofungin is coupled with the adaptation to other ECNs, such as micafungin and anidulafungin, in mutants with no ploidy change, but not in aneuploid mutants, which become more susceptible to micafungin and anidulafungin. Furthermore, we find that the initial mechanism of caspofungin adaptation determines the pattern of further adaptation as parentals with no ploidy change further adapt to all ECNs by relatively small decreases in susceptibility, whereas aneuploid parentals adapt to all ECNs, primarily by large decrease in susceptibilities. Our data suggest that either distinct or common mechanisms can govern adaptation to different ECNs.
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Physarum polycephalum belongs to Mycetozoans, a phylogenetic clade apart from the animal, plant and fungus kingdoms. Histones are nuclear proteins involved in genome organization and regulation and are among the most evolutionary conserved proteins within eukaryotes. Therefore, this raises the question of their conservation in Physarum and the position of this organism within the eukaryotic phylogenic tree based on histone sequences. We carried out a comprehensive study of histones in Physarum polycephalum using genomic, transcriptomic and molecular data. Our results allowed to identify the different isoforms of the core histones H2A, H2B, H3 and H4 which exhibit strong conservation of amino acid residues previously identified as subject to post-translational modifications. Furthermore, we also identified the linker histone H1, the most divergent histone, and characterized a large number of its PTMs by mass spectrometry. We also performed an in-depth investigation of histone genes and transcript structures. Histone proteins are highly conserved in Physarum and their characterization will contribute to a better understanding of the polyphyletic Mycetozoan group. Our data reinforce that P. polycephalum is evolutionary closer to animals than plants and located at the crown of the eukaryotic tree. Our study provides new insights in the evolutionary history of Physarum and eukaryote lineages.
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Considerable progress has been made recently in defining the interactions of linker histones (H1s) within nucleosomes. Major advancements include atomic resolution structures of the globular domain of full-length H1s in the context of nucleosomes containing full-length linker DNA. Although these studies have led to a detailed understanding of the interactions and dynamics of H1 globular domains in the canonical on-dyad nucleosome binding pocket, more information regarding the intrinsically disordered N-terminal and C-terminal domains is needed. In this review, we highlight studies supporting our current understanding of the structures and interactions of the N-terminal, globular, and C-terminal domains of linker histones within the nucleosome.
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Histonas , Nucleosomas , ADN/genética , ADN/metabolismo , Histonas/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: The vast majority of methods available to characterize genome-wide chromatin structure exploit differences in DNA accessibility to nucleases or chemical crosslinking. We developed a novel method to gauge genome-wide accessibility of histone protein surfaces within nucleosomes by assessing reactivity of engineered cysteine residues with a thiol-specific reagent, biotin-maleimide (BM). RESULTS: Yeast nuclei were obtained from cells expressing the histone mutant H2B S116C, in which a cysteine resides near the center of the external flat protein surface of the nucleosome. BM modification revealed that nucleosomes are generally equivalently accessible throughout the S. cerevisiae genome, including heterochromatic regions, suggesting limited, higher-order chromatin structures in which this surface is obstructed by tight nucleosome packing. However, we find that nucleosomes within 500 bp of transcription start sites exhibit the greatest range of accessibility, which correlates with the density of chromatin remodelers. Interestingly, accessibility is not well correlated with RNA polymerase density and thus the level of gene expression. We also investigated the accessibility of cysteine mutations designed to detect exposure of histone surfaces internal to the nucleosome thought to be accessible in actively transcribed genes: H3 102, is at the H2A-H2B dimer/H3-H4 tetramer interface, and H3 A110C, resides at the H3-H3 interface. However, in contrast to the external surface site, we find that neither of these internal sites were found to be appreciably exposed. CONCLUSIONS: Overall, our finding that nucleosomes surfaces within S. cerevisiae chromatin are equivalently accessible genome-wide is consistent with a globally uncompacted chromatin structure lacking substantial higher-order organization. However, we find modest differences in accessibility that correlate with chromatin remodelers but not transcription, suggesting chromatin poised for transcription is more accessible than actively transcribed or intergenic regions. In contrast, we find that two internal sites remain inaccessible, suggesting that such non-canonical nucleosome species generated during transcription are rapidly and efficiently converted to canonical nucleosome structure and thus not widely present in native chromatin.
