Uncovering Molecular Mechanisms of Epigenetic Regulation and Gene Expression
Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Chemistry
First Advisor
Simon, Matthew
Abstract
RNAs not only serve as the conduit between the genetic information stored in DNA and protein expression, but also exhibit non-coding biological roles. These functions are often mediated through the intricate secondary and tertiary structures of the RNA, and dysregulation can lead to a variety of diseases. As such, RNAs have become a target for small molecule therapeutic development. However, the screening methods developed thus far generally require a well-characterized RNA and do not provide information on how much of the transcriptome can be targeted. Here I describe work using metabolic labeling with 4-thiouridine (s4U) and disulfide tethering as a transcriptome-wide screening platform to identify small molecule binders of RNA. I show that a disulfide tether can protect s4U from oxidation and is compatible with downstream nucleotide recoding. I demonstrate that biotinylated disulfides can tether to and directly enrich s4U-labeled RNA, and sequencing can be used as a readout for small molecule binding. Taken together, disulfide tethering is one of the first general techniques to screen RNAs transcriptome-wide and has the potential to elucidate novel druggable pockets. In eukaryotic cells, DNA is packaged into the nucleus by wrapping around histone proteins and condensing into chromatin. These histones can be post- translationally modified as a mechanism of gene regulation, and the functional consequences are dependent on both the chemical moiety and the residue that is modified. Acetylation and methylation of lysine are two of the most foundational and well-studied histone post-translational modifications. Despite their importance, however, acetylation and methylation have thus far been considered mutually exclusive. Here I describe work on the identification and functional characterization of acetyl-methyllysine (Kacme), a previously undescribed histone post- translational modification in which the epsilon amine of lysine is both acetylated and monomethylated. I show that Kacme is a conserved post-translational modification that exists on histone H4 across species and tissues. Kacme marks transcription start sites in fly and human chromatin, is associated with increased rates of transcription, and functions at the level of transcription initiation. I demonstrate that Kacme can be affected by acetylation and methylation pathways both in vitro and in cells. Additionally, Kacme can be recognized by chromatin-associated proteins, and I show the crystal structure of H4Kacme bound to the bromodomain BRD2. In all, this work describes the first post- translational modification with two chemical groups that coexist on the same lysine residue and demonstrates that Kacme exhibits functions distinct from acetylation and methylation. I also describe my work further investigating the biological role of Kacme. I expand the scope of reagents and assays available to study Kacme by developing additional specific antisera and demonstrating compatibility with CUT&RUN and immunofluorescence. I further characterize enzymes capable of depositing and removing Kacme using in vitro assays and inhibitor screens. Finally, I demonstrate that Kacme is dynamic during development and differentiation and show preliminary data investigating the role of Kacme in transcriptional reactivation after mitosis. Together, this work supports that Kacme is a biologically relevant histone post-translational modification and lays the groundwork for further exploration of the function of Kacme.
Recommended Citation
Connor, Leah Julie, "Uncovering Molecular Mechanisms of Epigenetic Regulation and Gene Expression" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1246.
https://elischolar.library.yale.edu/gsas_dissertations/1246
