Structural Investigation of Chromatin Regulatory Complexes Using Electron Microscopy

Reyes, Alexis Amaris

doi:https://doi.org/10.21985/n2-hsvw-3e28

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Structural Investigation of Chromatin Regulatory Complexes Using Electron Microscopy

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Eukaryotic genomes are organized into chromatin, which acts to regulate access to the organism’s genetic material. A large and diverse class of proteins, known as chromatin modifiers and remodelers, are responsible for regulating the composition and structure of chromatin by monitoring nucleosomes. Chromatin remodelers are involved in multiple cellular processes, such as transcription and DNA repair, by controlling access to genomic DNA through the repositioning of nucleosomes. Four families of ATP-dependent chromatin remodelers have been identified in yeast, each with non-redundant roles within the cell. There has been a recent surge in structural models of chromatin remodelers in complex with their nucleosome substrates. These structural studies provide new insight into the mechanism of action for individual chromatin remodelers. The switch/sucrose nonfermentable (SWI/SNF) remodeling family act to regulate chromatin accessibility near promoters and play essential roles in multiple cellular processes. A high frequency of mutations has been found in SWI/SNF family subunits by exome sequencing in human cancer, and multiple studies support its role in tumor suppression. Recent structural studies of yeast SWI/SNF, RSC (Remodeling Structure of Chromatin), and the human homolog, BAF (BRG1/BRM associated factor), have provided a model for their complex assembly and their interaction with mono-nucleosomes, revealing the molecular function of individual subunits as well as the potential impact of cancer-associated mutations on the remodeling function. Using cryo-electron microscopy, we have solved the structure of yeast SWI/SNF, investigated the architecture of RSC, and compare these structures to other ATP-dependent chromatin remodelers. The histones that compose the nucleosome core play a large role in chromatin regulation. Each histone has an N-terminal tail that can be post-translationally modified with a variety of chemical groups, such as methyl and acetyl groups. The diverse groups of histone tail modifications make up the histone code, which refers to the hypothesis that histone tail modifications regulate chromatin states and transcription. It is generally accepted that acetylated nucleosomes mark for open chromatin and up-regulate transcription, while methylated nucleosomes mark for closed chromatin and down-regulate transcription. The proteins that catalyze the addition and removal of these histone marks are generally referred to as chromatin modifiers. The SET-domain protein family consists of a diverse group of chromatin modifiers; most of which act as methyltransferases with a few exceptions. Here we investigate the structure and function of the yeast histone deacetylase complex, SET3C. The SET3 complex (SET3C) is a seven-subunit histone deacetylase complex capable of transcriptional regulation. Methylated Histone 3 (H3) marks recruit SET3C to the nucleosome, and the SET3C catalytic subunits deacetylate H3 and H4 (Histone 4) tails. There is very limited structural knowledge of the SET3C subunits, with most subunits having unknown structures or functions. A catalytically active SET3 complex was endogenously purified from Saccharomyces cerevisiae and utilized for negative stain electron microscopy (EM) to determine an apo model for the holo-complex. The negative stain EM 3D model revealed a three-lobe architecture with each lobe extending from a central point.

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