RESUMO
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.
Assuntos
Microscopia Crioeletrônica , Fator de Transcrição GATA4 , Fator 3-alfa Nuclear de Hepatócito , Nucleossomos , Ligação Proteica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Nucleossomos/ultraestrutura , Animais , Fator de Transcrição GATA4/metabolismo , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/química , Camundongos , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Histonas/química , Sítios de Ligação , DNA/metabolismo , DNA/genética , DNA/química , Montagem e Desmontagem da Cromatina , HumanosRESUMO
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.
Assuntos
Cromatina , Nucleossomos , Fator 3 de Transcrição de Octâmero , Proteínas de Ligação a RNA , Humanos , Sequência de Bases , Reprogramação Celular , Cromatina/genética , DNA/metabolismo , Nucleossomos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismoRESUMO
Together with core histones, which make up the nucleosome, the linker histone (H1) is one of the five main histone protein families present in chromatin in eukaryotic cells. H1 binds to the nucleosome to form the next structural unit of metazoan chromatin, the chromatosome, which may help chromatin to fold into higher-order structures. Despite their important roles in regulating the structure and function of chromatin, linker histones have not been studied as extensively as core histones. Nevertheless, substantial progress has been made recently. The first near-atomic resolution crystal structure of a chromatosome core particle and an 11 Å resolution cryo-electron microscopy-derived structure of the 30 nm nucleosome array have been determined, revealing unprecedented details about how linker histones interact with the nucleosome and organize higher-order chromatin structures. Moreover, several new functions of linker histones have been discovered, including their roles in epigenetic regulation and the regulation of DNA replication, DNA repair and genome stability. Studies of the molecular mechanisms of H1 action in these processes suggest a new paradigm for linker histone function beyond its architectural roles in chromatin.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Sequência de Aminoácidos , Animais , Cromatina/química , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA/química , DNA/genética , DNA/metabolismo , Reparo do DNA , Replicação do DNA , Epigênese Genética , Variação Genética , Instabilidade Genômica , Histonas/química , Histonas/genética , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
DNA/química , Histonas/química , Nucleossomos/química , Microscopia Crioeletrônica , DNA/ultraestrutura , Humanos , Nucleossomos/ultraestrutura , Isoformas de Proteínas/químicaRESUMO
Linker histones bind to the nucleosome and regulate the structure of chromatin and gene expression. Despite more than three decades of effort, the structural basis of nucleosome recognition by linker histones remains elusive. Here, we report the crystal structure of the globular domain of chicken linker histone H5 in complex with the nucleosome at 3.5 Å resolution, which is validated using nuclear magnetic resonance spectroscopy. The globular domain sits on the dyad of the nucleosome and interacts with both DNA linkers. Our structure integrates results from mutation analyses and previous cross-linking and fluorescence recovery after photobleach experiments, and it helps resolve the long debate on structural mechanisms of nucleosome recognition by linker histones. The on-dyad binding mode of the H5 globular domain is different from the recently reported off-dyad binding mode of Drosophila linker histone H1. We demonstrate that linker histones with different binding modes could fold chromatin to form distinct higher-order structures.
Assuntos
Proteínas de Drosophila/química , Histonas/química , Nucleossomos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Drosophila melanogaster , Modelos Moleculares , Dados de Sequência Molecular , Nucleossomos/fisiologia , Ligação ProteicaRESUMO
It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh affinity (equilibrium dissociation constant KD of â¼2 × 10-12 M) measured using a single-molecule Förster resonance energy transfer method. It was hypothesized that the ultrahigh affinity and extreme disorder may be required for the chaperone function of ProTα, in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα-H1.0 complex using isothermal titration calorimetry and report a KD value of (4.6 ± 0.5) × 10-7 M. In addition, we show that ProTα facilitates the formation of the H1.0-nucleosome complex in vitro. The results of our study contrast with those of the previous report and provide new insights into the chaperone function of ProTα. Possible causes for the observed discrepancy in binding affinity are discussed.
