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.

Cryoelectron tomography reveals the multiplex anatomy of condensed native chromatin and its unfolding by histone citrullination.

Nucleosome chains fold and self-associate to form higher-order structures whose internal organization is unknown. Here, cryoelectron tomography (cryo-ET) of native human chromatin reveals intrinsic folding motifs such as (1) non-uniform nucleosome stacking, (2) intermittent parallel and perpendicular orientations of adjacent nucleosome planes, and (3) a regressive nucleosome chain path, which deviates from the direct zigzag topology seen in reconstituted nucleosomal arrays. By examining the self-associated structures, we observed prominent nucleosome stacking in cis and anti-parallel nucleosome interactions, which are consistent with partial nucleosome interdigitation in trans. Histone citrullination strongly inhibits nucleosome stacking and self-association with a modest effect on chromatin folding, whereas the reconstituted arrays undergo a dramatic unfolding into open zigzag chains induced by histone citrullination. This study sheds light on the internal structure of compact chromatin nanoparticles and suggests a mechanism for how epigenetic changes in chromatin folding are retained across both open and condensed forms.

Probing the Interaction Between Chromatin and Chromatin-Associated Complexes with Optical Tweezers.

Single-molecule force spectroscopy is a powerful tool to analyze the architecture and interaction of large macromolecular assemblies that are refractory to high-resolution structural interrogations. Here, we describe an optical tweezers-based platform for extracting the mechanical fingerprints of individual nucleosome arrays bound with chromatin-associated complexes, such as the Polycomb repressive complex 2 (PRC2). This platform comprehensively characterizes the diverse binding modes of PRC2 on chromatin, measures their mechanical strengths, and is broadly applicable to the studies of other epigenetic machineries.

Histone H3 core domain in chromatin with different DNA linker lengths studied by 1H-Detected solid-state NMR spectroscopy.

Chromatin, a dynamic protein-DNA complex that regulates eukaryotic genome accessibility and essential functions, is composed of nucleosomes connected by linker DNA with each nucleosome consisting of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. Magic angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy can yield unique insights into histone structure and dynamics in condensed nucleosomes and nucleosome arrays representative of chromatin at physiological concentrations. Recently we used J-coupling-based solid-state NMR methods to investigate with residue-specific resolution the conformational dynamics of histone H3 N-terminal tails in 16-mer nucleosome arrays containing 15, 30 or 60 bp DNA linkers. Here, we probe the H3 core domain in the 16-mer arrays as a function of DNA linker length via dipolar coupling-based 1H-detected solid-state NMR techniques. Specifically, we established nearly complete assignments of backbone chemical shifts for H3 core residues in arrays with 15-60 bp DNA linkers reconstituted with 2H,13C,15N-labeled H3. Overall, these chemical shifts were similar irrespective of the DNA linker length indicating no major changes in H3 core conformation. Notably, however, multiple residues at the H3-nucleosomal DNA interface in arrays with 15 bp DNA linkers exhibited relatively pronounced differences in chemical shifts and line broadening compared to arrays with 30 and 60 bp linkers. These findings are consistent with increased heterogeneity in nucleosome packing and structural strain within arrays containing short DNA linkers that likely leads to side-chains of these interfacial residues experiencing alternate conformations or shifts in their rotamer populations relative to arrays with the longer DNA linkers.

Linker DNA Length is a Key to Tri-nucleosome Folding.

The folding of a nucleosome array has long been one of the fundamental and unsolved problems in chromatin biology. In this study, we address how nucleosome array folding depends on the length of linker DNA. We performed molecular dynamics simulations of a tri-nucleosome, a minimal model of chromatin folding, with various linker lengths (LLs) ranging from 20 to 40 base pairs (bps). We found that the tri-nucleosome folding strongly depends on LLs, and classified the structure ensemble into five classes, named from trinuc-1 to trinuc-5. As a function of LL, the different classes appear, on average, every 2 bps with a period of 10 bps, and are characterized by distinct inter-nucleosome interactions. The trinuc-1 conformation corresponds to LL ~ 10n, where n is an integer, and is stabilized by the tight packing between the first and the third nucleosomes, consistent with a zigzag fiber form. Structures of the other four classes are more diverse and distributed continuously in the space of possible configurations. Histone-DNA electrostatic interactions in the tri-nucleosome are further analyzed.

