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The hierarchical packaging of chromatin fibers plays a critical role in gene regulation. The 30-nm chromatin fibers, a central-level structure bridging nucleosomal arrays to higher-order organizations, function as the first level of transcriptional dormant chromatin. The dynamics of 30-nm chromatin fiber play a crucial role in biological processes related to DNA. Here, we report a 3.6-angstrom resolution cryogenic electron microscopy structure of H5-bound dodecanucleosome, i.e., the chromatin fiber reconstituted in the presence of linker histone H5, which shows a two-start left-handed double helical structure twisted by tetranucleosomal units. An atomic structural model of the H5-bound chromatin fiber, including an intact chromatosome, is built, which provides structural details of the full-length linker histone H5, including its N-terminal domain and an HMG-motif-like C-terminal domain. The chromatosome structure shows that H5 binds the nucleosome off-dyad through a three-contact mode in the chromatin fiber. More importantly, the H5-chromatin structure provides a fine molecular basis for the intra-tetranucleosomal and inter-tetranucleosomal interactions. In addition, we systematically validated the physiological functions and structural characteristics of the tetranucleosomal unit through a series of genetic and genomic studies in Saccharomyces cerevisiae and in vitro biophysical experiments. Furthermore, our structure reveals that multiple structural asymmetries of histone tails confer a polarity to the chromatin fiber. These findings provide structural and mechanistic insights into how a nucleosomal array folds into a higher-order chromatin fiber with a polarity in vitro and in vivo.
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Mouse FOXA1 and GATA4 are prototypes of pioneer factors, initiating liver cell development by binding to the N1 nucleosome in the enhancer of the ALB1 gene. Using cryoelectron microscopy (cryo-EM), we determined the structures of the free N1 nucleosome and its complexes with FOXA1 and GATA4, both individually and in combination. We found that the DNA-binding domains of FOXA1 and GATA4 mainly recognize the linker DNA and an internal site in the nucleosome, respectively, whereas their intrinsically disordered regions interact with the acidic patch on histone H2A-H2B. FOXA1 efficiently enhances GATA4 binding by repositioning the N1 nucleosome. In vivo DNA editing and bioinformatics analyses suggest that the co-binding mode of FOXA1 and GATA4 plays important roles in regulating genes involved in liver cell functions. Our results reveal the mechanism whereby FOXA1 and GATA4 cooperatively bind to the nucleosome through nucleosome repositioning, opening chromatin by bending linker DNA and obstructing nucleosome packing.
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Microscopía por Crioelectrón , Factor de Transcripción GATA4 , Factor Nuclear 3-alfa del Hepatocito , Nucleosomas , Unión Proteica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-alfa del Hepatocito/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Nucleosomas/ultraestructura , Animales , Factor de Transcripción GATA4/metabolismo , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/química , Ratones , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Histonas/química , Sitios de Unión , ADN/metabolismo , ADN/genética , ADN/química , Ensamble y Desensamble de Cromatina , HumanosRESUMEN
In recent decades, the atmospheric moisture capacity has increased globally in concert with global warming, with a particularly notable warming trend in Arctic regions. However, due to limited observational data, the variation and causes of polar precipitation, especially large-scale precipitation events associated with Arctic cyclones, remain unclear. In this paper, GPM satellite data are compared with ERA5 reanalysis data to explore the characteristics of summer precipitation at the northern margin of the Eurasian region (NMER) and the influence of cyclone activity on precipitation. It is revealed that high precipitation values in the Arctic region, as indicated by the GPM and ERA5 data, are mainly concentrated at the NMER. However, the GPM data show an overall larger precipitation amount, while the station observations more closely agree with the ERA5 precipitation changes at the NMER. The cyclone identification results indicate that summer cyclones at the NMER are mainly distributed in the Barents, Kara and Laptev Seas, and the precipitation contribution rate of ERA5-derived cyclones is 37.35%, which is significantly higher than that of GPM-derived cyclones (29.47%). Furthermore, high cyclone activity results in more intense precipitation, with the top 5% of the strongest cyclones contributing 60% (GPM) and 40% (ERA5) to the total cyclonic precipitation.
