Characterization of Cbf11 and Mga2 interactions in the fission yeast

• Název práce v češtině: Charakterizace interakcí proteinů Cbf11 a Mga2 v poltivé kvasince
• Název v anglickém jazyce: Characterization of Cbf11 and Mga2 interactions in the fission yeast
• Klíčová slova: metabolismus lipidů, transkripční faktor, protein-proteinové interakce, poltivá kvasinka
• Klíčová slova anglicky: lipid metabolism, transcription factor, protein-protein interactions, fission yeast
• Akademický rok vypsání: 2021/2022
• Typ práce: diplomová práce
• Jazyk práce: angličtina
• Ústav: Katedra buněčné biologie (31-151)
• Vedoucí / školitel: RNDr. Martin Převorovský, Ph.D.
• Řešitel: skrytý - zadáno a potvrzeno stud. odd.
• Datum přihlášení: 13.10.2021
• Datum zadání: 13.10.2021
• Datum potvrzení stud. oddělením: 13.10.2021
• Datum odevzdání elektronické podoby: 09.08.2023
• Datum proběhlé obhajoby: 14.09.2023
• Oponenti: RNDr. Michal Čáp, Ph.D.

Předběžná náplň práce

Lipids serve as cellular energy sources, membrane building blocks, and signaling molecules. Their metabolism is extensively regulated at multiple levels. In the fission yeast Schizosaccharomyces pombe, three transcription factors have been identified that regulate genes involved in the production of triacylglycerols (Cbf11, Mga2) [1,2] and sterols (Sre1) [2]. Interestingly, there appears to be crosstalk between all these TFs. We have recently found that Cbf11 and Mga2 share target genes, both are required for mitotic fidelity, and our genetic and preliminary proteomic data suggest that these TFs form a complex. This would represent a novel regulatory paradigm, bringing together a member of the CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1 transcription factor family (Cbf11) with a functional analog of the mammalian SREBP-1 transcription factor (Mga2).
The primary aims of this MSc thesis project are to:
1) Validate the physical interaction between Cbf11 and Mga2 using co-immunoprecipitation and western blots.
2) Determine how Cbf11 and Mga2 protein levels and posttranslational modifications change under different growth conditions (nitrogen source, temperature) using western blots and proteomics.
3) Identify other potential interaction partners of Cbf11 and Mga2, and how their interactions change under different growth conditions, using protein affinity purification and proteomics.
All resources (tagged yeast strains, antibodies etc.) required for the completion of the project are readily available in the Laboratory of Microbial Genomics, and project funding will be covered by the GAUK grant no. 248120. Furthermore, the project will benefit from data produced in related projects in the laboratory (genome-wide DNA occupancies of the TFs, mitotic defects in TF mutants, crosstalk with nitrogen metabolism).
If time permits, the project can be extended with the following additional aims:
4) Identify the protein regions of Cbf11 and Mga2 responsible for their physical interaction using various truncated and point-mutated constructs and co-IP + western blots.
5) Determine the phylogenetic distribution of Cbf11 and Mga2 by mining publicly available genome sequence databases.
Overall, the project will help characterize a novel TF complex regulating fundamental aspects of metabolism in a widely used model organism. Furthermore, the results may be relevant to other fungal species of clinical, agricultural or biotechnological importance.

References
[1] CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast. Převorovský M, Oravcová M, Zach R, Jordáková A, Bähler J, Půta F, Folk P. Cell Cycle. 2016 Nov 16;15(22):3082-3093
[2] Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors. Burr R, Stewart EV, Espenshade PJ. J Biol Chem. 2017 Mar 31;292(13):5311-5324.