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Histonas , Nucleosomas , Cromatina/genética , Ensamble y Desensamble de Cromatina , Histonas/genética , Histonas/metabolismo , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
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Histonas/química , Proteínas Intrínsecamente Desordenadas/química , Acetilación , ADN/química , Glutamina/química , Histonas/genética , Histonas/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Lisina/metabolismo , Modelos Moleculares , Nucleosomas/metabolismo , Dominios Proteicos , Eliminación de SecuenciaRESUMEN
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.
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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.
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Secuenciación de Inmunoprecipitación de Cromatina/métodos , Cromatina/metabolismo , Histonas/análisis , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , ADN de Hongos/metabolismo , Estudios de Factibilidad , Genoma Fúngico , Histonas/genética , Histonas/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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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 â¼60-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 â¼3-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.
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Cromatina/metabolismo , ADN/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Acetilación , Animales , Ensamble y Desensamble de Cromatina/fisiología , Xenopus laevisRESUMEN
Candida albicans is a diploid fungus and a predominant opportunistic human pathogen. Notably, C. albicans employs reversible chromosomal aneuploidies as a means of survival in adverse environments. We previously characterized transcription on the monosomic chromosome 5 (Ch5) that arises with adaptation to growth on the toxic sugar sorbose in the mutant Sor125(55). We now extend this analysis to the trisomic hybrid Ch4/7 within Sor125(55) and a diverse group of three mutants harboring a single Ch5. We find a similar pattern of transcriptional changes on either type of aneuploid chromosome within these mutants wherein expression of many genes follows chromosome ploidy, consistent with a direct mechanism to regulate genes important for adaptation to growth. In contrast, a significant number of genes are expressed at the disomic level, implying distinct mechanisms compensating for gene dose on monosomic or trisomic chromosomes consistent with maintaining cell homeostasis. Finally, we find evidence for an additional mechanism that elevates expression of genes on normal disomic Ch4 and Ch7 in mutants to levels commensurate with that found on the trisomic Ch4/7b in Sor125(55). Several of these genes are similarly differentially regulated among mutants, suggesting they play key functions in either maintaining aneuploidy or adaptation to growth conditions.
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Adaptación Biológica , Aneuploidia , Candida albicans/genética , Cromosomas Fúngicos , Regulación de la Expresión Génica , Sorbosa/toxicidad , Transcripción Genética , Candida albicans/efectos de los fármacosRESUMEN
Combinatorial polyvalent contacts of histone-binding domains or readers commonly mediate localization and activities of chromatin-associated proteins. A pair of readers, the PHD fingers of the protein CHD4, has been shown to bivalently recognize histone H3 tails. Here we describe a mechanism by which these linked but independent readers bind to the intact nucleosome core particle (NCP). Comprehensive NMR, chemical reactivity, molecular dynamics, and fluorescence analyses point to the critical roles of intra-nucleosomal histone-DNA interactions that reduce the accessibility of H3 tails in NCP, the nucleosomal DNA, and the linker between readers in modulating nucleosome- and/or histone-binding activities of the readers. We show that the second PHD finger of CHD4 initiates recruitment to the nucleosome, however both PHDs are required to alter the NCP dynamics. Our findings reveal a distinctive regulatory mechanism for the association of paired readers with the nucleosome that provides an intricate balance between cooperative and individual activities of the readers.
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Histonas/metabolismo , Nucleosomas/metabolismo , Sitios de Unión , ADN/metabolismo , Polarización de Fluorescencia , Histonas/química , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Espectroscopía de Resonancia Magnética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Simulación de Dinámica Molecular , Nucleosomas/químicaRESUMEN
After the publication of this work [1], it was noticed that an initial was missing from the author name: Jeffrey Hayes. His name should be written as: Jeffrey J. Hayes.
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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.