Across eukaryotes, homopolymeric repeats of amino acids are enriched in regulatory proteins such as transcription factors and chromatin remodelers. These domains play important roles in signaling, binding, prion formation, and functional phase separation. Azf1p is a prion-forming yeast transcription factor that contains two homorepeat domains, a polyglutamine and a polyasparagine domain. In this work, we report a new phenotype for Azf1p and identify a large set of genes that are regulated by Azf1p during growth in glucose. We show that the polyasparagine (polyN) domain plays a subtle role in transcription but is dispensable for Azf1p localization and prion formation. Genes upregulated upon deletion of the polyN domain are enriched in functions related to carbon metabolism and storage. This domain may therefore be a useful target for engineering yeast strains for fermentation applications and small molecule production. We also report that both the polyasparagine and polyglutamine domains vary in length across strains of S. cerevisiae and propose a model for how this variation may impact protein function.

Across eukaryotes, homopolymeric repeats of amino acids are enriched in regulatory proteins such as transcription factors and chromatin remodelers. These domains play important roles in signaling, binding, prion formation, and functional phase separation. Azf1p is a prion-forming yeast transcription factor that contains two homorepeat domains, a polyglutamine and a polyasparagine domain. In this work, we report a new phenotype for Azf1p and identify a large set of genes that are regulated by Azf1p during growth in glucose. We show that the polyasparagine (polyN) domain plays a subtle role in transcription but is dispensable for Azf1p localization and prion formation. Genes upregulated upon deletion of the polyN domain are enriched in functions related to carbon metabolism and storage. This domain may therefore be a useful target for engineering yeast strains for fermentation applications and small molecule production. We also report that both the polyasparagine and polyglutamine domains vary in length across strains of S. cerevisiae and propose a model for how this variation may impact protein function.

Repetitive regions within the genome can code for proteins that have repetitive amino acid sequences. These genomic regions can expand or contract and, in the few cases studied, these changes can affect protein function. This project tests how variation in the repetitive regions of the prion‐forming protein Azf1p, cause changes in gene expression and prion formation. Azf1p has two repetitive regions, a polyQ and a polyN, and is a transcriptional activator. We used CRIPSR‐Cas9 gene editing technology to cut within repetitive regions and allow the inherent DNA repair mechanisms in budding yeast, S. cerevisiae, to fix the break and create expansions or contractions. Subsequently we tested the effect of the repeat variation in different assays, including spotting assays, growth assays, phenotypic assays and qPCR analysis. We generated several variants in the polyQ domain of the AZF1 gene which were confirmed via sequencing. The research presented here will assess how variation within these regions affects various functions of AZF1, including growth on different carbon sources, colony morphology and changes in target gene expression. This work hopes to both create ways of using CRISPR‐Cas9 on repetitive regions that can be useful in the study of the function of these repeats and identify new functional domains within an important transcription factor, AZF1.

The C-terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. Despite being an essential domain within an essential gene, we have previously demonstrated that the CTD coding region is genetically unstable. Furthermore, yeast with a truncated or mutated CTD sequence are capable of promoting spontaneous genetic expansion or contraction of this coding region to improve fitness. We investigated the mechanism by which the CTD contracts using a tet-off reporter system for RPB1 to monitor genetic instability within the CTD coding region. We report that contractions require the post-replication repair factor Rad5p but, unlike expansions, not the homologous recombination factors Rad51p and Rad52p. Sequence analysis of contraction events reveals that deleted regions are flanked by microhomologies. We also find that G-quadruplex forming sequences predicted by the QGRS Mapper are enriched on the noncoding strand of the CTD compared to the body of RPB1. Formation of G-quadruplexes in the CTD coding region could block the replication fork, necessitating post-replication repair. We propose that contractions of the CTD result when microhomologies misalign during Rad5p-dependent template switching via fork reversal.

Asthma is a disease characterized by an excess of free-radical nitric oxide, NO, which leads to airway inflammation and obstruction. S-nitrosothiols (SNO’s) are an important regulator in NO signaling, helping to relieve oxidative stress as well as bronchodilate and relax airway smooth muscle cells. SNO’s exert most of their effects through the NO-cysteine interactions found particularly in S-nitrosocysteine (CSNO). The particular S-nitrosocysteine of interest in this paper is L-CSNO due to its ability to inhibit potassium channels associated with poor lung function, thereby improving ventilation in asthmatic patients. Potassium channels, amongst other ion channels as well, are a new target for therapeutic asthma treatment due to their ability to regulate vascular smooth muscle cells. KCN genes from bronchoscopy data from asthma and control patients were sequenced and analyzed against several measures for asthma and general lung function: asthma severity, FeNO, FEV1%, and androgen receptor gene expression. The goal of this study was to determine which KCN genes, and subsequent Kv channels, are associated with better lung function and which are associated with worse lung function/more severe asthma. The most beneficial KCN genes were found to be KCNA1 and KCNA4, whereas the KCN gene associated with the worst lung function was the KCNK6 gene family. Thus, a potential novel signaling pathway for asthma regulation may involve the binding of L-CSNO to the Kv channels encoded by the KCNK6 gene family in order to inactivate them and improve ventilation and overall lung function.