Showing papers on "Histone binding published in 2020"
TL;DR: The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain–histone PTM interactions.
Abstract: Plant homeodomain (PHD) fingers are central “readers” of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete. We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk. The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain–histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.
53 citations
TL;DR: It is shown that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially, and that dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer.
Abstract: How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication-linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.
50 citations
TL;DR: Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage, and as compared with WT mice, Clec2D-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model.
41 citations
TL;DR: Molecular dynamics simulations of chromatosomes with the globular domain of two linker histone variants, generic H1 (genGH1) and H1.0), to determine how their differences influence chromatosome structures, energetics and dynamics show that both unbound linker Histone variants adopt a single compact conformation.
Abstract: Linker histones are epigenetic regulators that bind to nucleosomes and alter chromatin structures and dynamics. Biophysical studies have revealed two binding modes in the linker histone/nucleosome complex, the chromatosome, where the linker histone is either centered on or askew from the dyad axis. Each has been posited to have distinct effects on chromatin, however the molecular and thermodynamic mechanisms that drive them and their dependence on linker histone compositions remain poorly understood. We present molecular dynamics simulations of chromatosomes with the globular domain of two linker histone variants, generic H1 (genGH1) and H1.0 (GH1.0), to determine how their differences influence chromatosome structures, energetics and dynamics. Results show that both unbound linker histones adopt a single compact conformation. Upon binding, DNA flexibility is reduced, resulting in increased chromatosome compaction. While both variants enthalpically favor on-dyad binding, energetic benefits are significantly higher for GH1.0, suggesting that GH1.0 is more capable than genGH1 of overcoming the large entropic reduction required for on-dyad binding which helps rationalize experiments that have consistently demonstrated GH1.0 in on-dyad states but that show genGH1 in both locations. These simulations highlight the thermodynamic basis for different linker histone binding motifs, and details their physical and chemical effects on chromatosomes.
32 citations
TL;DR: Near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone suggest that nucleosomes chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.
Abstract: Chromosome structure at the multi-nucleosomal level has remained ambiguous in spite of its central role in epigenetic regulation and genome dynamics. Recent investigations of chromatin architecture portray diverse modes of interaction within and between nucleosome chains, but how this is realized at the atomic level is unclear. Here we present near-atomic resolution crystal structures of nucleosome fibres that assemble from cohesive-ended dinucleosomes with and without linker histone. As opposed to adopting folded helical '30 nm' structures, the fibres instead assume open zigzag conformations that are interdigitated with one another. Zigzag conformations obviate extreme bending of the linker DNA, while linker DNA size (nucleosome repeat length) dictates fibre configuration and thus fibre-fibre packing, which is supported by variable linker histone binding. This suggests that nucleosome chains have a predisposition to interdigitate with specific characteristics under condensing conditions, which rationalizes observations of local chromosome architecture and the general heterogeneity of chromatin structure.
23 citations
TL;DR: It is argued that such computations on multiple spatial and temporal scales will be increasingly important for the integration of genomic, epigenomic, and biophysical data on chromatin structure and related cellular processes.
Abstract: Chromatin structure, dynamics, and function are being intensely investigated by a variety of methods, including microscopy, X-ray diffraction, nuclear magnetic resonance, biochemical crosslinking, chromosome conformation capture, and computation. A range of experimental techniques combined with modeling is clearly valuable to help interpret experimental data and, importantly, generate configurations and mechanisms related to the 3D organization and function of the genome. Contact maps, in particular, as obtained by a variety of chromosome conformation capture methods, are of increasing interest due to their implications on genome structure and regulation on many levels. In this perspective, using seven examples from our group's studies, we illustrate how molecular modeling can help interpret such experimental data. Specifically, we show how computed contact maps related to experimental systems can be used to explain structures of nucleosomes, chromatin higher-order folding, domain segregation mechanisms, gene organization, and the effect on chromatin structure of external and internal fiber parameters, such as nucleosome positioning, presence of nucleosome free regions, histone posttranslational modifications, and linker histone binding. We argue that such computations on multiple spatial and temporal scales will be increasingly important for the integration of genomic, epigenomic, and biophysical data on chromatin structure and related cellular processes.