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BACKGROUND: Numerous models have been developed to predict acute kidney injury (AKI) after noncardiac surgery, yet there is a lack of independent validation and comparison among them. METHODS: We conducted a systematic literature search to review published risk prediction models for AKI after noncardiac surgery. An independent external validation was performed using a retrospective surgical cohort at a large Chinese hospital from January 2019 to October 2022. The cohort included patients undergoing a wide range of noncardiac surgeries with perioperative creatinine measurements. Postoperative AKI was defined according to the Kidney Disease Improving Global Outcomes creatinine criteria. Model performance was assessed in terms of discrimination (area under the receiver operating characteristic curve, AUROC), calibration (calibration plot), and clinical utility (net benefit), before and after model recalibration through intercept and slope updates. A sensitivity analysis was conducted by including patients without postoperative creatinine measurements in the validation cohort and categorising them as non-AKI cases. RESULTS: Nine prediction models were evaluated, each with varying clinical and methodological characteristics, including the types of surgical cohorts used for model development, AKI definitions, and predictors. In the validation cohort involving 13,186 patients, 650 (4.9%) developed AKI. Three models demonstrated fair discrimination (AUROC between 0.71 and 0.75); other models had poor or failed discrimination. All models exhibited some miscalibration; five of the nine models were well-calibrated after intercept and slope updates. Decision curve analysis indicated that the three models with fair discrimination consistently provided a positive net benefit after recalibration. The results were confirmed in the sensitivity analysis. CONCLUSIONS: We identified three models with fair discrimination and potential clinical utility after recalibration for assessing the risk of acute kidney injury after noncardiac surgery.
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Lesión Renal Aguda , Complicaciones Posoperatorias , Humanos , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/etiología , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/epidemiología , Medición de Riesgo/métodos , Estudios Retrospectivos , Estudios de Cohortes , Creatinina/sangre , Procedimientos Quirúrgicos Operativos/efectos adversos , Persona de Mediana Edad , Masculino , Femenino , Factores de Riesgo , AncianoRESUMEN
Pioneer transcription factors are vital for cell fate changes. PU.1 and C/EBPα work together to regulate hematopoietic stem cell differentiation. However, how they recognize in vivo nucleosomal DNA targets remains elusive. Here we report the structures of the nucleosome containing the mouse genomic CX3CR1 enhancer DNA and its complexes with PU.1 alone and with both PU.1 and the C/EBPα DNA binding domain. Our structures reveal that PU.1 binds the DNA motif at the exit linker, shifting 17 bp of DNA into the core region through interactions with H2A, unwrapping ~20 bp of nucleosomal DNA. C/EBPα binding, aided by PU.1's repositioning, unwraps ~25 bp of entry DNA. The PU.1 Q218H mutation, linked to acute myeloid leukemia, disrupts PU.1-H2A interactions. PU.1 and C/EBPα jointly displace linker histone H1 and open the H1-condensed nucleosome array. Our study unveils how two pioneer factors can work cooperatively to open closed chromatin by altering DNA positioning in the nucleosome.
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Proteína alfa Potenciadora de Unión a CCAAT , Nucleosomas , Ratones , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ADN/químicaRESUMEN
Centromeric chromatin plays a crucial role in kinetochore assembly and chromosome segregation. Centromeres are specified through the loading of the histone H3 variant CENP-A by the conserved chaperone Scm3/HJURP. The N-terminus of Scm3/HJURP interacts with CENP-A, while the C-terminus facilitates centromere localization by interacting with the Mis18 holocomplex via a small domain, called the Mis16-binding domain (Mis16-BD) in fission yeast. Fungal Scm3 proteins contain an additional conserved cysteine-rich domain (CYS) of unknown function. Here, we find that CYS binds zinc in vitro and is essential for the localization and function of fission yeast Scm3. Disrupting CYS by deletion or introduction of point mutations within its zinc-binding motif prevents Scm3 centromere localization and compromises kinetochore integrity. Interestingly, CYS alone can localize to the centromere, albeit weakly, but its targeting is greatly enhanced when combined with Mis16-BD. Expressing a truncated protein containing both Mis16-BD and CYS, but lacking the CENP-A binding domain, causes toxicity and is accompanied by considerable chromosome missegregation and kinetochore loss. These effects can be mitigated by mutating the CYS zinc-binding motif. Collectively, our findings establish the essential role of the cysteine-rich domain in fungal Scm3 proteins and provide valuable insights into the mechanism of Scm3 centromere targeting.
