Author ORCID Identifier
Date of Graduation
Masters of Science (MS)
Ambro van Hoof
Enhancers are the central genetic elements controlling cell-type and state specific transcription programs to dictate cell fates during development. Mechanistic understanding of enhancer action is important for both biology and disease research. In the human genome, more than 60k human enhancers were found to produce non-coding transcripts named enhancer RNAs (eRNAs). These created a new challenge to understand enhancer functions, which now are not only DNA elements that promote transcription but also RNA-producing transcription units themselves. Importantly, deregulation of eRNAs was associated with various diseases such as cancer, immune disorders, and neurodegeneration. However, the direct role of eRNAs in transcriptional regulation and enhancer-promoter looping remains debatable, and in cases that eRNAs may bear functions the mechanisms are incompletely understood. One important problem is that currently used perturbation methods of enhancer/eRNAs are not acute and cannot distinguish direct from indirect effects.
To reveal the dynamics of eRNA-regulated target gene transcription and epigenetic features, I attempted to establish acute and controllable systems to manipulate eRNA levels with high temporal precision. Three perturbation methods were tested on a testbed eRNA, including dCas9 based CRISPR activation or inhibition system and antisense oligonucleotide (ASO) based RNA inhibition system. I found that these CRISPR based systems show variable effects and sub-optimal acute perturbation of target eRNA, while ASOs are more acute for target perturbation. My study benchmarked several methods and established an acute perturbation system for controlling eRNAs as well as other types of RNAs, which could potentially overcome the current confounding issues of potential secondary effects. These results provided a foundation for further studies of fine-scale dynamics of eRNA-mediated transcriptional regulation and chromatin organization.
enhancer RNA, acute perturbation, transcriptional regulation, CRISPRi/a, antisense oligonucleotide
Available for download on Monday, July 29, 2024