5-hydroxymethylcytosine (5hmC) is a DNA modification studied in mammalian cells and tissues that has been implicated in embryonic stem cell differentiation, neuronal development, and formation of tumors. However, much is still unknown about the regulatory function of 5hmC and its genomic localization in diverse cell types. Recently, a novel, enzyme-based method was developed for 5hmC identification, APOBEC-Coupled Epigenetic Sequencing (ACE-seq), that is highly accurate, requires low input, and does not degrade DNA as commonly used bisulfite treatment methods do. ACE-seq is especially relevant to cellular environments that are epigenetically dynamic, including brain tissue and embryonic cell cultures. My project uses ACE-seq to generate high fidelity 5hmC characterizations in the Elf1 cell line across culture conditions that mimic early human embryonic development in which dramatic changes in DNA methylation occur. ACE-seq will be used on three human embryonic stem cell (hESC) conditions that mimic the transition from before embryonic implantation, naïve hESCs (two conditions), to near the time of embryonic implantation, primed hESCs. We hope to gain insight into 5hmC localization and regulatory function during this physiologically significant developmental event occurring during early embryogenesis. A more robust understanding of 5hmC regional abundance during this transition will help us elucidate the regulatory circuitry underlying early development. Knowledge gained in this project is especially relevant to the field of personalized medicine, as thorough understanding of pluripotency transitions will be significant to future applications of stem cell based therapies and precision healthcare. 

DNA is often correctly referred to as one of the building blocks of life. Understanding the combination of adenine, thymine, guanine, and cytosine that ultimately defines our characteristics is crucial to many genomic applications ranging from synthetic biology applications to our understanding of health and diseases. Modeling and understanding the causes and consequences of these base pair level interactions can impact the development of DNA origami and our understanding of genome stability in different health/disease states. DNA is widely known for its complex structures and uncovering, modeling, and predicting the structure and formation of DNA can provide valuable insights into various aspects of genomics including visualization and probe design. Modeling DNA’s inter-strand and intra-strand interactions has been the core focus of genomics for some time. Specifically, understanding intra-strand secondary structure formation across genomes has largely been unexplored due to computational limits. To further our understanding of DNA, I present the use of thermodynamic partition functions to model and predict intra-strand behavior of secondary structure formation. I have efficiently calculated secondary structure formation across hg38 at three relevant thermodynamic conditions and their corresponding length scales. With this information, I examine the distribution of the entire genome while also cross examining this information against specific regions with known biological features such as genic vs non-genic regions. Additionally, I apply the same predictive model to fluorescent in situ hybridization (FISH) probe design and provide additional quantitative metrics and insights in the development of optimal probe design. This research has the potential to further our understanding of the causes and consequences of non-random genes. Having additional information about the distribution of secondary structure formation of certain regions can be vital information about learning more about the selection of base pair sequences and its macro effects.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is caused by the death of dopamine-secreting neurons in the midbrain. The onset of symptoms, including progressively worsening tremors, movement difficulty, and dementia are thought to be caused by protein aggregates called Lewy bodies, mitochondrial defects, and neuroinflammation. Mutations in the GBA gene, which codes for the enzyme glucocerebrosidase which breaks down the lipid glucosylceramide, accounts for 5-10% of all PD cases. Our lab has a GBA mutant fly model that features symptoms similar to human PD – neurodegeneration, shortened lifespan, motor deficits, and increased protein aggregation. We hypothesize that GBA mutants fail to break down glucosylceramide, a lipid that is common in membranes of many pathogens. This glucosylceramide accumulation is responsible for triggering the innate immune response system, in turn causing inflammation leading to neural death. I explore innate immune system activation in the Toll, lmd, and Jak/Stat pathways and determine if a GBA mutation causes glucosylceramide accumulation to trigger the activation causing neuron loss, and the associated phenotypes. To do this, I will use reporters to test expression of these transgenic lines in a chemical assay, observe gene activation and protein expression following manipulation of glucosylceramide production, and use RNA interference to understand the effect of specific genetic perturbations in mutants. It is extremely valuable to understand the pathway of neuroinflammation to neurodegeneration, the mechanisms behind this, and the cascading influences they may have. My novel research on the impact of innate immune response pathways streamlines our comprehension of the mechanistic influences of PD and many other neurodegenerative diseases, leading to treatment and prevention development.

Although fertilization is a crucial process for sexually reproducing organisms, the molecular mechanisms mediating this process in humans and other animals remain unknown. In part, this is due to reproductive proteins evolving rapidly between closely related species. Sequence diversification and gene duplication makes establishing gene orthology (the same genetic locus in different species) difficult. The step of fertilization that I study is the sperm binding to the egg coat. ZP3 is an egg-coat protein and ZP3r is its sperm binding partner in mice. My research has shown that the human ortholog of mouse ZP3r has been misidentified as C4BPA and is instead the pseudogene C4BPAP1. This misidentification is likely due to difficulties in determining orthology when the gene is surrounded by duplicates. C4BPAP1 shows testes-specific expression despite being a pseudogene, consistent with a function in fertilization. However, pseudogenizing mutations in the C4BPAP1 locus have accumulated in humans and other great apes, indicating this gene may no longer play a role in fertilization in these species. Phylogenetic analysis of loci performed by maximum likelihood (Phylip) and synteny analysis shows that C4BPAP1 is the human ortholog of mouse ZP3r. By comparing protein sequences across species, we have found that ZP3r/C4BPAP1 varies in the number of CCP domains. The ancestral form of this gene in mammals contains 8 CCP domains, meanwhile mice contain 7. During my next two quarters, we will examine the evolution of the ZP3r gene family, look at ZP3r’s conservation across mammals, and determine whether ZP3r is undergoing positive selection, a characteristic common to functional fertilization genes. Our research is refining the identification of genetic loci implicated in mammalian fertilization. The precise identification of these loci is important for the development of novel contraceptives and for studies of infertility.
 

Copy number variants (CNVs) are typically a result of chromosomal duplications and deletions, making them a well-known form of genetic diversity and associated with several human disorders. Little is known about CNVs within humans and insight into CNV mechanisms would help scientists better understand, and potentially treat, many genome-based diseases. A particular form of CNV within humans is the inverted triplication of a gene without any chromosomal deletions. A similar phenomenon is observed at the SUL1 gene in Saccharomyces cerevisiae yeast cells, providing a model for studying such CNVs. The Brewer lab proposed a replication error mechanism responsible for this specific amplification described as Origin Dependent Inverted Replication Amplification (ODIRA). What impacts the initiation of this mechanism is unknown, but the proximity of SUL1 to the telomere raises the possibility that properties of the telomere may stimulate replication errors responsible for the triplication. I conducted a literature review analyzing 11 articles discussing various CNV mechanisms and telomeric influence on replication to establish their relationship. Through my review, I found a likely method to test whether the telomere does affect ODIRA. I propose utilizing a CRISPR-Cas9 based method to first circularize and eliminate the telomeres of the chromosome. Subsequently, the chromosome would be linearized at a location distant from the original telomere sites, effectively moving the entire SUL1 site away from potential telomeric influence. This research design allows for a comparison of SUL1 amplification events within the original and the restructured chromosomes and would reveal whether the telomeric region influences inverted SUL1 amplification formation. An observed reduction in rates of SUL1 amplification events with the reconstructed chromosomes would indicate telomeric influence on the amplification mechanism prompting further examinations within that genomic region. Attaining a greater understanding of this CNV mechanism yields information for future implications in genetic disease research.