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Proteínas Portadoras , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas Portadoras/genética , Centrómero/genética , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cisteína/metabolismo , Cinetocoros/metabolismo , Chaperonas Moleculares/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Zinc/metabolismoRESUMEN
Background: Intraoperative hypotension (IOH) is a common complication occurring in surgical practice. This study aims to comprehensively review the collaboration and impact of countries, institutions, authors, journals, keywords, and critical papers on intraoperative hypotension from the perspective of bibliometric, and to evaluate the evolution of knowledge structure clustering and identify research hotspots and emerging topics. Methods: Articles and reviews related to IOH published from 2004 to 2022 were retrieved from the Web of Science Core Collection. Bibliometric analyses and visualization were conducted on Excel, CiteSpace, VOSviewer, and Bibliometrix (R-Tool of R-Studio). Results: A total of 1,784 articles and reviews were included from 2004 to 2022. The number of articles on IOH gradually increased in the past few years, and peaked in 2021. These publications were chiefly from 1,938 institutions in 40 countries, led by America and China in publications. Sessler Daniel I published the most papers and enjoyed the highest number of citations. Analysis of the journals with the most outputs showed that most journals concentrated on perioperative medicine and clinical anesthesiology. Delirium, acute kidney injury and vasoconstrictor agents are the current and developing research hotspots. The keywords "Acute kidney injury", "postoperative complication", "machine learning", "risk factors" and "hemodynamic instability" may also become new trends and focuses of the near future research. Conclusion: This study uses bibliometrics and visualization methods to comprehensively review the research on intraoperative hypotension, which is helpful for scholars to better understand the dynamic evolution of IOH and provide directions for future research.
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Pioneer transcription factors possess the unique ability to access DNA within tightly packed chromatin structures, playing pivotal roles in cell differentiation and reprogramming. However, their precise mechanism for recognizing nucleosomes has remained mystery. Recent structural and biochemical investigations into the binding interactions between the human pioneer factor OCT4 and the LIN28B nucleosome by Sinha et al.1 and Guan et al.2 have yielded conflicting results regarding nucleosome positioning, nucleosomal DNA unwrapping, binding cooperativity, and the role of N-terminal tail of OCT4. In this study, we undertook a comparative analysis of these two research efforts and delved into the factors contributing to the observed discrepancies. Our investigation unveiled that the utilization of human and Xenopus laevis core histones, along with a discrete two-step salt dialysis method, led to distinct positioning of DNA within reconstituted LIN28B nucleosomes. Additionally, our reanalysis of the electrophoretic mobility shift assay data showed that H3 K27 acetylation did not increase OCT4 binding to the internal sites of the nucleosome when normalized to input; instead, it promoted sample aggregation. Thus, the available experimental data support the notion that the human LIN28B nucleosome is pre-positioned for efficient binding with multiple OCT4s, and there is no compelling evidence for its regulation by histone modifications.
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Pioneer transcription factors are vital for cell fate changes. PU.1 and C/EBPα work together to regulate hematopoietic stem cell differentiation. However, how they recognize in vivo nucleosomal DNA targets remain elusive. Here we report the structures of the nucleosome containing the mouse genomic CX3CR1 enhancer DNA and its complexes with PU.1 alone and with both PU.1 and the C/EBPα DNA binding domain. Our structures reveal that PU.1 binds the DNA motif at the exit linker, shifting 17 bp of DNA into the core region through interactions with H2A, unwrapping ~20 bp of nucleosomal DNA. C/EBPα binding, aided by PU.1's repositioning, unwraps ~25 bp entry DNA. The PU.1 Q218H mutation, linked to acute myeloid leukemia, disrupts PU.1-H2A interactions. PU.1 and C/EBPα jointly displace linker histone H1 and open the H1-condensed nucleosome array. Our study unveils how two pioneer factors can work cooperatively to open closed chromatin by altering DNA positioning in the nucleosome.
