Targeted Epigenetic Modification of the SLC9C1 Gene

Sodium Hydrogen Exchangers (NHEs) are a family of proteins that utilize the electrochemical gradient of Na+ influx to export intracellular H+ ions1. The NHE10 protein, which has its expression localized to the principal piece of sperm, has been shown to be essential for sperm motility2. Mice with the NHE10 encoding gene, SLC9C1, knocked-out are completely infertile due to immotile sperm. The tissue specific expression of NHE10 may be due to a variety of potential epigenetic factors. One of these, DNA methylation, is an epigenetic mechanism involving the addition of a methyl group to a Cytosine in a CpG dinucleotide. This DNA feature has been implicated to have significant regulatory effects on gene expression. Our lab has identified the cell line HeLa as having a highly methylated SLC9C1 promoter region. After expression analysis, we revealed that this cell line has low expression of this gene. We investigated if demethylating the promoter region of the SLC9C1 gene can increase its expression. We plan to use a dCas9 fused to a Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) guided by a designed sgRNA to achieve targeted demethylation.

We will next perform co-transfection with sgRNA plasmid + dCas9-TET1 plasmid on HeLa cells.

We can then test the efficacy of the complex to demethylate the SLC9C1 promoter using bisulfite sequencing to measure the methylation status.

We may then measure the expression of SLC9C1 using ddPCR to compare any changes resulting from the demethylation.

1. Pedersen SF, Counillon L. The SLC9A-C mammalian Na+/H+ exchanger family: Molecules, mechanisms, and physiology. Physiol Rev. 2019;99(4):2015-2113.

doi:10.1152/physrev.00028.2018

2. Cavarocchi E, Whitfield M, Chargui A, et al. The sodium/proton exchanger SLC9C1 (sNHE) is essential for human sperm motility and fertility. Clin Genet. 2021. doi:10.1111/cge.13927

Institutional Biosafety Committee (IBC) approval:  IBC #: Miami0051_James_2025_04_16