Unraveling the multiplex folding of nucleosome chains in higher order chromatin.

The DNA of eukaryotic chromatin and chromosomes is repeatedly supercoiled around histone octamers forming 'beads-on-a-string' chains of nucleosomes. The extent of nucleosome chain folding and DNA accessibility vary between different functional and epigenetic states of nuclear chromatin and change dramatically upon cell differentiation, but the molecular mechanisms that direct 3D folding of the nucleosome chain in vivo are still enigmatic. Recent advances in cell imaging and chromosome capture techniques have radically challenged the established paradigm of regular and hierarchical chromatin fibers by highlighting irregular chromatin organization and the importance of the nuclear skeletal structures hoisting the nucleosome chains. Here, we argue that, by analyzing individual structural elements of the nucleosome chain - nucleosome spacing, linker DNA conformations, internucleosomal interactions, and nucleosome chain flexibility - and integrating these elements in multiplex 3D structural models, we can predict the features of the multiplex chromatin folding assemblies underlying distinct developmental and epigenetic states in living cells. Furthermore, partial disassembly of the nuclear structures suspending chromatin fibers may reveal the intrinsic mechanisms of nucleosome chain folding. These mechanisms and structures are expected to provide molecular cues to modify chromatin structure and functions related to developmental and disease processes.

Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

Structure and Dynamics in the Nucleosome Revealed by Solid-State NMR.

Eukaryotic chromatin structure and dynamics play key roles in genomic regulation. In the current study, the secondary structure and intramolecular dynamics of human histone H4 (hH4) in the nucleosome core particle (NCP) and in a nucleosome array are determined by solid-state NMR (SSNMR). Secondary structure elements are successfully localized in the hH4 in the NCP precipitated with Mg2+ . In particular, dynamics on nanosecond to microsecond and microsecond to millisecond timescales are elucidated, revealing diverse internal motions in the hH4 protein. Relatively higher flexibility is observed for residues participating in the regulation of chromatin mobility and DNA accessibility. Furthermore, our study reveals that hH4 in the nucleosome array adopts the same structure and show similar internal dynamics as that in the NCP assembly while exhibiting relatively restricted motions in several regions consisting of residues in the N-terminus, Loop 1, and the α3 helix region.

Cis and trans internucleosomal interactions of H3 and H4 tails in tetranucleosomes.

Chromatin structure and the transcriptional state of a gene can be modulated by modifications made on H3 and H4 tails of histones. Elucidating the internucleosomal interactions of these tails is vital to understanding epigenetic regulation. Differentiation between cis (intra-nucleosomal) and trans (inter-nucleosomal) interactions is often difficult with conventional techniques since H3 and H4 tails are flexible. The distinction, however, is important because these interactions model short- and long-range chromatin interactions respectively and have different bearings in biological processes. Combining FCS and PCH analysis, we can decouple the contribution of histone tails to cis and trans effects. A Mg(2+) gradient was employed to facilitate compaction and oligomerization of tetranucleosomes with H3 and/or H4 tail truncations. H3 tails were found to play a multifunctional role and exhibit the ability to partake in both attractive cis and trans interactions simultaneously. H4 tails partake in attractive cis and repulsive trans interactions at low [Mg(2+)]. These interactions are diminished at higher [Mg(2+)]. Simultaneous H3 and H4 tail truncation inhibited array oligomerization but maintained local array compaction at relatively high [Mg(2+)]. The established experimental approach can be extended to study the detailed molecular interactions mediated by epigenetic modification of flexible histone tails.