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Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA sequences. Two use their POUS domains while the other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap â¼25 base pair DNA. Our analysis of previous genomic data and determination of the ESRRB-nucleosome-OCT4 structure confirmed the generality of these structural features. Moreover, biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Thus, our study suggests a mechanism of how OCT4 can target the nucleosome and open closed chromatin.
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Cromatina , Nucleosomas , Factor 3 de Transcripción de Unión a Octámeros , Proteínas de Unión al ARN , Humanos , Secuencia de Bases , Reprogramación Celular , Cromatina/genética , ADN/metabolismo , Nucleosomas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
Human acetyltransferases MOZ and MORF are implicated in chromosomal translocations associated with aggressive leukemias. Oncogenic translocations involve the far amino terminus of MOZ/MORF, the function of which remains unclear. Here, we identified and characterized two structured winged helix (WH) domains, WH1 and WH2, in MORF and MOZ. WHs bind DNA in a cooperative manner, with WH1 specifically recognizing unmethylated CpG sequences. Structural and genomic analyses show that the DNA binding function of WHs targets MORF/MOZ to gene promoters, stimulating transcription and H3K23 acetylation, and WH1 recruits oncogenic fusions to HOXA genes that trigger leukemogenesis. Cryo-EM, NMR, mass spectrometry and mutagenesis studies provide mechanistic insight into the DNA-binding mechanism, which includes the association of WH1 with the CpG-containing linker DNA and binding of WH2 to the dyad of the nucleosome. The discovery of WHs in MORF and MOZ and their DNA binding functions could open an avenue in developing therapeutics to treat diseases associated with aberrant MOZ/MORF acetyltransferase activities.
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Acetiltransferasas , Histona Acetiltransferasas , Leucemia , Humanos , Acetilación , Acetiltransferasas/metabolismo , Islas de CpG/genética , Histona Acetiltransferasas/metabolismo , Leucemia/genética , Translocación GenéticaRESUMEN
Pioneer transcription factors are essential for cell fate changes by targeting closed chromatin. OCT4 is a crucial pioneer factor that can induce cell reprogramming. However, the structural basis of how pioneer factors recognize the in vivo nucleosomal DNA targets is unknown. Here, we determine the high-resolution structures of the nucleosome containing human LIN28B DNA and its complexes with the OCT4 DNA binding region. Three OCT4s bind the pre-positioned nucleosome by recognizing non-canonical DNA motifs. Two use their POUS domains by forming extensive hydrogen bonds. The other uses the POUS-loop-POUHD region; POUHD serves as a wedge to unwrap âË»25 base pair DNA. Biochemical studies suggest that multiple OCT4s cooperatively open the H1-condensed nucleosome array containing the LIN28B nucleosome. Our study suggests a mechanism whereby OCT4s target the LIN28B nucleosome by forming multivalent interactions with nucleosomal motifs, unwrapping nucleosomal DNA, evicting H1, and cooperatively open closed chromatin to initiate cell reprogramming.
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The family Channidae, members of which are commonly known as snakehead fish, includes 53 Channa species and three Parachanna species. In this study, we characterized mitochondrial genomes (mitogenomes) of five Channa species (C. andrao, C. bleheri, C. ornatipinnis, C. pulchra, and C. stewartii) for the first time. We compared the mitogenomes with the mitogenomes of 11 other Channidae fish. The newly sequenced mitogenomes were 16,714 - 16,895 bp in length and contained 37 typical genes [13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs) and 22 transfer RNA genes (tRNAs)]. Positive AT-skews and negative GC-skews were found in the mitogenomes of Channidae. Most PCGs started with the conventional start codon, ATN; however, the sequence of the stop codon was variable. There was no essential difference in relative synonymous codon usage (RSCU) among the Channidae mitogenomes. The fastest-evolving gene atp8 and slowest-evolving gene cox1 were identified using Ka/Ks and pairwise relative genetic distance (p-distance). The displacement loop (D-loop) regions showed great variability, which affected the size of Channa mitogenomes. One origin of replication on the light strand (OL) region was found between trnN and trnC in the mitogenomes of Channidae. Phylogenetic analysis revealed three new sister pairs (C. andrao + C. bleheri, C. ornatipinnis + C. pulchra, and C. stewartii + C. gachua). Phylogenetic relationships established between the five Channa species based on mitogenomes were also supported by their morphological characteristics and geographical distribution. The novel information we obtained about these mitogenomes will contribute to elucidating the complex relationships among Channa species.