17 citations
TL;DR: This work shows that Swc5 uses a tandem DEF/Y motif within an intrinsically disordered region to engage the A-B dimer, providing the molecular basis for a general strategy used repeatedly during eukaryotic evolution to mobilize histones in various genomic functions.
Abstract: The SWR complex edits the histone composition of nucleosomes at promoters to facilitate transcription by replacing the two nucleosomal H2A-H2B (A-B) dimers with H2A.Z-H2B (Z-B) dimers. Swc5, a subunit of SWR, binds to A-B dimers, but its role in the histone replacement reaction was unclear. In this study, we showed that Swc5 uses a tandem DEF/Y motif within an intrinsically disordered region to engage the A-B dimer. A 2.37-A X-ray crystal structure of the histone binding domain of Swc5 in complex with an A-B dimer showed that consecutive acidic residues and flanking hydrophobic residues of Swc5 form a cap over the histones, excluding histone-DNA interaction. Mutations in Swc5 DEF/Y inhibited the nucleosome editing function of SWR in vitro. Swc5 DEF/Y interacts with histones in vivo, and the extent of this interaction is dependent on the remodeling ATPase of SWR, supporting a model in which Swc5 acts as a wedge to promote A-B dimer eviction. Given that DEF/Y motifs are found in other evolutionary unrelated chromatin regulators, this work provides the molecular basis for a general strategy used repeatedly during eukaryotic evolution to mobilize histones in various genomic functions.
17 citations
TL;DR: This work combines extensive molecular dynamics simulations, Markov state modeling and structural data to reveal a novel and transiently formed state that is conserved across all BD families and suggests that this novel state is an allosteric regulatory switch for BDs, potentially related to a recently unveiled BD-DNA binding mode.
Abstract: Bromodomains (BDs) are small protein modules that interact with acetylated marks in histones. These post-translational modifications are pivotal to regulate gene expression, making BDs promising targets to treat several diseases. While the general structure of BDs is well known, their dynamical features and their interplay with other macromolecules are poorly understood, hampering the rational design of potent and selective inhibitors. Here we combine extensive molecular dynamics simulations, Markov state modeling and structural data to reveal a novel and transiently formed state that is conserved across all BD families. It involves the breaking of two backbone hydrogen bonds that anchor the ZA-loop with the αA helix, opening a cryptic pocket that partially occludes the one associated with histone binding. Our results suggest that this novel state is an allosteric regulatory switch for BDs, potentially related to a recently unveiled BD-DNA binding mode.
11 citations
TL;DR: The findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency, and surprise, sustained H3K36me3 deposition is dependent onHP1γ.
Abstract: The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm. Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency.
10 citations
TL;DR: It is demonstrated that LLPS of Swi6 contributes to the emergent mechanical properties of nuclei and highlights that phase-separated domains can do mechanical work.
Abstract: Liquid-liquid phase separation (LLPS) has emerged as a major driver of cellular organization However, it remains unexplored whether the mechanical properties of LLPS domains are functionally important The heterochromatin protein HP1-α (and the orthologous Swi6 in S pombe) is capable of LLPS in vitro and promotes formation of LLPS heterochromatin domains in vivo Here, we demonstrate that LLPS of Swi6 contributes to the emergent mechanical properties of nuclei Using nuclear fluctuation analysis in live cells and force spectroscopy of isolated nuclei, we find that disrupting histone H3K9 methylation or depleting Swi6 compromises nuclear stiffness, while heterochromatin spreading through loss of the H3K9 demethylase, Epe1, increases nuclear stiffness Leveraging a separation−of−function allele, we demonstrate that phase separation of Swi6−not only its histone binding or dimerization−is essential for Swi6′s mechanical role These findings demonstrate that altering chromatin state has mechanical consequences and highlights that phase-separated domains can do mechanical work
9 citations
TL;DR: This article details how Size Exclusion Chromatography coupled to Multi-Angle Light Scattering (MALS) can be used to study histone chaperones and their complexes and finds SEC-MALS to be a robust and user-friendly approach for characterizing histone-binding proteins and their complex.
TL;DR: It is suggested that a complex of Tif1 family members, each of which possesses different specificity and efficiency, contributes to the silencing of retroviral sequences in ESC.
