Jennifer J. Ottesen

Histone fold modifications control nucleosome unwrapping and disassembly

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.

A Pyrrolysine Analogue for Site‐Specific Protein Ubiquitination

(Scheme 1) to generate a product with a native backbone. Specifically, a reversible transthioesterification in the presence of an exogenous thiol RSH gives an intermediate 3, which in turn undergoes irreversible intramolecular S!N acyl transfer to give the final peptide 4. Herein, we introduce a genetically encoded pyrrolysine analogue that places a ligation handle directly into a recombinant protein. We used NCL at this internal ligation site to generate a semisynthetic ubiquitinated protein. The cellular machinery for the incorporation of pyrrolysine (5, Scheme 2), 4] the 22nd genetically encoded amino acid, is sufficiently flexible to enable a number of other lysine derivatives to read through the amber stop codon. We previously described 6, a stable THF-based analogue of 5. We also introduced 7, which, owing to the presence of a terminal alkyne functionality, can be used as a chemical handle to label proteins through click chemistry. To further expand the range of available pyrrolysine analogues with unique and useful reactivity, we decided to test whether the d-cysteine-based analogue (S,S)-8 (d-Cys-e-Lys) could read through the UAG codon. We focused our attention on this cysteine isomer because our related readthrough studies of simple pyrrolysine analogues for protein click chemistry indicated that the presence of a lysine acyl substituent with the analogous sense of chirality to that found in 5 has a profound and beneficial influence on incorporation efficiency. For comparison purposes, however, we also included in our studies the diastereomeric analogue (R,S)-8 (l-Cys-e-Lys). The target pyrrolysine analogue (S,S)-8 was prepared by coupling the N,S-protected cysteine derivative (S)-9 with BocLys-OtBu (10) to provide amide (S,S)-11 in excellent yield (96 %, d.r.> 99.9:0.1; Scheme 3). Full deprotection with trifluoroacetic acid (TFA)/Et3SiH furnished (S,S)-8 as its TFA salt. The diastereomer (R,S)-8 was prepared in an analogous manner (see the Supporting Information). Scheme 1. Cysteine-based NCL. Scheme 2. Pyrrolysine (5) and analogues 6–8.

Acetylation of Histone H3 at the Nucleosome Dyad Alters DNA-Histone Binding

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 → 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.

Regulation of the nucleosome unwrapping rate controls DNA accessibility

Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2–hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry–exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.

Nucleosome remodeling by hMSH2-hMSH6

DNA nucleotide mismatches and lesions arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains approximately 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 whereby hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlight unanticipated strengths of the Molecular Switch Model for mismatch repair (MMR).

Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling

Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA–histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2–hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.

A Reversible Protection Strategy To Improve Fmoc-SPPS of Peptide Thioesters by the N-Acylurea Approach

C‐terminal peptide thioesters are an essential component of the native chemical ligation approach for the preparation of fully or semisynthetic proteins. However, the efficient generation of C‐terminal thioesters by Fmoc solid‐phase peptide synthesis remains a challenge. The recent N‐acylurea approach to thioester synthesis relies on the deactivation of one amine of 3,4‐diaminobenzoic acid (Dbz) during Fmoc SPPS. Here, we demonstrate that this approach results in the formation of side products through the over‐acylation of Dbz, particularly when applied to Gly‐rich sequences. We find that orthogonal allyloxycarbonyl (Alloc) protection of a single Dbz amine eliminates these side products. We introduce a protected Fmoc‐Dbz(Alloc) base resin that may be directly used for synthesis with most C‐terminal amino acids. Following synthesis, quantitative removal of the Alloc group allows conversion to the active N‐acyl‐benzimidazolinone (Nbz) species, which can be purified and converted in situ to thioester under ligation conditions. This method is compatible with the automated preparation of peptide–Nbz conjugates. We demonstrate that Dbz protection improves the synthetic purity of Gly‐rich peptide sequences derived from histone H4, as well as a 44‐residue peptide from histone H3.

Histone Core Phosphorylation Regulates DNA Accessibility

Background: Transcription and DNA replication are regulated by histone core phosphorylation. Results: Histone phosphorylation near the DNA entry-exit region of the nucleosome increases DNA unwrapping and accessibility, which are further enhanced when combined with histone acetylation. Conclusion: Histone core phosphorylation regulates DNA accessibility. Significance: Histone phosphorylation and acetylation function together to regulate occupancy of DNA regulatory complexes. Nucleosome unwrapping dynamics provide transient access to the complexes involved in DNA transcription, repair, and replication, whereas regulation of nucleosome unwrapping modulates occupancy of these complexes. Histone H3 is phosphorylated at tyrosine 41 (H3Y41ph) and threonine 45 (H3T45ph). H3Y41ph is implicated in regulating transcription, whereas H3T45ph is involved in DNA replication and apoptosis. These modifications are located in the DNA-histone interface near where the DNA exits the nucleosome, and are thus poised to disrupt DNA-histone interactions. However, the impact of histone phosphorylation on nucleosome unwrapping and accessibility is unknown. We find that the phosphorylation mimics H3Y41E and H3T45E, and the chemically correct modification, H3Y41ph, significantly increase nucleosome unwrapping. This enhances DNA accessibility to protein binding by 3-fold. H3K56 acetylation (H3K56ac) is also located in the same DNA-histone interface and increases DNA unwrapping. H3K56ac is implicated in transcription regulation, suggesting that H3Y41ph and H3K56ac could function together. We find that the combination of H3Y41ph with H3K56ac increases DNA accessibility by over an order of magnitude. These results suggest that phosphorylation within the nucleosome DNA entry-exit region increases access to DNA binding complexes and that the combination of phosphorylation with acetylation has the potential to significantly influence DNA accessibility to transcription regulatory complexes.

Segmental Isotopic Labeling for Structural Biological Applications of NMR

This chapter describes the preparation of precursor domains for the formation of multidomain segmentally labeled proteins by protein ligation.

Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.