USING HISTONE REPLACEMENT TO STUDY THE ROLES OF H3K36 IN REGULATION OF GENE EXPRESSION AND RNA PROCESSING Public Deposited

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  • March 20, 2019
Creator
  • Meers, Michael
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • The transcription of deoxyribonucleic acid (DNA) into a ribonucleic acid (RNA) intermediate, conventionally known as gene expression, is subject to extensive regulation at multiple phases before, during, and after the physical process of gene transcription. Importantly, each phase of transcription interfaces intimately not only with DNA itself, but also with the proteins to which it is bound in the cell, known as histones. Histones are a highly conserved class of globular proteins whose positive surface charges are able to neutralize the negatively charged phosphate groups in the DNA backbone. Thus, the otherwise rigid DNA helix is able to physically wrap around the histone core particle to form nucleosomes, the units of chromatin that serve to compact DNA into the spatially limited nuclear compartment. Each of the four histone subunits that contribute to the histone core particle contains an N-terminal unstructured tail region that is decorated with myriad post-translational modifications (PTMs), including methylation and acetylation of lysine residues, and phosphorylation of serine and threonine residues, among others. Different combinations of histone PTMs are known to be associated with functionally distinct regions of the genome, such as “active” genic regions in areas of high gene expression, or “silent” regions where transcription is rare. For this reason, it is thought that histone PTMs may represent heritable epigenetic information that can directly influence levels of local gene transcription. However, the “histone code hypothesis”, as it is known, has not been extensively tested for most PTMs outside of observing phenotypes after ablating the enzymes that catalyze them, which cannot rule out contributing effects from non-histone substrates. Here I present the design of a genetic system known as “histone replacement”, in which the endogenous histone genes in Drosophila melanogaster are deleted and replaced with transgenic copies bearing mutations at residues of interest that are normally post-translationally modified. We show that histone replacement is a viable strategy for direct interrogation of a host of chromatin and histone-related biological phenomena. I also use the histone replacement system to study a non-modifiable mutation of histone H3 lysine 36-to-arginine (H3K36R). H3K36 is methylated in coding regions and towards the 3’ ends of genes, and therefore is thought to interface directly with transcription elongation, alternative splicing, and mRNA processing. Using my system, I am able to confirm roles for H3K36 methylation in suppressing local histone acetylation, challenge its role in suppressing cryptic transcription initiation in coding regions, and posit a new role for it in post-transcriptional RNA processing. Together, these results represent a significant step forward in experimentally testing the histone code hypothesis, and indicate the possibility of elucidating new biology related to histones and chromatin that may be relevant to diseases in which global gene expression is altered, including most cancers.
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Rights statement
  • In Copyright
Advisor
  • Sethupathy, Praveen
  • Marzluff, William
  • Strahl, Brian
  • Matera, Arnold
  • Duronio, Robert
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2017
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