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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2023)

Guanine quadruplex (G4) structures are nonconical DNA conformations that play crucial roles in various cellular processes, including DNA replication and transcription regulation. G4 structures are frequently found in promoter regions of oncogenes, which render G4 structures promising therapeutic targets. However, the mechanisms through which G4 structures regulate biological functions remain underexplored. This dissertation focuses on the development of novel quantitative proteomic methods to identify putative G4-binding proteins, to characterize novel G4-binding proteins, to explore cellular modifiers of G4 stability, and to discover of novel biomarkers in breast cancer cells. In chapter 2, we employed an affinity-based quantitative proteomics analysis to identify novel G4-binding proteins. By utilizing three different biotinylated G4-forming oligonucleotides as probes, I achieved a comprehensive analysis of proteins interacting with various G4 conformations, which led to the discovery of over 30 G4-binding proteins. Building upon the pull-down experiments, I introduced, in Chapter 3, the use of photo-crosslinking G4 probes to capture G4-binding proteins. This approach enabled the capture of weak and transient interactions, where harsh washing conditions eliminate protein-protein interactions during the pull-down process. By Using this method, I identified 99 putative G4-binding proteins, and I also characterized one of these proteins, HELLS as a novel G4 helicase. In Chapter 4, I demonstrated, for the first time, that G4 DNA structures can undergo phase separation. By utilizing immunofluorescence microscopy and ChIP-seq analysis, I observed that phase separation modulates the stabilities of G4 structures in vitro and in cells. This discovery provides new insights into factors that modulate the formation and stabilities of G4 structures in cells. In Chapter 5, I identified novel small GTPases as biomarkers of radioresistance in breast cancer cells through a multiple-reaction monitoring-based targeted proteomics analysis. The study revealed ARFRP1 as a novel radioresistance biomarker in breast cancer cells, where its downregulation promotes radioresistance. In conclusion, this dissertation presents novel proteomic approaches for the identification of G4-binding proteins, understanding G4 stability modulation, and the discovery of potential biomarkers in breast cancer cells. These findings enhance our understanding of G4 biology and offer new avenues for therapeutic interventions and biomarker-driven cancer research.

    Cover page: Chemical Biology of DNA Guanine Quadruplex and its Binding Proteins
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2021)
    DNA sequences of high guanine (G) content have the potential to form G quadruplex (G4) structures. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of G4 structures by employing different sequences of G4 DNA derived from the human telomere and the promoters of c-MYC and c-KIT genes. Our results led to the identification of a number of candidate G4-interacting proteins, many of which were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and 78 other proteins that can bind selectively to one or two of the three DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 structure derived from the c-MYC promoter. Our quantitative proteomic screening also led to the identification of a number of candidate antireader proteins of G4 DNA. Together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance of understanding fully the G4-interaction proteome.
      Cover page: A Quantitative Proteomic Approach for the Identification of DNA Guanine Quadruplex-Binding Proteins.
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2022)
      DNA polymerase η (Pol η) catalyzes accurate bypass of ultraviolet light-induced cyclobutane pyrimidine dimers, and it also functions in several other related processes, including bypassing DNA with unusual structures. Here, we performed unbiased proteome-wide profiling of Pol η-interacting proteins by using two independent approaches, i.e., proximity labeling and affinity pull-down followed by LC-MS/MS analysis. We identified several helicases, including DHX9, as novel Pol η-interacting proteins. Additionally, ChIP-Seq analysis showed that Pol η is enriched at guanine quadruplex (G4) structure sites in chromatin. Moreover, Pol η promotes the recruitment of DHX9 to G4 structure loci in chromatin and facilitates DHX9-mediated unwinding of G4 structures. Deficiency in Pol η or DHX9 leads to attenuated replication across G4 regions in genomic DNA. Together, we unveiled the interaction between Pol η and DHX9 and demonstrated that the interaction promotes the replicative bypass of G4 structures in chromatin.
        Cover page: Polymerase η Recruits DHX9 Helicase to Promote Replication across Guanine Quadruplex Structures.
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2023)
        In the presence of monovalent alkali metal ions, G-rich DNA sequences containing four runs of contiguous guanines can fold into G-quadruplex (G4) structures. Recent studies showed that these structures are located in critical regions of the human genome and assume important functions in many essential DNA metabolic processes, including replication, transcription, and repair. However, not all potential G4-forming sequences are actually folded into G4 structures in cells, where G4 structures are known to be dynamic and modulated by G4-binding proteins as well as helicases. It remains unclear whether there are other factors influencing the formation and stability of G4 structures in cells. Herein, we showed that DNA G4s can undergo phase separation in vitro. In addition, immunofluorescence microscopy and ChIP-seq experiments with the use of BG4, a G4 structure-specific antibody, revealed that disruption of phase separation could result in global destabilization of G4 structures in cells. Together, our work revealed phase separation as a new determinant in modulating the formation and stability of G4 structures in human cells.