Assuntos
Histonas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Sequência de Aminoácidos , Calorimetria , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Histonas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Timosina/química , Timosina/genética , Timosina/metabolismoRESUMO
BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental disorder in which genetics plays a key aetiological role. The gene encoding NAD(P)H steroid dehydrogenase-like protein (NSDHL) is expressed in developing cortical neurons and glia, and its mutation may result in intellectual disability or congenital hemidysplasia. CASE PRESENTATION: An 8-year-old boy presented with a 260-kb NSDHL-containing duplication at Xq28 (151,868,909 - 152,129,300) inherited from his mother. His clinical features included defects in social communication and interaction, restricted interests, attention deficit, impulsive behaviour, minor facial anomalies and serum free fatty acid abnormality. CONCLUSION: This is the first report of an ASD patient with a related NSDHL-containing duplication at Xq28. Further studies and case reports are required for genetic research to demonstrate that duplication as well as mutation can cause neurodevelopmental diseases.
Assuntos
3-Hidroxiesteroide Desidrogenases/genética , Transtorno do Espectro Autista/genética , Duplicação Cromossômica , Cromossomos Humanos Par 10/química , Herança Materna , Adulto , Transtorno do Espectro Autista/sangue , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/fisiopatologia , Criança , Ácidos Graxos não Esterificados/sangue , Feminino , Dosagem de Genes , Expressão Gênica , Humanos , MasculinoRESUMO
The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3, 4). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3-H4)(2) heterotetramer and are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.
Assuntos
Centrômero/química , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Motivos de Aminoácidos , Sequência de Aminoácidos , Autoantígenos/química , Autoantígenos/metabolismo , Sítios de Ligação , Centrômero/metabolismo , Proteína Centromérica A , Sequência Conservada , DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismoRESUMO
Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1-nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.
Assuntos
Histonas/química , Substâncias Macromoleculares/química , Modelos Moleculares , Conformação Molecular , Nucleossomos/química , Sequência de Aminoácidos , Calorimetria , Histonas/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação ProteicaRESUMO
Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, "stapling" the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.
Assuntos
Proteína HMGN2/química , Nucleossomos/química , Sequência de Aminoácidos , Sítios de Ligação , DNA/química , DNA/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Técnicas In Vitro , Cinética , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Nucleossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT , Nucleossomos , Camundongos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Transativadores/genética , Transativadores/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , DNA/químicaRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
Objective: Early identification and intervention for children with global developmental delay (GDD) can significantly improve their prognosis and reduce the possibility of developing intellectual disability in the future. This study aimed to explore the clinical effectiveness of a parent-implemented early intervention program (PIEIP) for GDD, providing a research basis for the extended application of this intervention strategy in the future. Methods: During the period between September 2019 and August 2020, children aged 3 to 6 months diagnosed with GDD were selected from each research center as the experimental group and the control group. For the experimental group, the PIEIP intervention was conducted for the parent-child pair. Mid-term and end-stage assessments were performed, respectively, at 12 and 24 months of age, and parenting stress surveys were completed. Results: The average age of the enrolled children was 4.56 ± 1.08 months for the experimental group (n = 153) and 4.50 ± 1.04 months for the control group (n = 153). The comparative analysis of the variation in the progress between the two groups by independent t-test showed that, after the experimental intervention, the developmental quotient (DQ) of locomotor, personal-social, and language, as well as the general quotient (GQ) of the Griffiths Mental Development Scale-Chinese (GDS-C), the children in the experimental group demonstrated higher progress than those in the control group (P < 0.05). Furthermore, there was a significant decrease in the mean standard score of dysfunctional interaction, difficult children and the total level of parental stress in the term test for the experimental groups (P < 0.001 for all). Conclusions: PIEIP intervention can significantly improve the developmental outcome and prognosis of children with GDD, especially in the areas of locomotor, personal-social, and language.
RESUMO
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.
Assuntos
Acetiltransferases , Histona Acetiltransferases , Leucemia , Humanos , Acetilação , Acetiltransferases/metabolismo , Ilhas de CpG/genética , Histona Acetiltransferases/metabolismo , Leucemia/genética , Translocação GenéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA , Chaperonas de Histonas , Histonas , Cromatina/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Humanos , Ligação Proteica , Marcadores de SpinRESUMO
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).
Assuntos
Cromatina , Microscopia Crioeletrônica/métodos , Imagem Individual de Molécula/métodos , Anticorpos de Cadeia Única/química , Animais , Cromatina/química , Cromatina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismoRESUMO
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.