Single-molecule techniques allow for picoNewton manipulation and nanometer accuracy measurements of single chromatin fibers. However, the complexity of the data, the heterogeneity of the composition of individual fibers and the relatively large fluctuations in extension of the fibers complicate a structural interpretation of such force-extension curves. Here we introduce a statistical mechanics model that quantitatively describes the extension of individual fibers in response to force on a per nucleosome basis. Four nucleosome conformations can be distinguished when pulling a chromatin fiber apart. A novel, transient conformation is introduced that coexists with single wrapped nucleosomes between 3 and 7 pN. Comparison of force-extension curves between single nucleosomes and chromatin fibers shows that embedding nucleosomes in a fiber stabilizes the nucleosome by 10 kBT. Chromatin fibers with 20- and 50-bp linker DNA follow a different unfolding pathway. These results have implications for accessibility of DNA in fully folded and partially unwrapped chromatin fibers and are vital for understanding force unfolding experiments on nucleosome arrays.

Torsional stress generated during DNA replication and transcription has been suggested to facilitate nucleosome unwrapping and thereby the progression of polymerases. However, the propagation of twist in condensed chromatin remains yet unresolved. Here, we measure how force and torque impact chromatin fibers with a nucleosome repeat length of 167 and 197. We find that both types of fibers fold into a left-handed superhelix that can be stabilized by positive torsion. We observe that the structural changes induced by twist were reversible, indicating that chromatin has a large degree of elasticity. Our direct measurements of torque confirmed the hypothesis of chromatin fibers as a twist buffer. Using a statistical mechanicsbased torsional spring model, we extracted values of the chromatin twist modulus and the linking number per stacked nucleosome that were in good agreement with values measured here experimentally. Overall, our findings indicate that the supercoiling generated by DNAprocessing enzymes, predicted by the twin-supercoiled domain model, can be largely accommodated by the higher-order structure of chromatin.

Archaeal chromatin proteins share molecular and functional similarities with both bacterial and eukaryotic chromatin proteins. These proteins play an important role in functionally organizing the genomic DNA into a compact nucleoid. Cren7 and Sul7 are two crenarchaeal nucleoid-associated proteins, which are structurally homologous, but not conserved at the sequence level. Co-crystal structures have shown that these two proteins induce a sharp bend on binding to DNA. In this study, we have investigated the architectural properties of these proteins using atomic force microscopy, molecular dynamics simulations and magnetic tweezers. We demonstrate that Cren7 and Sul7 both compact DNA molecules to a similar extent. Using a theoretical model, we quantify the number of individual proteins bound to the DNA as a function of protein concentration and show that forces up to 3.5 pN do not affect this binding. Moreover, we investigate the flexibility of the bending angle induced by Cren7 and Sul7 and show that the protein–DNA complexes differ in flexibility from analogous bacterial and eukaryotic DNA-bending proteins.

DNA responds to small changes in force and torque by over- or undertwisting, forming plectonemes, and/or melting bubbles. Although transitions between either twisted and plectonemic conformations or twisted and melted conformations have been described as first-order phase transitions, we report here a broadening of these transitions when the size of a topological domain spans several kilobasepairs. Magnetic tweezers measurements indicate the coexistence of three conformations at subpicoNewton force and linking number densities ∼-0.06. We present a statistical physics model for DNA domains of several kilobasepairs by calculating the full partition function that describes this three-state coexistence. Real-time analysis of short DNA tethers at constant force and torque shows discrete levels of extension, representing discontinuous changes in the size of the melting bubble, which should reflect the underlying DNA sequence. Our results provide a comprehensive picture of the structure of underwound DNA at low force and torque and could have important consequences for various biological processes, in particular those that depend on local DNA melting, such as the initiation of replication and transcription.

The maintenance of gene repression is crucial for the prevention of cancer development and progression. PRC2 (Polycomb repressive complex 2) regulates the transcriptional repression of oncogenes through the tri-methylation of histone H3 at lysine 27 (H3K27me3), which is accomplished by the PRC2 catalytic subunit EZH2. Aberrant expression of EZH2 can lead to the onset of several highly aggressive cancers including prostate, breast, lung, melanoma, lymphoma, and pancreatic cancer. Efficient methyltransferase activity of EZH2 in vivo is dependent on the presence of the PHD finger protein 1 (PHF1), however the mechanism by which PHF1 regulates EZH2 is poorly understood. We hypothesize that PHF1 interacts directly with EZH2 at gene promoters and stimulates tri-methylation of H3K27, thereby facilitating silencing at key genes. Here we report our findings from binding studies of the EZH2-PHF1 complex and the effects of PHF1 on EZH2 methyltransferase activity.

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We studied the structure of nucleosomes, the assembly of nucleosomes and how these properties are altered by DNA methylation and histone acetylation based on single molecule and ensemble fluorescence measurements. Our study revealed that a compact and rigid nucleosome structure is induced by CpG methylation in both internal and terminal regions of nucleosomal DNA. Real-time monitoring of nucleosome assembly with NAP1 revealed that there are at least 3 stable intermediate states during the assembly. The kinetic stabilities of the intermediate states are significantly elevated upon CpG methylation. These results suggest that CpG methylation stabilizes the nucleosome structure and inhibits the disassembly by destabilizing the transition states. We also characterized effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and dinucleosomes. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies a topology change. We proposed a structural model for a dinucleosome in chromatin based on the structure of a dinucleosome spontaneously formed by two mononucleosomes in solution, which depends strongly on Mg2þ concentration and histone acetylation state. Mainly rendered by single molecule observations, these results suggest that structural changes of nucleosomes induced upon DNA methylation and histone acetylation may contribute to the regulation of genome activities.