Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Cellular, Molecular and Biomedical Sciences

First Advisor

Stephanie McKay

Second Advisor

Matthew Wargo


Stable epigenetic markers, including DNA methylation and hydroxymethylation, have distinctive impacts on transcription and subsequently have the potential to impact phenotypic variation. DNA 5-hydroxymethylcytosine (5-hmC) is a stable epigenetic mark formed from the oxidation of the methyl group of 5-methylcytosine (5-mC) and has different biological impacts than 5-mC. While the specific impact of hydroxymethylation on transcription has not been fully explored, 5-hmC has been associated with many cellular processes, including neuron development and transcription. Hydroxymethylation has been identified in all tissues in mammalian genomes but is predominant in central nervous system tissues and abundant in the brain. However, hydroxymethylation is tissue specific and to elucidate the function of 5-hmC, we must first determine genomic distribution of 5-hmC in various tissues. Previous 5-hmC work in humans and mice provide some insight into the distribution and potential function of 5-hmC in various brain tissues, but investigation of hydroxymethylation in livestock is notably lacking. We sought to examine 5-hmC in cattle and sheep brain tissues to characterize hydroxymethylation within and between species in multiple brain tissues. A combination of whole genome bisulfite sequencing (WGBS) and oxidative reduced representation bisulfite sequencing (oxRRBS) was utilized to identify 5-hmC sites at a single nucleotide resolution. In cattle, 5-hmC distribution is tissue-specific, and the prefrontal cortex (PFC) has increased 5-hmC sites compared to the hippocampus (Hi) and periaqueductal gray (PAG). Hydroxymethylation also varies by docility phenotype, with differentially hydroxymethylated cytosines (DhMCs) identified in all four tissues. Bovine chromosome 27 was identified as a hot spot for 5-hmC in all four tissues, and the Hi and PFC contained DhMCs on gene RN18S1, which plays a crucial role in protein production. When hydroxymethylation in the sheep cerebellum was compared in adult and fetal sheep, a higher number of 5-hmC sites were found in fetal sheep compared to adults, although 52 5-hmC sites were hydroxymethylated in all animals. Furthermore, 343 DhMCs were identified between fetal and adult sheep and were distributed genome wide. Gene FGF14, a fibroblast growth factor, was highlighted as being hydroxymethylated in all eight animals and differentially hydroxymethylated between fetal and adult groups and is associated with cerebellar ataxia, which affects motor control in animals. Hydroxymethylation in adult sheep cerebellum was also compared to adult cattle cerebellum and showed that the highest hydroxymethylated chromosomes in cattle (BTA25) and sheep (OAR24) were orthologous, indicating similarities in the genome-wide view of hydroxymethylation. Additionally, a detailed comparison of 5-hmC sites on highly hydroxymethylated genes was performed and included genes with roles in epigenetic regulation, such as KMT2C, GSE1, and CUX1, suggesting an interconnectedness between 5-hmC and other forms of epigenetic regulation. Collectively, this work provides new insights into the role of 5-hmC in cattle and sheep brain tissues and has implications for future studies on livestock production and animal health. These data showed that 5-hmC was highly tissue-specific and varied by species and age, highlighting the need for individual analysis of central nervous system tissues. The genome-wide distribution and dynamic function of 5-hmC in both cattle and sheep brain tissues necessitates further studies into the role of 5-hmC with respect to economically important traits including animal health traits as well as selection and domestication.



Number of Pages

207 p.

Available for download on Friday, April 18, 2025