My name is Jasmine Zhao, and I am a rising senior at the University of California, Los Angeles majoring in Molecular, Cell, and Developmental Biology (MCDB) and minoring in biomedical research. Having the opportunity to work in different labs these past three years has not only increased my fascination with the applications of research in the treatment of diseases but also helped me develop as a young scientist. Currently, I am especially interested in fields such as regenerative medicine and developmental biology.
My first introduction to research was when I joined Dr. Amander Clark’s lab during my freshman year at UCLA. The major goal of the Clark lab is to understand the cell and molecular mechanisms underlying germline development. Primordial germ cells (PGCs), which are cells that give rise to the sperm or egg, are especially sensitive to radiation and chemotherapy. Thus, in order to improve reproductive health and rebuild tissues after cancer therapy, our lab uses stem cell models to understand this early period of human development. One of the projects the lab is working on is developing more efficient in vitro methods to generate human primordial germ cell-like cells (PGCLCs) that can be used to study gametogenesis.
My project in the Clark lab focuses on germ cell tumors (GCTs), which are a heterogenous group of tumors hypothesized to arise from a common cell of origin, the PGC (Stevens, 1967). Although GCTs are considered rare, they account for 15% of malignancies in the adolescent to young adult population (15-40 years), with testicular GCTs being the most common malignancy for males in this age group (Calaminus, 2016). The cause and progression of this disease is not well understood. However, it is speculated that improper nuclear reprogramming or the failure of differentiation of PGCs may result in the development of GCTs. Recently, PRDM14, a transcription factor, has been associated with a variety of human diseases and was identified as a susceptibility locus for testicular cancer and intracranial germ cell tumors (Ruark et al., 2013; Terashima et al., 2013). Using immunofluorescence and immunohistochemistry, we found that PRDM14 is expressed in a variety of patient GCT samples but is not detectable in differentiated tissues such as teratomas, a tumor with all three germ layers. Intracranial GCTs, which are tumors located in the brain, also have a molecular signature resembling pre-gonadal or late-stage PGCs, a time period in which PRDM14 should start to be repressed. Therefore, we hypothesized that the failure to repress PRDM14 may function to enhance the malignant transformation of GCTs, driving PGCs to diverge from the normal developmental pathway. Our results suggest that the overexpression of PRDM14 can alter both differentiation and proliferation of PGCs.
Optimizing the Allotopic Expression of ATP6 to Mitochondria in Mutant Cells
This summer, my project will be conducted under the mentorship of Dr. Amutha Boominathan and Dr. Matthew O’Connor at the SRF Research Center in Mountain View. The goal of this project is to design and test different constructs that can potentially improve the allotopic expression of ATP6 to mitochondria in mutant cell lines. Mitochondria are double-membrane bound organelles that provide energy in the form of ATP to power the biochemical reactions of a cell. Unlike other organelles, however, mitochondria have their own DNA separate from the nucleus, and 13 out of those 37 genes encode for oxidative phosphorylation complex proteins. Due to possible leakage of the high-energy electrons of the respiratory chain, which results in the formation of reactive oxygen species, the oxidative stress mitochondrial-DNA (mtDNA) is subjected to can lead to mutations, aging, and cell death. For instance, the ATP6 gene encodes for subunit a of the Fo structural domain of ATP synthase, also known as Complex V. The Fo structural domain is embedded in the inner membrane of the mitochondria and contains the membrane proton channel that allows for the synthesis of ATP. Mutations of ATP6 have been implicated in different human diseases that affect neural development, vision, and motor movement such as Leigh syndrome and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP).
Figure 1. Design of constructs to improve the allotopic expression of the mitochondrial ATP6 gene.