Abstract: Embryonic stem cells (ESC) have the ability to epigenetically silence endogenous and exogenous retroviral sequences. Trim28 plays an important role in establishing this silencing, but less is known about the role other Trim proteins play. The Tif1 family is a sub-group of the Trim family, which possess histone binding ability in addition to the distinctive RING domain. Here, we have examined the interaction between three Tif1 family members, namely Trim24, Trim28 and Trim33, and their function in retroviral silencing. We identify a complex formed in ESC, comprised of these three proteins. We further show that when Trim33 is depleted, the complex collapses and silencing efficiency of both endogenous and exogenous sequences is reduced. Similar transcriptional activation takes place when Trim24 is depleted. Analysis of the H3K9me3 chromatin modification showed a decrease in this repressive mark, following both Trim24 and Trim33 depletion. As Trim28 is an identified binding partner of the H3K9 methyltransferase ESET, this further supports the involvement of Trim28 in the complex. The results presented here suggest that a complex of Tif1 family members, each of which possesses different specificity and efficiency, contributes to the silencing of retroviral sequences in ESC.
TL;DR: Results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation.
Abstract: Pygopus 2 (Pygo2) is a co-activator of Wnt/β-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing It remains unknown whether the Pygo2- H3K4me2/3 association has a functional relevance in breast cancer progression in vivo To investigate the functional relevance of histone binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due in part to decreased canonical Wnt/β-catenin signaling RNA and ATAC sequencing analyses of tumor-derived cell lines revealed downregulation of TGFβ signaling and upregulation of differentiation pathways such as PDGFR signaling Increased differentiation correlated with a luminal cell fate which could be reversed by inhibition of PDGFR activity Mechanistically, the Pygo2-histone interaction potentiated Wnt/ β-catenin signaling in part by repressing the expression of Wnt signaling antagonists Furthermore, Pygo2 and β-catenin regulated the expression of miR-29 family members which in turn repressed PDGFR expression to promote de- differentiation of wildtype Pygo2 mammary epithelial tumor cells Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving de-differentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways which attenuate primary tumor growth and metastasis formation Interfering with the Pygo2- H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer
TL;DR: The N-terminal regions of histone proteins (tails) are dynamic elements that protrude from the nucleosome and are involved in many aspects of chromatin organization and post-translational modifications present on these regions contribute to transcriptional regulation.
TL;DR: Molecular dynamics simulations of octa-nucleosome arrays with and without the H1 linker histone are studied to better understand the mechanisms dictating the structure of the chromatin fiber, suggesting that H1-saturated chromatin fibers are better suited to transferring long-range epigentic information.
Abstract: Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction. Significance Linker histones dynamically bind to DNA in chromatin fibers and serve as epigentic regulators. However, the extent to which they influence the gamut of chromatin architecture is still not well understood. Using molecular dynamics simulations, we studied compact octa-nucleosome arrays with and without the H1 linker histone to better understand the mechanisms dictating the structure of the chromatin fiber. Inclusion of H1 results in stabilization of the compact chromatin structure, while its removal results in a major conformational change towards an untwisted ladder-like state. The increased rigidity and correlations within the H1-bound array suggests that H1-saturated chromatin fibers are better suited to transferring long-range epigentic information.
TL;DR: It is demonstrated that ZMYND8 mediates all its anti-tumor roles through a common dual-histone mark binding to H4K16Ac and H3K36Me2, and is proposed as a potential therapeutic candidate for differentiation therapy in breast cancer.
Abstract: Accumulating evidences indicate the involvement of epigenetic deregulations in cancer. While some epigenetic regulators with aberrant functions in cancer are targeted for improving therapeutic outcome in patients, reinstating the functions of tumor-suppressor-like epigenetic regulators might further potentiate anti-cancer therapies. Epigenetic reader zinc-finger MYND-type-containing 8 (ZMYND8) has been found to be endowed with multiple anti-cancer functions like inhibition of tumor cell migration and proliferation. Here, we report another novel tumor suppressor role of ZMYND8 as an inducer of differentiation in breast cancer cells, by upregulating differentiation genes. Interestingly, we also demonstrated that ZMYND8 mediates all its anti-tumor roles through a common dual-histone mark binding to H4K16Ac and H3K36Me2. We validated these findings by both biochemical and biophysical analyses. Furthermore, we also confirmed the differentiation-inducing potential of ZMYND8 in vivo, using 4T1 murine breast cancer model in Balb/c mice. Differentiation therapy holds great promise in cancer therapy, since it is non-toxic and makes the cancer cells therapy-sensitive. In this scenario, we propose epigenetic reader ZMYND8 as a potential therapeutic candidate for differentiation therapy in breast cancer.
