Regulation of Nucleosome Dynamics: Mechanisms for Chromatin Accessibility and Metabolism

The DNA in eukaryotic cells is organized into a tightly-regulated structural polymer called chromatin that ultimately controls crucial functions of the genome, including gene expression, DNA synthesis, and repair. The basic unit of chromatin is the nucleosome in which 147bp of DNA wraps 1.7-times around eight ”core” histone proteins (two copies each of H2A, H2B, H3, H4). Repeats of this structural unit have been shown to fold into higher order structures, which play a central role in controlling DNA accessibility for transcription regulation. However, at the individual nucleosome level, DNA-histone interactions that wrap DNA into the nucleosome also control DNA accessibility. A significant number of factors have been shown to regulate nucleosome accessibility, including variants and posttranslational chemical modifications of to the core histone proteins, chromatin remodeling complexes that reposition and disassemble nucleosomes, and histone chaperones that deposit or remove histones. Ultimately, these chromatin regulatory factors must physically alter nucleosomes to change DNA accessibility to transcription, replication, and DNA repair machinery. This work encompasses a detailed study of the integral relationship between histone posttranslational modifications (PTMs) and DNA accessibility. There are more than 100 reported PTMs throughout the nucleosome, many of which serve as binding sites for chromatin regulatory proteins. However, a subset of these PTMs are buried beneath the DNA-histone interface and are seemingly inaccessible to regulatory proteins. Given that nucleosomes in vivo typically possess multiple PTMs, until recently it has been difficult to determine the precise function of PTMs residing in the DNA-histone interface. Using fully-synthetic and semi-synthetic protein ligation strategies in conjunction with the Ottesen Lab, we have ii engineered and incorporated histones bearing precise PTMs into nucleosomes for biophysical characterization. Additionally we have employed analogs and amino acid substitution mimics of these PTMs commonly used in biophysical and genetic screening assays. We find that PTMs located in the DNA-histone interface between the entry-exit region (where DNA enters and exits the nucleosome) and the Loss of Ribosomal Silencing (LRS) region 45 base pairs into the nucleosome function by controlling spontaneous, partial DNA unwrapping to increase DNA accessibility. Conversely, we find that PTMs located within the nucleosome dyad, which is the furthest point that DNA wraps into the nucleosomes, do not alter nucleosome unwrapping. Rather, these PTMs function to regulate DNA accessibility by increasing nucleosome mobility and decreasing nucleosome stability to facilitate disassembly. Additionally, we find that PTMs and DNA base pair sequence within the nucleosome entry-exit independently and additively regulate DNA unwrapping to enhance or suppress DNA accessibility. Moreover, we find that PTM mimics typically do not capture the biophysical function of the precise PTM. Finally, we find that PTMs in the DNA-histone interface function synergistically with the SWI/SNF chromatin remodelling complex and the MSH2/MSH6 DNA mismatch recognition complex to reposition and disassemble nucleosomes due to the reduction in DNA-histone binding and increased unwrapping imposed by the PTM. Taken together, our results suggest that PTMs in the DNA-histone interface can function independently or synergistically with other external factors to regulate transcription, replication, and DNA repair.