Nucleosomes are stable DNA-histone protein complexes that must be unwrapped and disassembled for genome expression, replication, and repair. Histone posttranslational modifications (PTMs) are major regulatory factors of these nucleosome structural changes, but the molecular mechanisms associated with PTM function remains poorly understood. Here we demonstrate that histone PTMs within distinct structured regions of the nucleosome directly regulate the inherent dynamic properties of the nucleosome. Precise PTMs were introduced into nucleosomes by chemical ligation. Single molecule magnetic tweezers measurements determined that only PTMs near the nucleosome dyad increase the rate of histone release in unwrapped nucleosomes. In contrast, FRET and restriction enzyme analysis reveal that only PTMs throughout the DNA entry-exit region increase unwrapping and enhance transcription factor binding to nucleosomal DNA. These results demonstrate that PTMs in separate structural regions of the nucleosome control distinct dynamic events, where the dyad regulates disassembly while the DNA entry-exit region regulates unwrapping. These studies are consistent with the conclusion that histone PTMs may independently influence nucleosome dynamics and associated chromatin functions.histone acetylation | chromatin dynamics | native chemical ligation

Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys 3 Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.All eukaryotic genomes are organized into strings of nucleosomes, where 147 bp of DNA are tightly wrapped around a histone protein octamer (1). Many biological processes are dependent on DNA-protein interactions. However, access to DNA-binding sites is often restricted by the nucleosome structure. Alterations in nucleosome structure, dynamics, and positioning have been hypothesized to play a gatekeeper role in regulating biological processes such as DNA replication, repair, and transcription (2).The post-translational modification (PTM) 3 of core histones (3) plays a central role in regulating the biological processing of eukaryotic genomes. Until recently, known histone PTMs were almost exclusively located on the unstructured histone tail regions, which extend from the structured core of the nucleosomes. PTMs in the histone tails can function to directly alter nucleosome (4 -6) and/or chromatin structure and stability (7,8) and function as protein-binding sites (9) in the "histone code" model (10).During the past 5 years over 30 additional histone PTMs were identified within structured regions of the nucleosome (11-13). Many of these modifications are buried within the nucleosome core and thus are not readily accessible for protein binding. Fifteen of these histone PTMs are located in the DNAhistone interface, where the histone octamer contacts the phosphate backbone of the wrapped DNA (14). Studies have suggested that only mild structural perturbations occur in coremodified nucleosomes, which implies that modifications buried beneath the DNA are unlikely to provide a protein-binding site (15). This has led to two additional models for the function of nucleosome core PTMs.Modifications such as Lys acetylation that reduce the positive charge of the histone octamer surface may reduce the bindi...

SUMMARY DNA nucleotide mismatches and lesion arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains ~146 bp DNA wrapped around an H2A, H2B, H3, and H4 histone octamer. We demonstrate that the mismatch recognition heterodimer hMSH2-hMSH6 disassembles a nucleosome. Disassembly requires a mismatch that provokes the formation of hMSH2-hMSH6 hydrolysis-independent sliding clamps, which translocate along the DNA to the nucleosome. The rate of disassembly is enhanced by actual or mimicked acetylation of histone H3 within the nucleosome entry-exit and dyad axis that occurs during replication and repair in vivo and reduces DNA-octamer affinity in vitro. Our results support a passive mechanism for chromatin remodeling where hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlights unanticipated strengths of the Molecular Switch Model for mismatch repair (MMR).

e Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as "readers," which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers. N ucleosomes, the basic building blocks of eukaryotic chromatin, impose a physical barrier to regulatory proteins and repress various DNA-mediated transactions (1). Despite spontaneous partial unwrapping and rewrapping of DNA near the entry/ exit site (2, 3), nucleosomes are quite stable and show limited mobility. Fourteen major histone-DNA contacts at 10.5-bp intervals primarily contribute to this stability, and about 14 kCal/mol of energy is required to break these contacts (4, 5). ATP-dependent chromatin remodelers like RSC and SWI/SNF reposition (6) or evict (7,8) nucleosomes by breaking these histone-DNA contacts during the course of remodeling. However, specific point mutations in the core histones can weaken key histone-DNA or histone-histone interactions in the nucleosome and partially alleviate the requirement for SWI/SNF (9). These mutations, termed SIN (SWI/SNF-independent) mutations, when present at the nucleosomal dyad or in the histone dimer-tetramer interface, decrease stability while increasing thermal mobility of nucleosomes and thereby may substitute for SWI/SNF function (10, 11).Incorporation of various posttranslational modifications (PTMs) into histones regulates nucleosome structure and dynamics. While the majority of PTMs reside in the unstructured N-terminal tail domain of histones, many have been identified in the ␣-helical histone fold motif that constrains the DNA superhelix to form the compact nucleosome core (12)(13)(14). Unlike the histone tail PTMs, those in the histone core are often buried and hence are less likely to be accessible for regulatory factor binding. Some of these nucleosome core PTMs are located in the histone-DNA interface (15,16). Several of them colocalize with known SIN mutations (15) and reduce DNA binding affinity (1...

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of RNA polymerase through the nucleosome. We develop a statistical-mechanics model of a nucleosome as a wormlike chain bound to a spool, incorporating fluctuations in the number of bases bound, the spool orientation, and the conformations of the unbound polymer segments. With the resulting free-energy surface, we perform dynamic simulations that permit a direct comparison with single-molecule experiments on a single nucleosome. This simple approach demonstrates that the experimentally observed structural states at nonzero tension are a consequence of the tension. Therefore, our model plays an important role in extrapolating the behavior to zero tension. This mechanism would arise in any system where the tether molecule is deformed in the transition state under the influence of tension. Using our statistical-mechanics model, we also study the translocation of RNA polymerase through a nucleosome. We consider RNA polymerase as a Brownian ratchet and model the translocation process using dynamic Monte Carlo simulation. We incorporate the effect of DNA elasticity on protein movement by considering the probability of RNA polymerase going into the pause state due to the force being applied by the bent DNA. Our theory suggests that RNA polymerase translocation is in pseudo-equilibrium with local DNA fluctuations and is not rate-limiting. Our theory predicts a very small change in translocation velocity of RNA polymerase in presence of TFIIS, suggesting that RNA polymerase generates sufficient forces to unravel the nucleosome in the absence of TFIIS.