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Genoma Mitocondrial , Perciformes , Animales , Filogenia , Peces/genética , Perciformes/genética , ARN de Transferencia/genética , GenómicaRESUMEN
Linker histone H1, facilitated by its chaperones, plays an essential role in regulating gene expression by maintaining chromatin's higher-order structure and epigenetic state. However, we know little about the structural mechanism of how the chaperones recognize linker histones and conduct their function. Here, we used biophysical and biochemical methods to investigate the recognition of human linker histone isoform H1.10 by the TAF-Iß chaperone. Both H1.10 and TAF-Iß proteins consist of folded cores and disordered tails. We found that H1.10 formed a complex with TAF-Iß in a 2:2 stoichiometry. Using distance restraints obtained from methyl-TROSY NMR and spin labels, we built a structural model for the core region of the complex. In the model, the TAF-Iß core interacts with the globular domain of H1.10 mainly through electrostatic interactions. We confirmed the interactions by measuring the effects of mutations on the binding affinity. A comparison of our structural model with the chromatosome structure shows that TAF-Iß blocks the DNA binding sites of H1.10. Our study provides insights into the structural mechanism whereby TAF-Iß functions as a chaperone by preventing H1.10 from interacting with DNA directly.
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Proteínas de Unión al ADN , Chaperonas de Histonas , Histonas , Cromatina/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Humanos , Unión Proteica , Marcadores de SpinRESUMEN
BACKGROUND: IgA nephropathy (IgAN), which has been reported as the most prevalent glomerulonephritis globally, is the major contributor to end-stage renal diseases. This bioinformatics study aimed to explore glomerulotubular crosstalk genes and dysregulated pathways relating to the pathogenesis of IgAN. METHODS: The microarray datasets from the Gene Expression Omnibus (GEO) database were searched. Weighted gene co-expression network analysis (WGCNA) and differentially expressed genes (DEGs) of both glomeruli and tubulointerstitium were conducted individually. The co-expression gene modules of glomeruli and tubulointerstitium were compared via gene function enrichment analysis. Subsequently, the crosstalk co-expression network was constructed via the STRING database and key genes were mined from the crosstalk network. Finally, key genes were validated using another GEO dataset (GSE99340) containing RNA-seq data of IgAN and lupus nephritis, and their potential diagnostic values were shown using receiver operating characteristic (ROC) analysis. RESULTS: Five hundred eighty-three DEGs and eight modules were identified in glomerular samples, while 272 DEGs and four modules were in tubulointerstitial samples. There were 119 overlapping DEGs between the two groups. Among the distinctive modules, four modules in glomeruli and one module in tubulointerstitium were positively associated with IgAN. While four modules in glomeruli and two modules in tubulointerstitium were negatively associated with IgAN. The top ten key genes screened by CytoHubba were ITGAM, ALB, TYROBP, ITGB2, CYBB, HCK, CSF1R, LAPTM5, FN1, and CTSS. Compared with lupus nephritis, there were significant differences in the expression levels of CYBB, CTSS and TYROBP (P < 0.05), while other key genes showed no significant difference. Meanwhile, CYBB, CTSS, and TYROBP demonstrated possible diagnostic significance. CONCLUSIONS: The crosstalk genes confirmed in this study may provide novel insight into the pathogenesis of IgAN. Immune-related pathways are associated with both glomerular and tubulointerstitial injuries in IgAN. The glomerulotubular crosstalk might perform a role in the pathogenesis of IgAN.
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Glomerulonefritis por IGA , Nefritis Lúpica , Biomarcadores , Biología Computacional , Femenino , Redes Reguladoras de Genes/genética , Glomerulonefritis por IGA/genética , Humanos , MasculinoRESUMEN
The chromatosome, a nucleosome bound to a histone H1, is the structural unit of metazoan chromatin. Determination of the high-resolution structure of the chromatosome is challenging due to the dynamic nature of H1 binding. Here, we present a protocol for purifying an optimized single-chain antibody variable fragment (scFv) that can be used to stabilize the chromatosome for single-particle cryo-EM studies. This protocol facilitates high-resolution cryo-EM structure determination of nucleosomes with a natural DNA sequence, chromatosomes, and other protein nucleosome complexes. For complete details on the use and execution of this protocol, please refer to Zhou et al. (2021).
