Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Genome-wide identification of PCBP1 binding sites in mouse using ExoChew technique Olivia Lewis 1 , OMS-I, Daniel Ross 1 , BS, Chris Syed, OMS-IV, Rachael Baker, BA, Rachel Daley, OMS-III, Krishna Patel, OMS-III, Levi Diggins, OMS-IV, Ramu Anandkrishnan, and Bidyut K Mohanty* Edward Via College of Osteopathic Medicine, 350 Howard St, Spartanburg, SC 29316

Introduction

Results

Results

DNA and DNA binding proteins play significant roles in cancer by regulating key metabolic pathways. Therefore, understanding DNA-protein interactions genome-wide that play roles in cancer can lead to identification of new drug targets. The genome is interspersed with polyguanine and their complimentary polycytosine (polyC) sequences that can form noncanonical secondary structures called G-quadruplexes (G4s) and i-motifs (iMs). G4s and iMs can affect various cellular processes including DNA replication, DNA repair, and transcription. The family of polycytosine-binding proteins (PCBPs, PCBP 1-4 and hnRNP K) includes DNA- and RNA-binding proteins that bind to single stranded DNA (ssDNA) and RNA sequences containing polyC rich sequences. Although extensive work has been carried out on their RNA binding, very less information is available on their DNA binding, especially to the DNA secondary structures. HYPOTHESIS: We predicted that the cancer-associated polyC rich DNA-binding protein PCBP1 can be used to identify i-motif forming sequences as polyC-rich DNAs form iMs. Here we test this hypothesis at a genome-wide scale.

Figure 4 . CD analysis of the DNA fragments binding to PCBP1. The peaks show that the DNA fragments with poly cytosine repeats form i-motif.

Figure 2. Location of Pulled down sequences within mouse genome. Majority of reads are found in the intergenic region, indicating PCBP1 could play a role in various transactions on DNA including replication, transcription and repair.

Discussion, Conclusions, and Future Directions

References 1. Patel, K., Lodha, C., Smith, C., Diggins, L., Kolluru, V., Ross, D., Syed, C., Lewis, O., Daley, R., and Mohanty, BK (corresponding author) (2023) ExoChew: An exonuclease technique to generate single-stranded DNA libraries. Biorxiv. doi: https://doi.org/10.1101/2023.10.02.560524. 2. Mohanty, B. K., Karam, J. A., Howley, B. V., Dalton, A., Grelet, S., Dincman, T., Streitfeld, W. S., Yoon, J-H, Balakrishnan, L., Chazin, W. J., Long, D. T. and Howe, P. H. (2021) Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity. Life Science Alliance. 16;4(9):e202000995. doi: 10.26508/lsa.202000995. PMID: 34272328. 3. Diggins, L., Ross, D., Bhanot, S., Corallo, R., Daley, R., Patel, K., Lewis, O., Donahue, S., Thaddeus, J., Hiers, L., Syed, C., Eagerton, D. and Mohanty, BK (corresponding author) (2024) CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences. Biophys Rep (NY) 5(1):100187. doi: https://doi.org/10.1016/j.bpr.2024.100187. 4. https://BioRender.com q Using our novel ExoChew technique we have identified genome wide PCBP1 binding sites in mouse genome. The sequences are located at various parts of the genome and are rich in polycytosine stretches. q CD spectroscopic analysis of a selected PCBP1 binding sequences identified from this search show that they form i-motifs. q Future studies will be directed towards: o Understanding the role of PCBP1 at the newly identified sequences. o Understanding the role of PCBP1 in linear DNA - I-motif dynamics. o Identifying genome-wide PCBP1-DNA interactions in human cells which will give us tools to target human cancers.

Methods

Table 1. Selected PCBP1-pulldown sequences . MME Seq 1 is a negative control, not containing any ‘CCC’ repetitions. Seq 2 contains ‘CCC’ repetitions, one that is 16 nucleotides in length. MME Seq 3 contains less ‘CCC’ repeats than Seq 2 or Seq 4. MME Seq 4 contains more ‘CCC’ repeats than Seq 2.

1. 10 µg of mouse genomic DNA was sonicated to generate a library of 200 base pair fragments of double stranded DNA. 2. The library was treated with T7 exonuclease that binds to 5' ends of double-stranded DNA and cleaves unidirectionally to generate a library of single-stranded DNA (ExoChew). 3. A GST-tagged PCBP1 protein was incubated with the single-stranded DNA library. 4. The PCBP1-bound DNA molecules were extracted and sequenced.

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Figure 3. Electrophoretic mobility shift assay (EMSA) showing PCBP1 binding to selected pulled down DNA sequences . Gels differ by pH and results are not significant between the two. MME Seq 1 did not bind to PCBP1 as there was full shift of DNA to bottom of gel. MME Seq 2 bound to PCBP1 with more affinity than MME Seq 3 or MME Seq 4.

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Acknowledgements

Figure 1. Flow chart of methods.

BKM was funded by VCOM REAP grants 1032453 and 1302559.

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