The objective of this study was to be able to identify the genes that are regulated by enhancers of our interest, which are associated with rheumatoid arthritis. Enhancers are short regions of DNA that increase the transcription of a particular gene. Identifying an enhancers location can be difficult, because some enhancers can be found many base pairs away from its target gene. The significance of these enhancers is that mutations in enhancers lead to changes in phenotypes and identifying a single nucleotide polymorphisms (SNPs) at an enhancer region can be crucial for gene regulation. Previous research in the lab identified novel enhancer variants associated with rheumatoid arthritis but their target genes are not known. The use of Capture Hi-C data helped identify genes that are interacting with enhancer variants. This information was taken and used to identify what would happen when those putative enhancers are deleted using CRISPR-Cas9. The successful deletion of the enhancer variants leads to changes in gene expression which helped identify possible target genes. Deletion of variant enhancer rs:8019130 had an optimal deletion efficiency of 24.1%, and enhancer rs: 11574636 had an optimal deletion efficiency of 14.1%. The results demonstrated how deletion did occur but further runs using the tracking of insertions or deletions (indels) by decomposition (TIDE) must be done with the analysis of a primer for a 700bp amplicon size. Through an examination of these deletions we can further analyze the impacts they have in the regulation of gene expression and help identify the role that those genes have with rheumatoid arthritis.

The kinetochore plays a key role in aligning chromosomes during mitosis and helps ensure proper separation of duplicated DNA molecules into daughter cells. Some proteins that associate with the kinetochore regulate attachment to microtubules by destabilizing weak attachments and stabilizing strong attachments. In the model organism Saccharomyces cerevisiae, the Ipl1 kinase and Stu2 protein have similar effects on regulating tension on the kinetochore. Since Ipl1 is a kinase, and Stu2 is phosphorylated, we believe Stu2 function may be regulated by Ipl1. To test if Stu2 function is dependent on phosphorylation by Ipl1, we mutated possible Ipl1 phosphorylation sites and observed its effects on Stu2 function. Four Ipl1 target consensus sites within the Stu2 protein were selected for this study. We used site-directed mutagenesis techniques to alter these potential Ipl1 phosphorylation sites on Stu2 by mutating serine codons to alanine codons. We successfully constructed S40A and S430A/S593A mutations in Stu2 using the megaprimer whole plasmid (MEGAWHOP) cloning technique. We then introduced the mutated version of stu2 into yeast and conducted phenotype tests to determine if inactivating these putative phosphorylation sites affected its function. Our initial results indicated that phosphorylation at sites S430 and S593 may not be required for proper Stu2 function. We suggest that further testing of Ipl1 phosphorylation sites could reveal possible mutations that lead to malfunction of Stu2 that then affect the kinetochores ability to release improper attachments to microtubules. This would give insight into how signaling between kinetochore proteins can help to ensure proper chromosome segregation and avoid aneuploidy.

Treatments for cystic fibrosis (CF) have extended patients’ lifespan, resulting in CF-related diabetes (CFRD) as a major CF complication affecting 30-50% of adults. A key pathological feature of CFRD is the deposition of islet amyloid polypeptide (IAPP) as amyloid in pancreatic islets. In type 2 diabetes, Islet amyloid is associated with decreased beta-cell mass and function. Current mouse models of CFRD do not develop all the pathological features observed in human CFRD, including islet amyloid. To generate a mouse model of CFRD which exhibits islet amyloid deposition, we crossbred a CF mouse (CftrF508del) and a human IAPP (hIAPP) transgenic mouse and compared amyloid deposition in cultured islets from the resulting offspring. Islets from each of the mice genotypes (NT.Cftrdel/del (n=2), NT.Cftr+/del (n=4), NT.Cftr+/+ (n=5), hIAPP.Cftrdel/del (n=2), hIAPP.Cftr+/del (n=5), hIAPP.Cftr+/+ (n=3)) were isolated and cultured for 7 days in high (16.7 mM) glucose to induce amyloid formation. Islets were fixed in neutral-buffered formalin, paraffin-embedded and sectioned. Sections were stained with thioflavin S (for amyloid) after which amyloid prevalence (% islets with amyloid), amyloid severity (% islet area occupied by amyloid) were quantified. Islets from hIAPP transgenic mice developed islet amyloid when cultured in vitro, while islets from non-transgenic mice did not. Among hIAPP transgenic mice with different CF genotypes (hIAPP.Cftrdel/del, hIAPP.Cftr+/del, hIAPP.Cftr+/+, respectively), islet amyloid prevalence (74±13%, 73±11%, 81±5 %; p=0.89 by ANOVA)[NE2] or severity (3.4 ± 1.7%, 2.0 ± 1.1%, 3.57 ± 0.4%; p=0.64 by ANOVA) did not differ. The presence of amyloid in islets of mice expressing hIAPP genotypes supports the utility of this new mouse model to study this aspect of islet pathology as seen in human CFRD.