          Cover page: Phase Separation Modulates the Formation and Stabilities of DNA Guanine Quadruplex.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2020)
          Vascular endothelial zinc finger 1 (VEZF1) plays important roles in endothelial lineage definition and angiogenesis. Vasohibins 1 and 2 (VASH1 and VASH2) can form heterodimers with small vasohibin-binding protein (SVBP) and were recently shown to regulate angiogenesis by acting as tubulin detyrosinases. Here, we showed that VEZF1 binds directly with DNA guanine quadruplex (G quadruplex, G4) structures in vitro and in cells, which modulates the levels of the two isoforms of VASH1 mRNA. Disruption of this interaction, through genetic depletion of VEZF1 or treatment of cells with G4-stabilizing small molecules, led to increased production of the long over short isoform of VASH1 (i.e. VASH1A and VASH1B, respectively) mRNA and elevated tubulin detyrosinase activity in cells. Moreover, disruption of VEZF1-G4 interactions in human umbilical vein endothelial cells resulted in diminished angiogenesis. These results suggest that the interaction between VEZF1 and G4 structures assumes a crucial role in angiogenesis, which occurs through regulating the relative levels of the two isoforms of VASH1 mRNA and the detyrosinase activity of the VASH1-SVBP complex. Together, our work revealed VEZF1 as a G4-binding protein, identified a novel regulatory mechanism for tubulin detyrosinase, and illustrated that the VEZF1- and VASH1-mediated angiogenesis pathways are functionally connected.
            Cover page: VEZF1-guanine quadruplex DNA interaction regulates alternative polyadenylation and detyrosinase activity of VASH1.
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2023)
            R-loops and guanine quadruplexes (G4s) are secondary structures of nucleic acids that are ubiquitously present in cells and are enriched in promoter regions of genes. By employing a bioinformatic approach based on overlap analysis of transcription factor chromatin immunoprecipitation sequencing (ChIP-seq) data sets, we found that many splicing factors, including U2AF1 whose recognition of the 3 splicing site is crucial for pre-mRNA splicing, exhibit pronounced enrichment at endogenous R-loop- and DNA G4-structure loci in promoter regions of human genes. We also revealed that U2AF1 binds directly to R-loops and DNA G4 structures at a low-nM binding affinity. Additionally, we showed the ability of U2AF1 to undergo phase separation, which could be stimulated by binding with R-loops, but not duplex DNA, RNA/DNA hybrid, DNA G4, or single-stranded RNA. We also demonstrated that U2AF1 binds to promoter R-loops in human cells, and this binding competes with U2AF1s interaction with 3 splicing site and leads to augmented distribution of RNA polymerase II (RNAPII) to promoters over gene bodies, thereby modulating cotranscriptional pre-mRNA splicing. Together, we uncovered a group of candidate proteins that can bind to both R-loops and DNA G4s, revealed the direct and strong interactions of U2AF1 with these nucleic acid structures, and established a biochemical rationale for U2AF1s occupancy in gene promoters. We also unveiled that interaction with R-loops promotes U2AF1s phase separation, and our work suggests that U2AF1 modulates pre-mRNA splicing by regulating RNAPIIs partition in transcription initiation versus elongation.
              Cover page: Promoter R-Loops Recruit U2AF1 to Modulate Its Phase Separation and RNA Splicing.
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2022)
              Radiation therapy benefits more than 50% of all cancer patients and cures 40% of them, where ionizing radiation (IR) deposits energy to cells and tissues, thereby eliciting DNA damage and resulting in cell death. Small GTPases are a superfamily of proteins that play critical roles in cell signaling. Several small GTPases, including RAC1, RHOB, and RALA, were previously shown to modulate radioresistance in cancer cells. However, there is no systematic proteomic study on small GTPases that regulate radioresistance in cancer cells. Herein, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled (SIL) peptides, to identify differentially expressed small GTPase proteins in two pairs of breast cancer cell lines, MDA-MB-231 and MCF7, and their corresponding radioresistant cell lines. We identified 7 commonly altered small GTPase proteins with over 1.5-fold changes in the two pairs of cell lines. We also discovered ARFRP1 as a novel regulator of radioresistance, where its downregulation promotes radioresistance in breast cancer cells. Together, this represents the first comprehensive investigation about the differential expression of the small GTPase proteome associated with the development of radioresistance in breast cancer cells. Our work also uncovered ARFRP1 as a new target for enhancing radiation sensitivity in breast cancer.
                Cover page: Targeted Proteomic Analysis of Small GTPases in Radioresistant Breast Cancer Cells.
                • Article
                • Peer Reviewed
                UC Riverside Previously Published Works (2021)
                The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.
                  Cover page: YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2014)
                  As a typical self-driven many-particle system far from equilibrium, traffic flow exhibits diverse fascinating non-equilibrium phenomena, most of which are closely related to traffic flow stability and specifically the growth/dissipation pattern of disturbances. However, the traffic theories have been controversial due to a lack of precise traffic data. We have studied traffic flow from a new perspective by carrying out large-scale car-following experiment on an open road section, which overcomes the intrinsic deficiency of empirical observations. The experiment has shown clearly the nature of car-following, which runs against the traditional traffic flow theory. Simulations show that by removing the fundamental notion in the traditional car-following models and allowing the traffic state to span a two-dimensional region in velocity-spacing plane, the growth pattern of disturbances has changed qualitatively and becomes qualitatively or even quantitatively in consistent with that observed in the experiment.
                    Cover page: Traffic Experiment Reveals the Nature of Car-Following
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