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Researchers
Researchers
Yousif Alshathir, Joey Cuppini, Rajat Thapa, Jason Abele, Paul F. James
Yousif Alshathir
Yousif Alshathir
Biology, Premed
Paul F. James
Paul F. James
Department of Biology, Miami University
Background / Abstract
Background / Abstract
Epigenetics is the study of how alterations to DNA structure without alteration to the genomic code alters gene expression. This field has expanded greatly in the last 30 years as we began to understand the importance of how surface and structural changes regulate gene expression. Examples of structural epigenetic factors include histone modifications, chromatin remodeling, and DNA methylation of cytosines commonly found in clusters upstream of the transcription start site known as CpG islands.
Our lab is currently focusing on the effects of DNA methylation on promoter and CpGIs of two subfamilies of transmembrane proteins known as SLC9C and SLC9B, each of which have two subfamily members. Previous research in our lab has shown that methylation of the CpGs in the promoter region of SLC9C1 causes reduced expression of the protein NHE10 causing infertility in male mice. Our goal is to determine if this is the case in a genomic context within a cell line to conduct further experiments and determine how demethylation of these regions will affect expression. To do this we need to first determine the methylation status in the promoter and CpGI regions of SLC9C1 as well as determining its expression of NHE10.
Methods
Methods
Figure 2: The outline for how methylation status and expression is determined.
Results
Results
Figure 3: The results of bisulfite sequencing of the promoter and CpGI regions in Hela cells. The bar above depicts the SLC9C1 gene in humans and the locations of the promoter and CpGI in relation to the transcription start site (TSS). Below is the methylation status of these regions, row is a individual sample with each circle depicting a CpG site where filled in circles are methylated.
Figure 4: This figure shows the RT-PCR result of NHE10 in Hela cells in comparison to a β-actin control. The figure on the left shows that NHE10 exhibits tissue specific expression in testis and not in Hela cells. No-RT internal controls were used to confirm the absence of contamination. The figure on the right shows that the testis and Hela both express β-actin, a protein expressed in all cells as a reference point to the amount of expression.
Conclusion / Future Directions
Conclusion / Future Directions
To show that our results are statistically significant we would like to have at least ten individual samples of each fragment for our methylation status data. Currently we only have four samples of the second promoter fragment, meaning there is a some more methylation profiling that needs to be done. With that being said, the partially completed methylation data suggests that the promoter and CpGI regions of SLC9C1 are highly methylated in Hela cells. RT-PCR expression analysis also indicates that NHE10 is not expressed. This data shows that Hela cells are a good model for studying the effects of demethylation on SLC9C1 expression.
The next step can be achieved by creating a Hela cell line expressing a dCas-TET protein and guide RNAs (gRNAs) targeting the promoter and CpGI regions. Doing this and repeating methylation and expression profiling will show to what degree methylation of the promoter and CpGI regions will have an effect on the expression of NHE10.
The following is an image of poster presented at the 2026 Undergraduate Research Forum
References
References
Gardner CC, James PF. 2023. Na+/H+ Exchangers (NHEs) in Mammalian Sperm: Essential Contributors to Male Fertility. International Journal of Molecular Sciences. 24(19):14981–14981. doi:https://doi.org/10.3390/ijms241914981.
Gardner CC, Abele JA, Winkler TJ, Reckers CN, Anas SA, James PF. 2024. Common as well as unique methylation-sensitive DNA regulatory elements in three mammalian SLC9C1 genes. Gene. 893:147897. doi:https://doi.org/10.1016/j.gene.2023.147897.
BioRender. 2025. BioRender. appbiorendercom. https://app.biorender.com/.
NACE Career Readiness Competencies
NACE Career Readiness Competencies
Through my research, I have developed teamwork, critical thinking, and technology skills by working together to troubleshoot experimental issues while also learning how to use molecular biology techniques and machines.
Acknowledgments
Acknowledgments
We would like to acknowledge funding support through the NIH grant R15 HD101985-01A1.
Institutional Biosafety Committee (IBC) approval: IBC #: Miami0051_James_2025_04_16
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