Abstract
The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.
Highlights
The eukaryotic genome is highly compacted into a proteinDNA complex called chromatin
Using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail
The force-dependent extension of the tether is obtained by the sum of the extensions of each nucleosome and the extension of the bare DNA handles at the flanks of the chromatin fiber, which are modeled by a wormlike chain
Summary
Chromatin with short NRL folds into a stiffer structure than chromatin with long NRL. H2A-H2B dissociation will result in less nucleosome-wrapped DNA (as tetrasomes only wrap a single turn of DNA) and fewer nucleosomes that stack on each other (for which the acidic patch on H2A-H2B is required) Both effects will increase the extension of the fiber at forces below the unstacking transition. These differences in fiber composition, do not impact the stiffness of WT and cross-linked MT fibers, which continue to reflect the stiffness characteristic for their NRL The force-extension curves of cross-linked and reduced MT fibers largely overlap at forces below the unstacking transition with the differences in stiffness between 167- and 197-bp NRL fibers being independent of the presence of cross-linking. Ical properties of the folded fiber but does inhibit the unstacking transition (Fig. 3), we can unequivocally attribute this unstacking transition in both 167- and 197-bp NRL fibers to the rupture of H4 tail-mediated nucleosome stacking rather than the gradual unwrapping of the nucleosomal DNA
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