Mechanistic Study Of Methionine Related Genes And Their Regulation In Budding Yeast
Restricted (Penn State Only)
- Author:
- Lee, James
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 28, 2024
- Committee Members:
- Joseph Reese, Major Field Member
Lu Bai, Chair & Dissertation Advisor
Santhosh Girirajan, Program Head/Chair
Marina Feric, Major Field Member
Janine Kwapis, Outside Unit & Field Member
Jean-Paul Armache, Major Field Member - Keywords:
- phase separation
biomolecular condensates
3D genome organization
transcription factories
chromatin hubs
gene clusters - Abstract:
- Eukaryotic genomes are compacted into chromosomes that are spatially organized in the nuclei. Within the nucleus, chromosomes form extensive intra and inter contacts that can occur over long linear genomic distances. A subset of these interactions, such as promoter-promoter or promoter-enhancer looping, are thought to play a role in gene regulation. In some cases, multiple genes come together in 3D space to form “multi-gene clusters”. These clusters often involve co-regulated genes responding to stress or other types of environmental cues, such as heat shock, starvation, virus infection, or developmental signals. Microscopic evidence suggests these gene clusters may be related to a phenomenon named as “transcription factories”, where at least a fraction of Pol II, mediator, and nascent transcripts coalesce into distinct foci. Proteomic analysis of transcription factories showed that they are also enriched with other components in the transcription pathway, including transcription factors (TFs), histone modification enzymes, and chromatin remodelers. Overall, these observations suggest that some co-regulated genes can physically cluster into sub-nuclear compartments with elevated local concentrations of transcription-related factors. Although multiple examples of multi-gene-clusters have been reported, their mechanism of formation, and the functional roles these clusters have in organizing the 3D genome and gene regulation have been elusive. In this study, I utilize the inducible co-regulated MET gene regulon in haploid budding yeast to study how the activation of the MET regulon leads to the formation of MET “transcriptional hotspot”, a multi-gene cluster in the nucleus of budding yeast cells. Using high-resolution live cell imaging, I show the TFs involved in MET gene activation (Met4, Met32) form puncta in vivo and Met32 forms liquid-liquid phase separated condensates in vitro, in which Met4 can partition with. These data provide a possible mechanism of formation for the MET transcriptional hotspots. Additionally, I studied how the formation of the MET transcriptional hotspot leads to the regulation of the genes involved. By performing genome wide ChIP-seq on the core Met TFs (Met4, Met32, Met28, Cbf1), I identified 34 genomic regions that were co-bound by the Met TFs. mRNA-seq of yeast cells in MET repressed (+met) and active (-met) conditions confirmed that MET genes nearby the ChIP-seq peaks were induced in -met conditions. By labeling of two MET genes (MET13, MET6) where the Met TFs are bound by tetO-tetR-mCherry, I show that these genomic locations co-localize with the Met4 condensates. Finally, to study how the Met TF puncta organize the chromatin contacts among its target genes, I utilized a novel chromosomal contact method (MTAC) to validate our hypothesis that upon MET gene activation, there is clustering of MET genes in 3D nuclear space. By performing MTAC with MET genes (MET6, MET13) as viewpoints (VPs), I show that five targets of Met TFs form intra and inter chromosomal contacts indicating that they are clustered in 3D nuclear space. These finding could shed new insights on how the formation of multigene clusters driven by TF molecular condensates could organize the 3D genome and impact gene regulation of the genes involved in the multigene clusters.