Ciljana modulacija CpG metilacije gena HNF1A upotrebom molekularnog alata CRISPR/dCas9- TET1 i utjecaj na njegovu transkripciju

master's thesis

• Title (english): Targeted modulation of HNF1A gene's CpG methylation using CRISPR/dCas9-TET1 molecular tool and effect on its transcription
• Author: Tomislav Barberić
• Mentor: Vlatka Zoldoš (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Domagoj Đikić (član povjerenstva)
• Committee member: Ana Previšić (član povjerenstva)
• Committee member: Mirta Tkalec (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-02-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

HNF1α je transkripcijski faktor koji sudjeluje u regulaciji ekspresije mreže gena uključenih u niz molekularnih i metaboličkih procesa u jetri, gušterači, crijevima i bubrezima. U β stanicama gušterače, mutacije u genu HNF1A vode do poremećaja otpuštanja inzulina uslijed stimulacije glukozom, a fenotip u ljudi je dijabetes tip MODY3. Pretpostavlja se da bi epigenetičko utišavanje gena HNF1A metilacijom moglo imati isti efekt na ekspresiju gena te fenotip kao i genska mutacija. Ovo istraživanje imalo je za cilj transfecirati staničnu liniju 1.1B4, model za istraživanje β stanica gušterače, fuzijskim proteinom TET1-dCas9 te istražiti dolazi li do promjene metilacije na 4 CpG mjesta koja su prijašnjim korelacijskim analizama otkrivena kao kandidat regulatorna mjesta za transkripcijsku aktivnost gena HNF1A. U tu svrhu naveo sam fuzijski protein TET1-dCas9 pomoću jedne molekule sgRNA na 4 kandidat CpG mjesta u 1. egzonu gena HNF1A. Nakon uspješne transfekcije analizirao sam metilaciju citozina pirosekvenciranjem nakon bisulfitne konverzije molekule DNA te sam potvrdio smanjenje stupnja metilacije na sva 4 CpG mjesta u periodu od 30 dana nakon transfekcije stanica 1.1B4. Također sam analizirao razinu transkripcije pomoću metode RT-qPCR upotrebom proba TaqMan te utvrdio povećanu razinu transkripcijske aktivnosti gena HNF1A u uspješno transfeciranim stanicama. Rezultati ovog istraživanja potvrđuju da epigenetička manipulacija 4 kandidat CpG mjesta dovodi do povećanja transkripcijske aktivnosti gena HNF1A što ukazuje na regulatornu ulogu ova 4 CpG mjesta u stanicama 1.1B4.

Abstract (english)

HNF1α is a transcription factor involved in regulating the expression of a network of genes involved in a number of molecular and metabolic processes in the liver, pancreas, intestines and kidneys. Mutations in the HNF1A gene lead to impaired glucose stimulated insulin secretion in pancreatic β cells, and the resulting phenotype in humans is MODY3 type diabetes. It is hypothesized that epigenetic silencing of the HNF1A gene could have the same effect on gene expression and phenotype as the gene mutation. The aim of this study was to transfect 1.1B4 cells, which serve as a model for pancreatic β cells, with a TET1-dCas9 fusion protein in order to change methylation level on the 4 CpG sites that previous corelation studies identified as candidate regulatory sites for the transcription activity of the HNF1A gene. To achieve this, TET1-dCas9 fusion protein was targeted to the 4 candidate CpG sites in the 1st exon of the HNF1A gene by using a single sgRNA. Analysis of DNA methylation by pirosequencing after bisulphate DNA conversion confirmed a decrease in the methylation levels at all 4 CpG sites during the 30 day period following transfection. RT-qPCR method using TaqMan probes revealed an increase of HNF1A transcriptional activity following the epigenetic manipulation with TET1-dCas9. The results of this study showed that epigenetic manipulation of the 4 candidate CpG sites leads to an increase in the HNF1A gene transcriptional activity, which confirms a regulatory role of these 4 CpG sites for HNF1A expression in 1.1B4 cells.

• Keywords: gen HNF1A; CRISPR-dCas9; TET1; demetilacija DNA; stanice 1.1B4
• Keywords (english): HNF1A gene; CRISPR-dCas9; TET1; DNA demethylation; 1.1B4 cells
• Language: croatian
• URN:NBN: urn:nbn:hr:217:218306
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-02-27
• Created on: 2020-07-13 07:22:47