TL;DR: In this paper, the authors provided proof of concept that the tandem PHD finger of nuclear receptor-binding SET (Su(var)3-9, Enhancer of zeste, Trithorax) domain protein 1 (PHDVC5HCHNSD1) is ligandable.
Abstract: PHD fingers are small chromatin binding domains, that alone or in tandem work as versatile interaction platforms for diversified activities, ranging from the decoding of the modification status of histone tails to the specific recognition of non-histone proteins. They play a crucial role in their host protein as mutations thereof cause several human malignancies. Thus, PHD fingers are starting to be considered as valuable pharmacological targets. While inhibitors or chemical probes of the histone binding activity of PHD fingers are emerging, their druggability as non-histone interaction platform is still unexplored. In the current study, using a computational and experimental pipeline, we provide proof of concept that the tandem PHD finger of Nuclear receptor-binding SET (Su(var)3-9, Enhancer of zeste, Trithorax) domain protein 1 (PHDVC5HCHNSD1) is ligandable. Combining virtual screening of a small subset of the ZINC database (Zinc Drug Database, ZDD, 2924 molecules) to NMR binding assays and ITC measurements, we have identified Mitoxantrone dihydrochloride, Quinacrine dihydrochloride and Chloroquine diphosphate as the first molecules able to bind to PHDVC5HCHNSD1 and to reduce its documented interaction with the Zinc finger domain (C2HRNizp1) of the transcriptional repressor Nizp1 (NSD1-interacting Zn-finger protein). These results pave the way for the design of small molecules with improved effectiveness in inhibiting this finger-finger interaction.
TL;DR: It is proposed that the novel lead compound NS5 has an inhibitory effect on BRD2 in glioblastoma.
Abstract: Glioblastoma (GBM) is the most common primary brain malignancy, rarely amenable to treatment with a high recurrence rate. GBM are prone to develop resistance to the current repertoire of drugs, including the first-line chemotherapeutic agents with frequent recurrence, limiting therapeutic success. Recent clinical data has evidenced the BRD2 and BRD4 of the BET family proteins as the new druggable targets against GBM. In this relevance, we have discovered a compound (pyrano 1,3 oxazine derivative; NSC 328111; NS5) as an inhibitor of hBRD2 by the rational structure-based approach. The crystal structure of the complex, refined to 1.5 A resolution, revealed that the NS5 ligand significantly binds to the N-terminal bromodomain (BD1) of BRD2 at the acetylated (Kac) histone binding site. The quantitative binding studies, by SPR and MST assay, indicate that NS5 binds to BD1 of BRD2 with a KD value of ∼1.3 µM. The cell-based assay, in the U87MG glioma cells, confirmed that the discovered compound NS5 significantly attenuated proliferation and migration. Furthermore, evaluation at the translational level established significant inhibition of BRD2 upon treatment with NS5. Hence, we propose that the novel lead compound NS5 has an inhibitory effect on BRD2 in glioblastoma.
TL;DR: The results show that the presence of PSO (no matter if there is ultra violet light treatment) can increase the overall probability of hist one binding to dsDNA while lowering the selectivity of histone binding to the specific DNA sequence in vitro.
Abstract: The investigation of the DNA-histone interactions and factors that affect such interactions in the nucleosome is essential for understanding the role of chromatin organization in all cellular processes involved in the repair, transcription, and replication of the eukaryotic genome. As a kind of photosensitive molecule, psoralen (PSO) is used in the treatment of skin disease with ultraviolet light (PSO and ultra violet light, type A). The effect of treatment is remarkable, but the side effect is also obvious. PSO can be embedded in a 5' TA sequence in double-stranded DNA (dsDNA), and dsDNA is mainly wrapped around a histone octamer to form a nucleosome structure in human cells. Therefore, it is very necessary to explore the influence of PSO on DNA-histone interactions. To this end, the binding specificity and mode of DNA and histone in the presence or absence of PSO are investigated systematically. The results show that the presence of PSO (no matter if there is ultra violet light treatment) can increase the overall probability of histone binding to dsDNA while lowering the selectivity of histone binding to the specific DNA sequence in vitro. In addition, the increase of solution ionic strength can lower the ratio of histone binding to nonspecific DNA.
