Author ORCID Identifier
Date Available
12-15-2024
Year of Publication
2023
Degree Name
Doctor of Philosophy (PhD)
Document Type
Doctoral Dissertation
College
Medicine
Department/School/Program
Molecular and Cellular Biochemistry
First Advisor
Dr. Yvonne Fondufe-Mittendorf
Second Advisor
Dr. Rebecca Dutch
Abstract
Fundamental to cellular differentiation, development, and physiology is the precise regulation of gene expression. At the center of this control lies chromatin, and disruptions in chromatin biology are key contributors to complex human diseases. By convention, gene expression analysis primarily focuses on transcript abundance, however the splicing of transcripts into distinct isoforms is also of biological relevance. One new form of alternative splicing, backsplicing, is responsible for generating circular RNAs (circRNAs), which are emerging as essential components to gene regulatory networks. However, unlike other forms of alternative splicing, the role that chromatin plays in backsplicing and circRNA formation remains poorly understood.
This study examines the impact of chromatin on circRNA biogenesis and function. Using a Drosophila model, we investigate how the PARP1-chromatin complex affects backsplicing. Through native elongating transcript sequencing (NETseq), we uncover the role of PARP1 in regulating RNA polymerase II (RNAPII) pausing across circRNA host genes. Our findings reveal that PARP1-chromatin influences circRNAs biogenesis by altering RNAPII pausing within gene bodies, with outcomes contingent upon the architectural context of host genes. We find PARP1 regulates backsplicing by controlling RNAPII pausing, and the outcomes of this regulation are determined by the context of host gene architecture. Furthermore, PARP1's influence extends to RNAPII pausing within introns and exons, influencing the direction of transcriptional output to favor either circRNA or mRNA.
We next explore the pathological implications of disrupted chromatin dynamics, particularly in the context of Rett Syndrome, arising from mutations in the chromatin-associated protein MECP2 a PARP1 nucleosome competitor. Employing a single-cell multi-omics approach within patient-derived cortical spheroids, we illuminate cell-type-specific alterations in chromatin accessibility and gene expression mediated by mutant MECP2. These changes correlate with disturbances in the maturation of MEIS2+ neurons and identify potential circRNA candidates associated with these cells in the context of Rett syndrome. The functional presence of these circRNAs in RTT-associated cell types is being tested. Future mechanistic studies will determine how these dysregulated circRNAs function to drive Rett pathology.
Finally, we asked how circRNA might function in an epigenetically driven disease—arsenic (iAs)-induced lung cancer. We show that the expression of SATB2, a 3D-chromatin organizer linking chromatin to the nuclear matrix, is upregulated by iAs exposure in bronchial epithelial cells along with its circRNA, circ3915. We show both SATB2 and circ3915 collaborate to promote cancer cell proliferation, migration, and carcinogenic gene expression pathways characterized by acquisition of oncogenic NFE2L2 and KRAS-like gene expression signature. We also show that circ3915 is translated in iAs-transformed cells, where it’s peptide associates with full-length SATB2 to modify chromatin associations in the nucleus. This discovery suggests that iAs triggers SATB2 expression, leading to alterations in chromatin states that govern gene expression to promote oncogenesis.
This work advances our understanding of how genome architecture can provide context to fundamental biological processes such as splicing. Our identification of a circRNA that can be translated is exciting and demonstrates how circRNA translation adds to protein biodiversity and function. These findings not only broaden our comprehension of the intricate connections between genome architecture and fundamental biological processes like splicing but also highlight the translational potential of circRNA and its significance in precision medicine as a source of biomarkers and therapeutic targets.
Digital Object Identifier (DOI)
https://doi.org/10.13023/etd.2023.451
Funding Information
This study was supported by the National Science Foundation under National Science Foundation under Grants no. NSF/MCB-517986 in 2017-2020, no. NSF/MCB-016515 in 2020-2022, NSF/MCB-2230470 in 2022-2023, and Graduate Student Fellowship (no.: GRF-1839289) in 2018. This study was also supported by the National Institutes of Health under grants no. R01- ES024478 from 2017-2021, and no. R01-ES034253 from 2022-2023. Finally, this study was supported by the West Michigan Neurodegenerative Disease Program (MiND) Catalytic Pilot Award no. GF000120 from 2022-2023.
Recommended Citation
Eleazer, Rebekah, "The Role of Chromatin Structure in Circular RNA Biogenesis, Function and Disease" (2023). Theses and Dissertations--Molecular and Cellular Biochemistry. 70.
https://uknowledge.uky.edu/biochem_etds/70
