Via Research Recognition Day Program VCOM-Carolinas 2025

Biomedical Research

Genome-Wide Comparison of DNA-Binding Sites Between Tumor Suppressor PCBP1 and Oncogene hnRNP K Christopher Syed OMS-IV, Krishna Patel OMS-III, Olivia Lewis OMS-I, Daniel Ross, Ramu Anadakrishnan, David Eagerton and Bidyut Mohanty Edward via College of Osteopathic Medicine, 350 Howard St. Spartanburg SC

Results

Results

Introduction • Various cellular proteins bind specific DNA or RNA sequences and regulate key processes, such as cancer genesis and development. • Two such proteins are poly(rC)-binding protein 1 ( PCBP1 ) and heterogeneous nuclear ribonucleoprotein K ( hnRNP K ), both of which bind polycytosine-rich sequences in single-stranded DNA (ssDNA) and RNA. • Despite their similar binding motifs, these proteins exert opposite effects . PCBP1 acts as a tumor suppressor , managing transcription and splicing across several oncogenic pathways; it is significantly downregulated in gastric, hepatocellular, pancreatic, and prostate cancers. In contrast, hnRNP K acts as an oncogene , with aberrant expression linked to tumorigenesis and disease progression in a number of different cancer types, including breast, colorectal, esophageal, lung, hepatocellular, and prostate cancers. • While the RNA-binding properties of these proteins have been extensively studied, the details of their DNA-binding remain unexplored. Notably, no comparative analysis of the binding sites of PCBP1 and hnRNP K has been conducted to date. • RESEARCH GOAL : This study aims to conduct a comprehensive genome wide analysis of the binding sites of PCBP1 and hnRNP K. • CLINICAL IMPORTANCE : By determining the distinct and shared DNA binding characteristics of these proteins, our findings may offer insights that could deepen our understanding of the molecular mechanisms underlying cancer progression. This research holds the potential to inform the development of novel therapeutics targeting these regulatory proteins.

CC nc st enra1p 2 DU sphp26a SI nmvgs 6 Dlg2 Ptprn2 Spata5 Mid1 Oca2 Nr2c2 Pard3b Hcn1 Rab11fip4 Nell1 Vwa8 Oxct1 Rn45s Sipa1l1 Fgf2 Rims2 Tead1 Atrnl1 Gm6468 hnRNP K

Chl1 Gm20754 Rarb Lsamp Speer4cos Zfp600 GGmrid221119 Anks1b Fstl5 Dlg2 Oca2 A2m Agbl1 Sri Cntnap2 Dmd Dpyd Fam83a Stx8 Pclo Speer8-ps1 Akap9 Zfp978 Mbnl1 PCBP1

5’ - CCC AGCACTG CCCC TCTGGA CCC GGT- 3’ No more than 9 bp apart At least 3 clusters of at least 3 C’s

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TABLE 1. Top 25 Genes Bound by PCBP1 and hnRNP K. Genes highlighted in red represent oncogenes, while genes in green represent tumor suppressors

FIGURE 2. Cytosine-rich DNA Fragments Bound by PCBP1 and hnRNP K C- rich DNA is represented by DNA with at least 3 clusters of at least 3 C’s, no more than 9 bp apart (2A). PCBP1 and hnRNP K both bind highly C-rich DNA (2B)

Conclusions

1.Patel, K., Lodha, C., Smith, C., Diggins, L., Kolluru, V., Ross, D., ... & Mohanty, B. K. (2023). ExoChew: An exonuclease technique to generate single-stranded DNA libraries. bioRxiv, 2023-10. 2.Ansa-Addo, E. A., Huang, H. C., Riesenberg, B., Iamsawat, S., Borucki, D., Nelson, M. H., ... & Li, Z. (2020). RNA binding protein PCBP1 is an intracellular immune checkpoint for shaping T cell responses in cancer immunity. Science advances, 6(22), eaaz3865. 3.Gallardo, M., Hornbaker, M. J., Zhang, X., Hu, P., Bueso-Ramos, C., & Post, S. M. (2016). Aberrant hnRNP K expression: all roads lead to cancer. Cell Cycle, 15(12), 1552 1557. 4.Nishigaki, M., Aoyagi, K., Danjoh, I., Fukaya, M., Yanagihara, K., Sakamoto, H., ... & Sasaki, H. (2005). Discovery of aberrant expression of R-RAS by cancer-linked DNA hypomethylation in gastric cancer using microarrays. Cancer research, 65(6), 2115-2124. 5.Chang, C. Y., Wu, K. L., Chang, Y. Y., Liu, Y. W., Huang, Y. C., Jian, S. F., ... & Hsu, Y. L. (2021). The downregulation of LSAMP expression promotes lung cancer progression and is associated with poor survival prognosis. Journal of Personalized Medicine, 11(6), 578. BKM was funded by VCOM REAP grants 1032453 and 1302559. • This study represents the first genome-wide comparison of the binding sites of PCBP1 and hnRNP K. • By determining the distinct and shared DNA-binding characteristics of these proteins, our findings may offer insights that could deepen our understanding of the molecular mechanisms underlying cancer progression. • Our findings form a framework with which we can build off to better understand the mechanism of binding of these proteins. • This research holds the potential to inform the development of novel therapeutics targeting these regulatory proteins. • Experiments are underway to repeat this experiment using human DNA from HeLa cell lines, as well performing PCR on DNA fragments before sequencing to ensure no data loss. • Future directions will focus on using PCBP1 and hnRNP K knockout cell lines to examine how the loss of these proteins affects expression of their target genes. References/ Acknowledgements

Methods

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FIGURE 3. Read Distribution Across Chromosomes The distribution of loci bound by PCBP1 and hnRNP K differs across chromosomes. The x-axis represents genomic positions, while the y-axis indicates the percentage of reads aligned to each genomic region.

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FIGURE 1. Methods. Exochew Technique (1A) and Protein Structure (1B) of hnRNP K (above) and PCBP1 (below)

FIGURE 4. Binding Profiles of PCBP1 and hnRNP K. Figures represent binding of PCBP1 (4A), hnRNP K (4B), and reference mouse genome (4C)

2025 Research Recognition Day

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