SAGINI, HANAN,ABDUALRAHMAN,M (2022) Characterisation of the bZIP Transcription Factor Hac1 Key Domains in Transcriptional Repression of Early Meiotic Genes. Doctoral thesis, Durham University.
|PDF (PhD Thesis ) - Accepted Version|
In Saccharomyces cerevisiae, accumulation of unfolded proteins in the endoplasmic reticulum promotes HAC1 mRNA splicing by the transmembrane kinase-endoribonuclease Ire1. Spliced Hac1 consists of 238 amino acids (Hac1i), compared to its unspliced form of 230 amino acids (Hac1u). Hac1i negatively regulates differentiation in response to nitrogen starvation, such as meiosis. Hac1i represses transcription of early meiotic genes (EMGs) under the control of URS1 promoter element. URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. Recruitment of Rpd3L histone deacetylase complex by Ume6-Ime1 to URS1 facilitates repression of EMGs by Hac1i in response to nitrogen starvation, interfering with sporulation.
The bZIP transcription factor Hac1i consists of a DNA-binding domain, leucine zipper domain, and a C-terminal transactivation domain. These domains share highly conserved residues with other well studied bZIP transcription factors, such as Gcn4. Here we observe the effect of different Hac1 constructs in regulating EMGs transcription through USR1 in nitrogen sensing. We constructed and characterised Hac1u, and three classes of point mutants in the different domains of the Hac1i transcription factor, Hac1i-N49L mutation in the DNA-binding domain, Hac1i-L67P/L74P/V81P mutants in the leucine zipper, and a S238A mutation in the transactivation domain requirement. Experiments with URS1-lacZ reporter genes demonstrated that Hac1i has higher repression potential than Hac1u and requires its transactivation domain for repression of EMGs in response to nitrogen starvation. Hac1i bZIP mutant residues partially disrupt repression of EMGs transcription by Hac1i; consequently, these point mutations interfere with binding of Hac1 to DNA, dimerisation of Hac1, and transcriptional activation by Hac1. Data from these T4C-URS1-lacZ reporter experiments were confirmed using northern analysis of EMGs such as IME2, HOP1, and SPO13. Further northern analysis eliminated several known constituents of Rpd3L histone deacetylase complex (DEP1, CTI6, RXT2, RXT3, PHO23, SDS3, SAP30), as transcriptional targets of Hac1i. In summary, Hac1i, its bZIP domain key residues, and its transactivation domain are required to repress transcription of EMG via URS1 in response to nutrient sensing. Overall, the findings provided novel insights into how Hac1 represses early meiotic genes via the bZIP characteristics in S. cerevisiae. Future work will investigate and develop methods to screen and identify Hac1i potential targets involved in negatively regulating EMGs in response to nitrogen starvation, and characterisation whether Hac1 inhibits nucleosomal acetylation in the promoters of EMGs.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||10 Aug 2022 15:54|