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. 2016 Aug 25;120(33):8642-53.
doi: 10.1021/acs.jpcb.6b03197. Epub 2016 Jun 16.

Mesoscale Modeling Reveals Hierarchical Looping of Chromatin Fibers Near Gene Regulatory Elements

Gavin D Bascom  1 , Karissa Y Sanbonmatsu  2 , Tamar Schlick  1   3
Affiliations

Mesoscale Modeling Reveals Hierarchical Looping of Chromatin Fibers Near Gene Regulatory Elements

Gavin D Bascom et al. J Phys Chem B. .

Abstract

While it is well-recognized that chromatin loops play an important role in gene regulation, structural details regarding higher order chromatin loops are only emerging. Here we present a systematic study of restrained chromatin loops ranging from 25 to 427 nucleosomes (fibers of 5-80 Kb DNA in length), mimicking gene elements studied by 3C contact data. We find that hierarchical looping represents a stable configuration that can effectively bring distant regions of the GATA-4 gene together, satisfying connections reported by 3C experiments. Additionally, we find that restrained chromatin fibers larger than 100 nucleosomes (∼20Kb) form closed plectonemes, whereas fibers shorter than 100 nucleosomes form simple hairpin loops. By studying the dependence of loop structures on internal parameters, we show that loop features are sensitive to linker histone concentration, loop length, divalent ions, and DNA linker length. Specifically, increasing loop length, linker histone concentration, and divalent ion concentration are associated with increased persistence length (or decreased bending), while varying DNA linker length in a manner similar to experimentally observed "nucleosome free regions" (found near transcription start sites) disrupts intertwining and leads to loop opening and increased persistence length in linker histone depleted (-LH) fibers. Chromatin fiber structure sensitivity to these parameters, all of which vary throughout the cell cycle, tissue type, and species, suggests that caution is warranted when using uniform polymer models to fit chromatin conformation capture genome-wide data. Furthermore, the folding geometry we observe near the transcription initiation site of the GATA-4 gene suggests that hierarchical looping provides a structural mechanism for gene inhibition, and offers tunable parameters for design of gene regulation elements.

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Figures

Figure 1:
Figure 1:
Nucleosome fiber model. a) Basic building block nucleosomes are represented as coarse-grained rigid cores with pseudo-charges approximating the electric field of the nucleosome core particle (PDB 1KX5) without histone tails. Linker DNA beads are drawn in red, while the linker histone globular head domain is shown in gold and the intrinsically disordered C-terminal domain of the linker histone is shown in teal. Histone tails are drawn in green, yellow, blue and red, corresponding to H4, H2A, H3 and H2B tails, respectively. b) Nine nucleosome fiber with tails and linker histones. c) Two nucleosome component with reference length and angles. Bending angles are labelled as β, while l0 refers to equilibrium distances and ϕNs refers to equilibrium torsions. DN terms are labelled in red, tail terms in blue, linker histone terms in teal, and indices are in black.
Figure 2a:
Figure 2a:
Space filling representation of restrained loop structures of 25, 50, and 75 nucleosomes (~.5, 10, 15Kb) for −LH, 12LH, +LH, and +LH+Mg2+ fibers. We find that for all loop lengths smaller than ~100 nucleosomes (~ 20Kb), restrained loops do not form higher order folds.
Figure 2b:
Figure 2b:
Space filling representation of restrained loop structures of 100, 125, 150 and nucleosomes (~ 20, 25, 30, 40 Kb) for−LH, 12LH, +LH, and +LH+Mg2+ fibers. We find that for all loop lengths larger than 100 nucleosomes (~ 20Kb), restrained fibers form twisted plectonemes, with interdigitation near the base and mini-loops near the distal part of the loop. These mini-loops can undergo further folding, which is modulated by increasing linker histone (LH) and divalent ion (+Mg2+) concentration.
Figure 3a:
Figure 3a:
Representative internucleosome interaction matrices for 50-nucleosome restrained-loop fibers with −LH, 12LH, +LH, and +LH+Mg2+. Local contacts cluster along the diagonal, where fibers show dominant zigzag organization, and simple hairpins are evident by regions perpendicular to the main diagonal. For both systems, divalent ions and linker histones decrease the strength (percentage of occurrence) of higher-order interactions, shown as weaker diagonal hairpins. Additional interaction matrices for other loops sizes are provided in Supporting Information.
Figure 3b:
Figure 3b:
Representative internucleosome interaction matrices for 125-nucleosome restrained-loop fibers with −LH, 12LH, +LH, and +LH+Mg2+. Local contacts cluster along the diagonal, where fibers show dominant zigzag organization, and simple hairpins are evident by regions perpendicular to the main diagonal. For both systems, divalent ions and linker histones decrease the strength (percentage of occurrence) of higher-order interactions, shown as weaker diagonal hairpin regions. Additional interaction matrices for other loop sizes are provided in Supporting Information.
Figure 4:
Figure 4:
Folding schematic of the GATA-4 mesoscale gene locus model. a) Linear GATA-4 gene locus (80Kb) with 3C loop regions reported by Tiwari et al. indicated in pink, blue, green, white, and purple (indicated with 1,2,3, and 4). Artificial restraints were placed on the fiber to bring each boundary region to within 50 nm of one another. b-d) Intermediate states drawn to show formation of hierarchical folds and e) final structure observed by mesoscale modeling. f) Same as e) but rotated left and right. g) Cartoon of the chromatin fiber axis showing formation of the fold. This folding scheme decreases total fiber length by >1,000 nm and blocks access to the transcription start site, located at the boundary of green and white loop regions.
Figure 5:
Figure 5:
Persistence lengths LP calculated for restrained loops ranging from 25–200 nucleosomes in length. LP is a measurement of bending probability, formally given as the length by which correlations of the vectors which are tangent to the fiber axis are lost. All fibers show increasing persistence length with increasing loop length. Linker histone depleted (−LH) fibers and linker histone and divalent ion saturated fibers (+LH+Mg2+) show a sharp increase in persistence length around 100–150 nucleosome loops, while subsaturated linker histone fibers (12LH) and saturated linker histone (+LH) fibers show more gradual increase in LP around the same regions.
Figure 6:
Figure 6:
Loop architecture with “Nucleosome Free Regions” (NFR) modeled by increasing the DNA linker length at that region. The NFR increases persistence length, especially for −LH fibers (left). This manifests as an opening of the otherwise condensed plectoneme, suggesting a possible structural role for nucleosome depleted region.
Figure 7:
Figure 7:
a) Space filling representations of the mesoscale GATA-4 gene locus models both with divalent ions (bottom row), and without divalent ions (top row). DNA is drawn in red and nucleosome cores are drawn in either blue or white. Histone tails are not shown. b) Linker histone and divalent ion (Mg2+) concentration affect global attributes of the gene-locus. Computed persistence lengths, packing ratios, and internal energy are presented in the order of decreasing potential energy (increasing linker histone and magnesium concentration). Energy/nucleosome steadily decreases with linker histone and divalent ion presence, while the compaction ratios increase. Persistence length, which varies by as much as 100 mm in linker histone depleted fibers, shows decreased variance for all other fibers, with an average value of 375nm.
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