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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. [Display omitted] • FOXA1 binds linker DNA, whereas GATA4 targets an internal site within the nucleosome • The IDRs of FOXA1 and GATA4 interact with the acidic patch in H2A/H2B • FOXA1 enhances GATA4 binding by nucleosome repositioning-mediated cooperativity • FOXA1 and GATA4 open chromatin by disrupting H1 binding and nucleosome packing FOXA1 and GATA4 are prototype pioneer factors. Zhou et al. determined the cryo-EM structures of the native N1 nucleosome bound to FOXA1 or GATA4 or both. The authors provide insights into the binding modes, illustrating that the two pioneer factors work cooperatively to open chromatin by disrupting nucleosome packing. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

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. • 2.8–3.1 Å resolution structures of chromatosomes containing human H1 isoforms • Human somatic H1 isoforms bind to the nucleosome on the dyad • C-terminal tails of the H1 isoforms control distinct flanking DNA orientations • Chromatosome H3 N-tails relocate and inhibit ISWI remodeler binding and activity Linker histones bind to the nucleosome to form chromatosomes. Zhou et al. determined the cryo-EM structures of the chromatosomes containing human H1.0, H1.4, and H1.10 isoforms, respectively, and investigated their dynamics using nuclear magnetic resonance and molecular dynamics simulation. The study reveals distinct structural and dynamic features of the chromatosomes. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • High-resolution structures of the LIN28B nucleosome bound to three OCT4s • All OCT4s recognize non-canonical DNA sequences in the nucleosome • One OCT4 POUHD serves as a wedge to unwrap ∼25 bp nucleosomal DNA • OCT4s work cooperatively to open H1-condensed nucleosome array Guan et al. determined the structures of the nucleosome containing a genomic DNA and its complexes with OCT4. They find that multiple OCT4 molecules bind the nucleosome and its two DNA binding domains work together to unwrap the nucleosomal DNA. The study suggests how OCT4 may open closed chromatin locally. [ABSTRACT FROM AUTHOR]

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)., Competing Interests: The authors declare no competing interests.