Via Research Recognition Day 2024 VCOM-Carolinas

Biomedical Studies

Dynamics of noncanonical DNA structures of c-Myc Oncogene Levi Diggins OMS-III, Rebecca Corallo OMS-II, Daniel Ross, Olivia Lewis, Krishna Patel OMS-II, Sundeep Bhanot OMS-I, Rachel Daley OMS-II and Bidyut K Mohanty. Edward via College of Osteopathic Medicine, 350 Howard St. Spartanburg SC

research design, study implementation, and data analysis and interpretation.

Abstract

Results

Conclusions

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Introduction and Methods Oncogenes are characterized as proto-oncogenes which have undergone a gain-of-function mutation which alters cellular processes and ultimately promotes cancer formation and growth. The mechanism and cellular environment for which oncogenes become mutated and subsequently add to cancer growth is dependent on a variety of physical, structural, chemical, and biological factors. Our goal is to elucidate the dynamics of noncanonical DNA structures formed at promoter-proximal region of c-Myc gene. Specifically, the guanine-rich DNA sequence, at this region of c-Myc promoter, which can form normal Watson Crick base pairing and double-stranded DNA with complementary sequence, can also form a noncanonical secondary structure called G-quadruplex (G4); across the sequence, its complementary cytosine-rich DNA also forms a noncanonical intercalating motif (i-motif or iM). These structures play important roles in cancer by affecting gene expression, DNA replication and other processes. Understanding the regulation of the dynamics between G4 and iM at Myc promoter, their stabilization or destabilization by proteins and ligands will help design drugs that can target these structures in cancer cells. The current work aims to understand the dynamics of G4 and iM by in vitro biophysical and biochemical techniques. C-Myc is aberrantly expressed in over 70% of human cancers. 1 Most notably, 70-80% of cases of Burkitt Lymphoma are associated with a t(8;14)(q24;q32) translocation causing a sequence of the Myc oncogene and one of its 2 promoters to be connected with a highly active IgG heavy chain enhancer . There are a variety of epistatic, epigenetics and post-translational mechanisms that can lead to Myc overexpression. 2 In-vivo formation of iM and G4 can play an important role as a genetic switch by facilitating or reducing cMyc expression. 3 Due to this, c Myc gene targeting therapy is promising for treating cancer. Methods: For this work, iM and G4 were formed in-vitro using buffers with varying pH which preferentially promoted the corresponding structure formation. The presence of these structures were then studied using Circular Dichroism (CD), a spectroscopic method that measures the absorption of right and left circularly polarized light in chiral molecules including DNA and DNA-protein complexes. Once the absorption peaks of G4 and iM were standardized the formation of these structures in various conditions could be analyzed. The formation of G4 and iM was studied in the presence of competing guanine and cytosine rich sequences as well as E. coli derived single strand binding protein (SSB) and hnRNP E1 protein. To further mimic in-vivo conditions, the capability of G4 formation in samples containing flanking sequences was studies. Electromobility shift assay was used to ensure protein-DNA complex formation and compare competition of the protein and complimentary sequences.

C-Myc encodes a transcription factor involved in cell differentiation, apoptosis, and metabolism. Due to its role in cellular regulation c-Myc is one of the most common oncogenes seen in cancer formation. Upstream to the c-Myc gene there lies a complementary cytosine and guanine rich sequence (Figure 1A). Within this sequence noncanonical iM and G4 structures can form altering gene expression. In G4 structures, four guanines form a Hoogsteen hydrogen bonding among them to put all four guanines in the same plane to generate the G-quartet or G-tetrad. In contrast, iM contains four strands of DNA in which two ‘Cs’ join to each other forming a C:C + pairing (Figure 1B). CD analysis showed the absorption peaks for G4 at ~260-263 nm and for iM at ~280-285 nm which is consistent with previous studies (Figure 2) 4. • Within the cell the formation of iM and G4 occurs in the presence of various DNA binding proteins such as SSB. SSB helps protect genome stability by coating single stranded DNA thus preventing DNA damage. • In the presence of SSB the formation of both iM and G4 decreased, this occurred to a greater degree with iM than G4 (Figure 3A and 3B). This is perhaps due to the increased binding strength of the guanine quartet of G4 compared to cytosine binding in iM. • The formation G4 was inhibited in the presence flanking sequences (Figure 3B). Thus, the flanking sequences may play a role in regulation of G4 formation. • When iM and G4 structures were treated with increasing concentrations of their complimentary strands there was a paradoxical increase in absorption peaks (Figure 4B and 4A). This was perhaps due to limitations of CD sensitivity as the absorption peaks of iM, G4 and double stranded are too close together to differentiate. • hnRNP E1 preferentially bind to cytosine rich sequences similar to those found in iM and c-MYC. In the presence of E1 there was a decrease in the formation of iM due to protein binding (Figure 5A). Increasing concentrations of the guanine rich sequence was able to displace E1 binding with the cytosine rich sequence in favor of double stranded DNA (Figure 5B).

