Chromatin - Projects

Bridging fiber to gene folding by hierarchical looping

Although it has long been assumed that chromatin folds into a 30nm fiber, many recent experiments reveal disordered fibers of various sizes. In particular, we know little about higher-order folding of such fibers in living systems. In collaboration with Sergei Grigoryev (Penn State) who developed Electron Microscope Assisted Nucleosome Interaction Capture (EMANIC) technique, our mesoscale modeling unraveled a new folding motif for higher-order chromatin: hierarchical looping, compatible with irregular `zigzag’ folding motifs and ubiquitous looping of chromatin (Grigoryev et al., 2016). Hierarchical looping is similar to rope ‘flaking’ as used in mountain climbing to pack rope and unravel it easily without tangling. Here, 10 nm zigzag chromatin fibers are compacted laterally into self-associating loops that then stack and fold in space. This mechanism brings together distant DNA elements in a manner that can be controlled by linker histone density and histone modifications. Thus, hierarchical looping has epigenetic and gene regulation implications, as well as packing consequences for larger genetic structures such as genes (See Figure 4). These findings are consistent with experiments that emphasize zigzag folding (Hsieh et al., 2015), and different densities of interacting fibers (Eagen et al., 2015; Ou et al., 2017).

Figure 4
Fig. 4: Chromatin folding spans many scales, from fibers to genes to chromosomes. Hierarchical looping may occur at the kb-scale, possibly bridging fibers to genes.

Our work continued to show the importance of hierarchical looping in gene elements. While much data is available describing the length of chromatin loops near specific gene elements, there is a paucity of data regarding the specific three dimensional orientation of chromatin loops on the kilobase scale, or chromatin loops near gene elements. In collaboration with Karissa Sanbonmatsu (Los Alamos National Labs), our study of restrained chromatin loops ranging from 25 to 427 nucleosomes (fibers of 5–80 kb DNA in length), to reveal twisted plectoneme dynamics and looping. To determine the three dimensional structure of chromatin looping near a gene element, we combined our model with chromatin conformation capture data, which reports 5 distinct loops near the repressed GATA-4 gene state (See Figure 5). We show that hierarchicallooping represents a stable configuration that can effectively bring distant regions of the GATA-4 gene together, consistent with contact data. The folding geometry observed near the transcription start site (TSS) of the GATA-4 gene suggests that hierarchical looping provides a structural mechanism for gene inhibition, by occluding the TSS. This structural insight offers tunable parameters for design of gene regulation elements and gene silencing (Bascom et al., 2016).


Figure 5

Figure 5: Hierarchical looping was found to accommodate 3C contacts in the GATA-4 gene, offering a possible gene silencing mechanism.


Role of histone tail acetylation in chromatin domain formation

Histone tails and their epigenetic modifications play crucial roles in gene expression regulation by altering the architecture of chromatin. However, the structural mechanisms by which histone tails influence the interconversion between active and inactive chromatin remain unknown. Given the technical challenges in obtaining detailed experimental characterizations of the structure of chromatin, multiscale computations offer a promising alternative to model the effect of histone tails on chromatin folding. To model both local charge modifications of the histone tails and global fiber conformational changes, we pioneered a multiscale approach to combine multi-microsecond atomistic molecular dynamics simulations of dinucleosomes and histone tails in explicit solvent and ions (with three different state-of-the-art force fields and validated by experimental NMR measurements), with coarse-grained Monte Carlo simulations of 24-nucleosome arrays (Collepardo-Guevara et al., 2015). Our analyses describe the conformational landscape of histone tails, their roles in chromatin compaction, and the impact of lysine acetylation, a widespread epigenetic change, on both (See Figure 6). We found that while the wild-type tails are highly flexible and disordered, the dramatic increase of secondary-structure order by lysine acetylation unfolds chromatin by decreasing tail availability for crucial fiber-compacting internucleosome interactions rather than charge modulation per se, as previously believed.


This molecular mechanism of the effect of histone tails and their charge modifications on chromatin folding explains the sequence sensitivity and underscores the delicate connection between local and global structural and functional effects. The approach also opens new avenues for multiscale processes of biomolecular complexes. As recently shown, acetylated tails localized to specific regions of the chromatin fiber can lead to spontaneous contact domain segregation, a key feature of chromatin structures in vivo (See Figure 7, Rao et al., 2017).

Figure 6
Fig. 6: Lysine acetylation decondenses chromatin fibers, a multiscale study.

Figure 7

Figure 7: Localized regions of tail folding leads to spontaneous domain segregation.


Role of nucleosome placement in kilobase pair range chromatin contacts

The formation and dynamics of long-range contacts in living-system chromatin, in relation to gene regulation, are not well understood, but are clearly essential for bringing together linearly-distant segments for biological regulation. Nucleosome placement, or linker-length patterns, in chromatin have evolved to yield specific spatial features in chromatin fibers. But how these nucleosome patterns promote specific structures for biological function remains unknown. We examined by mesoscale modeling the effects of nucleosome positions on long-range contacts and looping of long chromatin fibers (Bascom et al., 2017). We investigated linker lengths ranging from 18-45 bp, with linker length distributions modeled after living systems, linker length distributions modeled after living systems with frequent Nucleosome Free Regions (NFRs), and linker length distributions modeled after gene rich chromatin, and uniform short linkers follow the 5′ NFR (more randomly distributed linker lengths are near the 3′ NFR).


Our data show that the irregular patterns in chromatin modeled after living systems produce special structural characteristics when compared to generic chromatin fibers with uniform or alternating linker lengths (See Figure 8). Importantly, non-uniform distributions of linker lengths with NFRs decrease associated persistence lengths, thereby enhancing flexibility and encouraging kb range contacts. NFRs between neighboring gene segments diminish short-range contacts between flanking nucleosomes, while enhancing kb range contacts via hierarchical looping. Because contacts among kb range elements are crucial to gene activity, linker length distributions in living systems are important aspects of such regulation. Our data suggest that fiber stiffness may vary dramatically from fiber to fiber or from one gene encoding segment to another, underscoring the role of linker length in the folding propensity of chromatin fibers and thus the regulation of the cell cycle and gene activity.

Figure 8
Fig. 8: Kb scale contacts are affected by life-like nucleosome placement patterns.