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Cromatina , Microscopía por Crioelectrón/métodos , Imagen Individual de Molécula/métodos , Anticuerpos de Cadena Única/química , Animales , Cromatina/química , Cromatina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Histonas/química , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/metabolismoRESUMEN
Accurate chromosome segregation relies on the specific centromeric nucleosome-kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.
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Centrómero/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Cinetocoros/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Centrómero/genética , Centrómero/ultraestructura , Proteína A Centromérica/química , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Microscopía por Crioelectrón , ADN de Hongos/química , ADN de Hongos/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/química , Conformación de Ácido Nucleico , Nucleosomas/genética , Nucleosomas/ultraestructura , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Genomic DNA in eukaryotes is organized into chromatin through association with core histone proteins to form nucleosomes. To understand the structure and function of chromatin, we must determine the structures of nucleosomes containing native DNA sequences. However, to date, our knowledge of nucleosome structures is mainly based on the crystallographic studies of the nucleosomes containing non-native DNA sequences. Here, we discuss the technical issues related to the determination of the nucleosome structures and review the few structural studies on native-like nucleosomes. We show how an antibody fragment-aided single-particle cryo-EM can be a useful method to determine the structures of nucleosomes containing genomic DNA. Finally, we provide a perspective for future structural studies of some native-like nucleosomes that play critical roles in chromatin functions.
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ADN/ultraestructura , Heterocromatina/ultraestructura , Histonas/ultraestructura , Nucleosomas/ultraestructura , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleosomas/genética , Nucleosomas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismoRESUMEN
The repeating structural unit of metazoan chromatin is the chromatosome, a nucleosome bound to a linker histone, H1. There are 11 human H1 isoforms with diverse cellular functions, but how they interact with the nucleosome remains elusive. Here, we determined the cryoelectron microscopy (cryo-EM) structures of chromatosomes containing 197 bp DNA and three different human H1 isoforms, respectively. The globular domains of all three H1 isoforms bound to the nucleosome dyad. However, the flanking/linker DNAs displayed substantial distinct dynamic conformations. Nuclear magnetic resonance (NMR) and H1 tail-swapping cryo-EM experiments revealed that the C-terminal tails of the H1 isoforms mainly controlled the flanking DNA orientations. We also observed partial ordering of the core histone H2A C-terminal and H3 N-terminal tails in the chromatosomes. Our results provide insights into the structures and dynamics of the chromatosomes and have implications for the structure and function of chromatin.
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ADN/química , Histonas/química , Nucleosomas/química , Microscopía por Crioelectrón , ADN/ultraestructura , Humanos , Nucleosomas/ultraestructura , Isoformas de Proteínas/químicaRESUMEN
ATP-dependent chromatin remodeling factors of SWI/SNF2 family including ISWI, SNF2, CHD1 and INO80 subfamilies share a conserved but functionally non-interchangeable ATPase domain. Here we report cryo-electron microscopy (cryo-EM) structures of the nucleosome bound to an ISWI fragment with deletion of the AutoN and HSS regions in nucleotide-free conditions and the free nucleosome at â¼ 4 Å resolution. In the bound conformation, the ATPase domain interacts with the super helical location 2 (SHL 2) of the nucleosomal DNA, with the N-terminal tail of H4 and with the α1 helix of H3. Density for other regions of ISWI is not observed, presumably due to disorder. Comparison with the structure of the free nucleosome reveals that although the histone core remains largely unchanged, remodeler binding causes perturbations in the nucleosomal DNA resulting in a bulge near the SHL2 site. Overall, the structure of the nucleotide-free ISWI-nucleosome complex is similar to the corresponding regions of the recently reported ADP bound ISWI-nucleosome structures, which are significantly different from that observed for the ADP-BeFx bound structure. Our findings are relevant to the initial step of ISWI binding to the nucleosome and provide additional insights into the nucleosome remodeling process driven by ISWI.