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ƵĨĨĞƌ DLJĐ ' Figure 2 : CD analysis of i-motif and G quadruplex in sodium cacodylate (SCB, pH 5.5). Control (blue) contains 250 µL of SCB buffer. G4 (grey) Absorption peak ~260-263 nm. iM (orange) absorption peak ~280-285 nm. B. A. DLJĐ

Figure 3B: CD analysis showing the effect of SSB on the G4 structure in Guanine rich strand of C-Myc. Control (light blue): 250 µL of buffer. Sample 1 (orange): cMyc G strand DNA. Sample 3 (green): cMyc G strand DNA + SSB. Sample 4 (dark blue)L cMyc G strand DNA with its flanking sequence.

Figure 3A: CD analysis showing the effect of SSB on iM structure in cytosine rich strand of C-Myc. Control (blue): 250 µL of buffer. Sample 1 (orange): only cMyc C strand DNA. Sample 3 (green): cMyc DNA + SSB.

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Figure 4A: CD analysis of Guanine rich strand competition on cMyc C strand. Control (light green): 250 µL of SCB buffer. Sample 1 (dark green): cMyc C strand. Sample 2 (blue): cMyc C strand ½ concentration of G-rich strand. Sample 3 (Red): 1:1 ratio of cMyc C strand and G strand.

Figure 4B: CD analysis of Cytosine rich strand competition on G4. Control (blue): 250 µL of TrisKCL buffer. Sample 1 (grey): cMyc G4 strand. Sample 2 (orange): cMyc G strand + ½ concentration of C-rich strand. Sample 3 (green): 1:1 ratio of cMyc G strand and C strand.

1. Madden, Sarah K., et al. “Taking the Myc out of Cancer: Toward Therapeutic Strategies to Directly Inhibit C -Myc - Molecular Cancer.” BioMed Central , BioMed Central, 4 Jan. 2021, molecular cancer.biomedcentral.com/articles/10.1186/s12943-020-01291-6#:~:text=c Myc%20is%20a%20transcription%20factor%20that%20is%20constitutively,suggesting%20this%20to%20be%20a%20 viable%20therapeutic%20strategy. 2. Graham, Brittany, and David Lynch. Burkitt Lymphoma - Statpearls - NCBI Bookshelf , National Library of Medicine, www.ncbi.nlm.nih.gov/books/NBK538148/. Accessed 22 Jan. 2024. 3. Brown, Susie L., and Samantha Kendrick. “The I -Motif as a Molecular Target: More than a Complementary DNA Secondary Structure.” Pharmaceuticals , vol. 14, no. 2, Jan. 2021, p. 96, https://doi.org/10.3390/ph14020096. Accessed 3 Apr. 2021. 4. Wright, Elisé P., et al. “Identification of Multiple Genomic DNA Sequences Which Form I -Motif Structures at Neutral PH.” Nucleic Acids Research , vol. 45, no. 6, Feb. 2017, pp. 2951 – 59, https://doi.org/10.1093/nar/gkx090. Accessed 1 May 2022.

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Figure 5A: CD analysis showing the effect of E1 on the formation of iM. Control (blue): 250 µL of SCB buffer. Sample 1 (yellow) E1. Sample 2 (orange): cMyc C strand; Sample 3 (grey):cMyc C strand + E1.

Figure 5B: CD analysis of G-rich strand competition on iM+E1 complex. From left to right. Lane 1 (control) 2µL of cMyc C strand. Lane 2 cMyc C strand + E1. Lane 3 [cMyc C strand + E1] + 1µL G strand. Lane 4 [cMyc C strand + E1] + 2µL G strand. Lane 5 [cMyc C strand + E1] + 3µL G strand.

BKM was supported by VCOM’s REAP grant 1032453.

Figure 1A: C-MYC gene with rich cytosine and guanine strands

Figure 1B: G-quadruplex and i-Motif structure

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2024 Research Recognition Day