(A) Soluble gene sequence with V5 epitope tag to verify if the gene can be expressed in mammalian cells (B) Soluble gene sequence with V5 epitope tag that is appended to the ATP5G1 mitochondrial targeting sequence (MTS) and codon corrected ATP6 for location-specific targeting (C) ATP6 with the first transmembrane domain (TMD1) deleted and a DDK epitope tag (Constructs will be cloned into pCMV and pENTR vectors and transfected into mammalian cells)
Allotopic expression has been proposed as a gene therapy approach that can potentially treat mitochondrial-DNA diseases. This method aims to express a wild-type copy of an affected mitochondrial gene in the nucleus of a cell, target it to the mitochondria, and allow functional replacement of the defective protein. Dr. Boominathan et al. has previously shown that stable allotopic co-expression of ATP8 and ATP6 is able to rescue a patient cybrid cell line that is null for the ATP8 protein and has significantly lowered ATP6 protein levels (Boominathan et al., 2016). However, improving the exogenous amount of ATP6 that can be expressed or targeted to the mitochondria may be necessary in order to achieve complete restoration of ATP synthase activity and structure. Therefore, my project will investigate whether appending an additional gene sequence, the soluble tag, can help stabilize ATP6 and prevent unfolding before it is inserted into mitochondria. Derived from a thermophilic bacterium, this additional gene sequence might be able to enhance the expression of low but expressible proteins such as ATP6. Two constructs have been designed to address this hypothesis. As shown in Figure 1A and 1B, these two constructs will be cloned into pCMV and pENTR vectors and help us evaluate if the tag can be expressed in mammalian cells and if proper targeting and import of ATP6 to the mitochondria is possible, respectively. The last construct, which is depicted in Figure 1C will be focused on decreasing the mean hydrophobicity of the ATP6 protein. High mean hydrophobicity, especially in the first 100 amino acids is one of the largest barriers for successful allotopic expression of membrane proteins (Oca-Cossio et al. 2003). We hypothesize that the first transmembrane domain of ATP6 is not involved in critical functions for the protein and can be manipulated to diminish the mean hydrophobicity. As such, we also will utilize both deletion and site-directed mutagenesis of the first transmembrane domain of ATP6 to determine if ATP6 expression can be enhanced. If these constructs can provide more efficient expression of ATP6, similar methods can be applied to other mitochondrial genes to improve the rescue of mitochondrial function by allotopic expression.
Future Plans:
References:
Boominathan, A., Vanhoozer S., Basisty N., Powers K., Crampton, A.L., Wang X., Friedricks, N., Schilling, B., Brand, M.D., O’Connor, M.S. Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant. Nucleic Acids Research. 2016; 44(19):9342-9357.
Calaminus G, Joffe J. Germ Cell Tumors in Adolescents and Young Adults. Progress in tumor research 2016; 43:115-27.
Oca-Cossio, J., Kenyon, L., Hao, H., Moraes, C.T. Limitations of allotopic expression of mitochondrial genes in mammalian cells. Genetics. 2003; 165, 707-720.
Ruark, E., Seal, S., McDonald, H., Zhang, F., Elliot, A., Lau, K., Perdeaux, E., Rapley, E., Peto, J., Kote-Jarai, Z., Muir, K., Nsengimana, J., Shipley, J. Identification of nine new susceptibility loci for testicular cancer, including variants near DAZL and PRDM14. Nat Genet. 2013; 1038/ng.2635.
Stevens L. Origin of testicular teratomas from primordial germ cells in mice. J. Natl. Cancer Inst. 1967a; 38, 549–552.
Terashima, K., Yu, A., Chow, WY., Hsu, WC., Chen, P., Wong, S., Hung, YS., Suzuki, T., Nishikawa, R., Matsutani, M., Nakamura, H., Ng, HK., Allen, JC., Aldape, KD., Su, JM., Adesina, AM., Leung, HC., Man, TK., Lau, CC. (2013). Genome-wide analysis of DNA copy number alterations and loss of heterozygosity in intracranial germ cell tumors. Pediatr Blood Cancer. 2013; 61(4):594-600.