31 Jan 2020
TL;DR: Investigating the effect of peptide AEDL on chromatin structure in tobacco provides novel insight into the mechanism of gene regulation by short peptides and has implications for breeding more resistant or productive varieties of tobacco and other crops.
Abstract: Eukaryotic DNA is tightly packed into chromatin, a DNA–protein structure that exists as transcriptionally permissive euchromatin or repressive heterochromatin. Post-translational modification of histones plays a key role in regulating chromatin dynamics. Short peptides derived from various sources are known to function as epigenetic modulators; however, their mechanisms of action are poorly understood. We addressed this issued by investigating the effect of peptide AEDL on chromatin structure in tobacco (Nicotiana tabacum L.), a commercially important plant species. The chromatin of tobacco interphase cells is characterized by the presence of zones of transcriptionally active domains and particular domains of condensed chromatin of cells that partially coincide with heterochromatin zones. Chromatin decondensation and the formation of euchromatin, accompanied by the activation of genes expression activity, are a determining factor in responses to stressful effects. Our results show that plants grown in the presence of 10−7 M peptide AEDL transformed condensed chromatin domains from 45% in control cells to 25%. Histone modifications, which constitute the so-called histone code, play a decisive role in the control of chromatin structure. Fluorescence quenching experiments using fluorescein isothiocyanate-labeled histones revealed that the linker histone H1 and complexes of core H3 and H1 histones with DNA bound to peptide AEDL in a 1: 1 molar ratio. The peptide was found to bind to the N-terminal lysine residue of H1 and the lysine residue at position 36 of the H3 C terminus. These interactions of histones H1 and H3 with AEDL peptide loosened the tightly packed chromatin structure, getting transcriptionally active euchromatin. Our findings provide novel insight into the mechanism of gene regulation by short peptides and have implications for breeding more resistant or productive varieties of tobacco and other crops.
TL;DR: Since they are canonical histone transporters providing effective chromatin remodeling, NAP family proteins are involved in neuronal differentiation, spermatogenesis, and long-term memory formation, which indicates the importance of this family in ontogenesis.
Abstract: Histone chaperones are a class of proteins that bind and transport histones, preventing their chaotic aggregation when forming nucleosomes. Histone chaperones of the NAP (Nucleosome Assembly Protein) family contain a highly conserved central NAP domain, which is necessary for histone binding and nucleosome assembly. They are an essential component in creating and maintaining the eukaryotic chromatin dynamics on which the transcription of many genes depends. The review considers the NAP family of proteins and its specific representatives: NAP1, NAP2, and CG5017/Hanabi. Since they are canonical histone transporters providing effective chromatin remodeling, NAP family proteins are involved in neuronal differentiation, spermatogenesis, and long-term memory formation, which indicates the importance of this family in ontogenesis.
TL;DR: PhAST (Photochemical Analysis of Structural Transitions) is used to monitor at the base pair level, structural alterations induced all along DNA upon histone binding or release, offering the first consistent, detailed comparison of nucleosome assembly and disassembly in vitro.
Abstract: Nucleosome assembly and disassembly play a central role in the regulation of gene expression. Here we use PhAST (Photochemical Analysis of Structural Transitions) to monitor at the base pair level, structural alterations induced all along DNA upon histone binding or release. By offering the first consistent, detailed comparison of nucleosome assembly and disassembly in vitro, we are able to reveal similarities and differences between the two processes. We identify multiple intermediate states characterised by specific PhAST signatures; revealing a complexity that goes beyond the known sequential events involving (H3-H4)2 tetramer and H2A-H2B heterodimers. Such signatures localise and quantify the extent of the asymmetry of DNA/histone interactions with respect to the nucleosome dyad. This asymmetry is therefore defined by the localisation and amplitude of the signals. The localisation of the signal is consistent between assembly and disassembly and dictated by the DNA sequence. However, the amplitude component of this asymmetry not only evolves during the assembly and disassembly but does so differently between the two processes.