Structural mechanism of LIN28B nucleosome targeting by OCT4.

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 ∼25 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.

Emerging roles of linker histones in regulating chromatin structure and function.

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.

Distinct Structures and Dynamics of Chromatosomes with Different Human Linker Histone Isoforms.

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.

Structural Mechanisms of Nucleosome Recognition by Linker Histones.

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.

Binding Affinity and Function of the Extremely Disordered Protein Complex Containing Human Linker Histone H1.0 and Its Chaperone ProTα.

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 ∼2 × 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.

NSDHL-containing duplication at Xq28 in a male patient with autism spectrum disorder: a case report.

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.

Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3.

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.

Structural insights into the histone H1-nucleosome complex.

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.

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR.

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.

Structural mechanism of synergistic targeting of the CX3CR1 nucleosome by PU.1 and C/EBPα.

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.

Structural mechanism of synergistic targeting of the CX3CR1 nucleosome by PU.1 and C/EBPα.

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.

Structural mechanism of LIN28B nucleosome targeting by OCT4 for pluripotency.

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 ∻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.

The human LIN28B nucleosome is inherently pre-positioned for efficient binding of multiple OCT4s without H3 K27 acetylation.

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.

A multicenter clinical study on parent-implemented early intervention for children with global developmental delay.

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.

MORF and MOZ acetyltransferases target unmethylated CpG islands through the winged helix domain.

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.

Structural Mechanism of TAF-Iß Chaperone Function on Linker Histone H1.10.

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.

Structures of Native-like Nucleosomes: One Step Closer toward Understanding the Structure and Function of Chromatin.

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.

Preparation of scFv stabilized chromatosomes for single-particle cryo-EM structure determination.

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).

Structural and dynamic mechanisms of CBF3-guided centromeric nucleosome formation.

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.

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme.

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.