Deans’ Summer Research Fellowship (DSRF) Story: Jorge Mato Frontela

Author: Jorge Mato Frontela

What was your major research question for the DSRF project and where do you hope this project will go?

Cells need to adapt to changes in available carbon sources, which may require rapid changes in gene expression. This regulation starts at the genomic level, when genes are transcribed. In eukaryotic cells, genes are wrapped around multimeric proteins called histones, which help pack all the cellular DNA inside the nucleus. To initiate transcription, cells add a methyl group to lysine 4 of Histone 3, but this depends on the previous addition of a protein called ubiquitin to Histone 2B. Ubiquitin is added by the enzyme Rad6 and removed by Ubp8, which localizes to the nucleus when yeast cells ferment and to the mitochondria when they respire. My main question this summer was to understand the role that Ubp8 played when cells rewired their metabolism to respire, hypothesizing that the absence of Ubp8 in the nucleus would lead to increased levels of ubiquitinated Histone 2B (H2Bub). I found that H2Bub levels increased when cells grew in respiratory conditions. To assess the role of Ubp8, I deleted it from the genome and measured levels of H2Bub. I found that when ubp8Δ cells use fermentable carbon sources, H2Bub levels increase, but when they respire, H2Bub bands disappear. Then I asked if the absence of H2Bub was because ubp8- cells were unable to respire. Measuring mitochondria abundance through mitochondrial Porin1 levels showed that deletion of UBP8 led to decreased levels of mitochondria compared to WT. Additionally, growth assays indicated that these cells failed to grow in respiratory conditions. Since Porin1 levels were not completely depleted, I asked if other factors that likely impaired ubp8- cells' ability to respire. I hypothesized that the absence of H2Bub may have led to the absence of H3 methylation, thus impairing cells’ ability to initiate transcription through this mechanism. To test this, I measured H3 methylation at lysine 4 via western blot. Unexpectedly, H3 methylation persisted despite the absence of H2Bub, indicating Ubp8 regulation simultaneously impacts mitochondrial function and transcription methylation during metabolic rewiring. Together, these findings help understand the role of Ubp8 in connecting transcription regulation. They also challenge the notion that H3 methylation at lysine 4 depends on H2B ubiquitination, opening new avenues to reconsider how these histone marks interact.

What challenges did you encounter during your research and how did you work through these difficulties?

One challenge in this project was culturing yeast cells that had been deleted from UBP8 in respiratory conditions. These cells do not grow in media with a carbon source that they cannot ferment, such as glycerol or ethanol. However, I needed to culture these cells in this condition to explore the role of Ubp8 in chromatin regulation and metabolic rewiring. To solve this, I tried many different delusions and even wrote an R script to streamline the dilution calculation.

What sparked your interest in this field?

What I like most about biology is the complexity of biological systems, especially at the cellular and molecular level. Thousands of genes and even more proteins support life in a cell, and, although they may seem chaotic, there is order if you look closely enough. Finding that order is what I really enjoy about research in biology.

Curious about DSRF?

This project was made possible through the Deans’ Summer Research Fellowship (DSRF)—a unique opportunity for rising juniors and seniors in the Trinity College of Arts & Sciences to pursue funded summer research full-time!