Structural basis for linker histone H5-nucleosome binding and chromatin fiber compaction.

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.

Structural insights into the cooperative nucleosome recognition and chromatin opening by FOXA1 and GATA4.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

Atomic resolution cryo-EM structure of a native-like CENP-A nucleosome aided by an antibody fragment.

Genomic DNA in eukaryotes is organized into chromatin through association with core histones to form nucleosomes, each distinguished by their DNA sequences and histone variants. Here, we used a single-chain antibody fragment (scFv) derived from the anti-nucleosome antibody mAb PL2-6 to stabilize human CENP-A nucleosome containing a native α-satellite DNA and solved its structure by the cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. In comparison, the corresponding cryo-EM structure of the free CENP-A nucleosome could only reach 3.4 Å resolution. We find that scFv binds to a conserved acidic patch on the histone H2A-H2B dimer without perturbing the nucleosome structure. Our results provide an atomic resolution cryo-EM structure of a nucleosome and insight into the structure and function of the CENP-A nucleosome. The scFv approach is applicable to the structural determination of other native-like nucleosomes with distinct DNA sequences.

Chromatin structures condensed by linker histones.

In eukaryotic cells, genomic DNA exists in the form of chromatin through association with histone proteins, which consist of four core histone (H2A, H2B, H3, and H4) families and one linker histone (H1) family. The core histones bind to DNA to form the nucleosome, the recurring structural unit of chromatin. The linker histone binds to the nucleosome to form the next structural unit of chromatin, the chromatosome, which occurs dominantly in metazoans. Linker histones also play an essential role in condensing chromatin to form higher order structures. Unlike the core histones in the formation of the nucleosome, the role of linker histone in the formation of the chromatosome and high-order chromatin structure is not well understood. Nevertheless, exciting progress in the structural studies of chromatosomes and nucleosome arrays condensed by linker histones has been made in the last several years. In this mini-review, we discuss these recent experimental results and provide some perspectives for future studies.

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.

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.

Revisit of Reconstituted 30-nm Nucleosome Arrays Reveals an Ensemble of Dynamic Structures.

It has long been suggested that chromatin may form a fiber with a diameter of ~30 nm that suppresses transcription. Despite nearly four decades of study, the structural nature of the 30-nm chromatin fiber and conclusive evidence of its existence in vivo remain elusive. The key support for the existence of specific 30-nm chromatin fiber structures is based on the determination of the structures of reconstituted nucleosome arrays using X-ray crystallography and single-particle cryo-electron microscopy coupled with glutaraldehyde chemical cross-linking. Here we report the characterization of these nucleosome arrays in solution using analytical ultracentrifugation, NMR, and small-angle X-ray scattering. We found that the physical properties of these nucleosome arrays in solution are not consistent with formation of just a few discrete structures of nucleosome arrays. In addition, we obtained a crystal of the nucleosome in complex with the globular domain of linker histone H5 that shows a new form of nucleosome packing and suggests a plausible alternative compact conformation for nucleosome arrays. Taken together, our results challenge the key evidence for the existence of a limited number of structures of reconstituted nucleosome arrays in solution by revealing that the reconstituted nucleosome arrays are actually best described as an ensemble of various conformations with a zigzagged arrangement of nucleosomes. Our finding has implications for understanding the structure and function of chromatin in vivo.

Alterations in plasma cytokine levels in chinese children with autism spectrum disorder.

Genetic alterations, together with environmental risk factors during infancy and childhood, contribute significantly to the etiology of autism spectrum disorder (ASD), a heterogeneous neurodevelopmental condition characterized by impairments in social interaction and restricted, repetitive behaviors. Mounting evidence points to a critical contribution of immunological risk factors to the development of ASD. By affecting multiple neurodevelopmental processes, immune system dysfunction could act as a point of convergence between genetics and environmental factors in ASD. Previous studies have shown altered cytokine levels in individuals with ASD, but research in Asian populations are limited. Here, we measured the plasma levels of 11 candidate cytokines in ASD and typically developing (TD) children. The cohort included 41 TD children and 87 children with ASD, aged 1-6 years. We found that as compared to the TD group, children with ASD had higher plasma levels of Eotaxin, TGF-ß1 and TNF-α. The increase in TGF-ß1 level was most significant in males, while the increase in Eotaxin was most significant in females. Eotaxin level negatively correlated with the social affect score (SA) in ADOS, while TNF-α level positively correlated with total development quotient (DQ), measured using GMDS. These pilot findings suggest potentially important roles of Eotaxin, TGF-ß1 and TNF-α in ASD in the Chinese population. Autism Res 2018, 11: 989-999. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Alteration of immune system function is an important risk factor for autism spectrum disorder (ASD). Here we found that the levels of cytokines, including Eotaxin, TGF-ß1 and TNF-α, are elevated in Chinese children with ASD, as compared to typically developing children. The change in TGF-ß1 level was most prominent in boys, while that of Eotaxin was more significant in girls. These results provide evidence for changes in cytokine profile in Chinese children with ASD.