Date of Award

Summer 8-11-2023

Embargo Period

8-16-2028

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

College

College of Graduate Studies

First Advisor

Philip Howe

Second Advisor

Vamsi Gangaraju

Third Advisor

Viswanathan Palanisamy

Fourth Advisor

Denis Guttridge

Fifth Advisor

Antonis Kourtidis

Abstract

Metastasis, the spread of cancer cells from the tissue of origin to a secondary site(s), is the leading cause of cancer-related deaths. The molecular mechanisms underlying metastasis remain unclear. The epithelial-to-mesenchymal transition (EMT) is a transcriptionally driven phenotypic change in cell morphology, considered central to metastasis. Transcriptional dysregulation is a hallmark of cancerous cells. A single transcription factor can be activated by multiple signaling pathways. This redundancy leads to therapeutic resistance, as ultimately, genes that are downstream of therapeutic targets can still be activated by alternative pathways.

Transforming Growth Factor β (TGFβ) induces EMT by releasing cytoplasmic Polycytosine Binding Protein 1 (PCBP1) from a structurally conserved secondary structure found in the 3’UTR of pro-EMT mRNAs, the BAT (TGFβ activated translation) element, thereby allowing their translation. In addition to translational inhibition, PCBP1 regulates alternative splicing by binding to RNA secondary structures, also leading to inhibition of EMT. As PCBP1 is primarily localized to the nucleus, where key processes involve single-stranded nucleic acids prone to secondary structure formation, we began investigating PCBP1’s other potential nuclear roles.

PCBP1, an RNA and single-stranded DNA (ssDNA) binding protein, binds single-stranded polycytosine (poly(C)) tracts with high specificity. Poly(C) tracts are enriched in cancer-relevant genomic loci, and some form secondary structures: intercalated motifs (i-motifs) on the C-rich strand and G-quadruplexes (G4s) on the G-rich strand. At the start of this project, we discovered PCBP1’s binding to and potential regulation of i-motifs. As PCBP1 binding sites are enriched at gene promoters and PCBP1 knockdown results in transcriptional dysregulation, the goal of my research became to determine how and when PCBP1 binds to ssDNA to regulate transcription.

This work shows that PCBP1’s DNA binding sites are enriched at transcription start sites and that by binding to promoter regions, PCBP1 regulates transcription in addition to its previously known roles in splicing and translation. At PCBP1 target genes, PCBP1 interacts with several RNA/DNA hybrid (R-loop) associated G-quadruplex resolving helicases. Furthermore, our experiments show that PCBP1 interacts with RNA Helicase A (DHX9) to modulate transcription by regulating DHX9 accumulation and activity. PCBP1 depletion leads to defects in R-loop processing and dysregulation of transcriptional elongation of PCBP1 target genes. PCBP1’s high sequence specificity and interaction with helicases suggest that its mechanism in transcription involves guiding helicases to specific loci during transcription, thereby modulating their activity.

Rights

Copyright is held by the author. All rights reserved.

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Available for download on Wednesday, August 16, 2028

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