Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions

Abstract

The architecture of the chromatin fiber, which determines DNA accessibility for transcription and other template-directed biological processes, remains unknown. Here we investigate the internal organization of the 30-nm chromatin fiber, combining Monte Carlo simulations of nucleosome chain folding with EM-assisted nucleosome interaction capture (EMANIC). We show that at physiological concentrations of monovalent ions, linker histones lead to a tight 2-start zigzag dominated by interactions between alternate nucleosomes (i ؎ 2) and sealed by histone N-tails. Divalent ions further compact the fiber by promoting bending in some linker DNAs and hence raising sequential nucleosome interactions (i ؎ 1). Remarkably, both straight and bent linker DNA conformations are retained in the fully compact chromatin fiber as inferred from both EMANIC and modeling. This conformational variability is energetically favorable as it helps accommodate DNA crossings within the fiber axis. Our results thus show that the 2-start zigzag topology and the type of linker DNA bending that defines solenoid models may be simultaneously present in a structurally heteromorphic chromatin fiber with uniform 30 nm diameter. Our data also suggest that dynamic linker DNA bending by linker histones and divalent cations in vivo may mediate the transition between tight nucleosome packing within discrete 30-nm fibers and self-associated higher-order chromosomal forms.chromatin structure ͉ electron microscopy ͉ mesoscopic modeling ͉ Monte Carlo simulations ͉ linker histone T he DNA in eukaryotic chromatin is packed and functionally regulated by histones and nonhistone architectural proteins (1). The primary packing level is represented by an array of repeating units, the nucleosomes, where the DNA is wound around histone octamers (2). Further compaction is achieved through a hierarchy of folding levels, including the 30-nm chromatin fiber (secondary level) and more compact and self-associating tertiary and quaternary forms whose structures are unknown (3-6). Because DNA conformation in chromatin and nucleosome packing are intimately connected to DNA/protein recognition and gene regulation, there has been intense interest in understanding chromatin structure, energetics, and dynamics. Some experimental studies have suggested that nucleosomal arrays fold in a zigzag arrangement with relatively straight linkers and a 2-start nucleosome interaction pattern that brings each nucleosome in proximity to its second nearest neighbor (7-10) consistent with chromatin fibers observed in situ (11). Other evidence suggests that chromatin condensed with linker histones and divalent cations such as Mg 2ϩ can form either zigzag structures with various nucleosome topologies (12, 13) or solenoid-like arrangements. The latter class of structures has bent DNA linkers and predominant interactions between either nearest neighbor nucleosomes (14, 15) and/or between every fifth or sixth nucleosome along the chain (16,17).The picture has became more complex recently with our heighte...

From Macroscopic to Mesoscopic Models of Chromatin Folding

“…This intriguing suggestion for a hybrid compact fiber in divalent ion conditions was recently verified by a new experimental technique termed EMANIC which uses formaldehyde cross-linking followed by unfolding and EM visualization to capture internucleosomal patterns [47]. Such an ensemble of interchanging configurations with straight and bent linker DNAs is energetically advantageous since linker DNA bending can minimize repulsion at the fiber axis.…”

“…Fig.11 As Fig. 11 shows [47], without linker histones and a monovalent concentration of c s = 0.15M, the fiber organizes as a classic open zigzag with a sedimentation coefficient S 20,W of 39±1.2S (compared to 37S experimentally) and a packing ratio of 4 nucleosomes per 11nm. When the linker histone is added, sandwiching the entering and exiting linker DNA from each nucleosome, a rigid stem forms to allow closer contact; the fiber is markedly more compact (S 20,W =49±1.7S, compared to 55.6S experimentally, and packing ratio of 7 nucleosomes per 11nm).…”

Multiscale simulation of DNA

“…We can divide computational approaches of chromatin structure into three basic [156,160], ionic environment [157,161], presence of linker histones [163] and different intra-and inter-chain physical interactions [156,157,[160][161][162][163][164]. In the top-down models the chromatin structure is derived by implementing experimental restraints coming from chromosome conformation capture techniques into a simple model of the chromatin fiber.…”

Contributions of Sequence to the Higher-Order Structures of DNA

“…The approximate representation of local structure and dynamics (at levels ranging from individual to tens of basepairs) has proven useful for interpreting the configurational properties of short proteinmediated DNA loops (10-13) and well-defined chromatin constructs (14)(15)(16)(17)(18), including the long-range communication between different parts of model oligonucleosome chains and the spatial pathways captured with microscopic techniques. Mesoscale studies tend to ignore the influences of DNA sequence other than those associated with certain well-known nucleotide repeating patterns, such as the natural curvature of so-called A-tracts.…”

“…Models of DNA fibers deduced from x-ray diffraction studies raised questions about structural motifs (53) along the same lines as questions raised in recent years about the spatial disposition of nucleosomes in chromatin (7,8). While recent advances in single-particle cryogenic electron microscopy techniques (54) offer promise for the development of near-atomic-resolution models of chromatin in the future, studies that combine the cross-linking of chromatin with electron microscopic imaging (14) have resolved some of the controversy about nucleosomal organization in solution.…”