EFFECTS OF SIMULTANEOUS MANIPULATION OF DNA AND HISTONES ON THE EXPRESSION OF GENES INVOLVED IN EPITHELIAL-MESENCHYMAL TRANSITION USING CRISPR/dCas9 SYSTEM

doctoral thesis

• Title (croatian): UTJECAJ ISTOVREMENE MANIPULACIJE DNA I HISTONA NA EKSPRESIJU GENA UKLJUČENIH U EPITELNO-MEZENHIMSKU TRANZICIJU PRIMJENOM SUSTAVA CRISPR/dCas9
• Author: Nika Foglar
• Mentor: Vlatka Zoldoš (mentor)
• Committee member: Nataša Bauer (predsjednik povjerenstva)
• Committee member: Rosa Karlić (član povjerenstva)
• Committee member: Oliver Vugrek (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2024-03-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

Interplay between epigenetic modifications is essential for regulation of gene expression.
The adaptation of CRISPR/Cas9 technology for epigenetic research by introducing dead Cas9
(dCas9) fused to epigenetic effector domains (ED) has enabled significant advancements in this
field. I expanded the CRISPR/dCas9 modular system with five histone modifying domains
(G9a, G9a-me3, LSD1, KDM5A, macro1.1) and DNMT3L domain, as well as the multi-guide
module to accommodate up to 11 gRNAs. From a set of candidate genes involved in epithelial
to mesenchymal transition (EMT), ZEB1 and SNAI1 were selected for further epigenetic
manipulations in HepG2 cell line. Their CpG islands were simultaneously targeted with dCas-
DNMT3A combined with specific dCas9-ED fusions to understand the interplay of DNA
methylation and histone modifications. The combinations had synergistic effect on gene
expression and affected the EMT phenotype. Interplay between introduced modifications was
also observed as a locus specific synergistic increase in DNA methylation, and was not
disrupted by global histone acetylation increase after sodium butyrate treatment. The
combinatorial epigenetic effects were not replicated in A549 cell line, suggesting specificity of
epigenetic regulatory pathways.

Abstract (croatian)

Međudjelovanje epigenetičkih modifikacija je neophodno regulaciju ekspresije gena.
Korištenje CRISPR/Cas9 tehnologije u epigenetičkim istraživanjima pomoću inaktivnog Cas9
(dCas9) fuzioniranog s epigenetičkim efektorskim domenama (ED) omogućilo je značajan
napredak u ovom području. Proširila sam modularni sustav CRISPR/dCas9 s pet domena za
modifikaciju histona (G9a, G9a-me3, LSD1, KDM5A, makro1.1) i domenom DNMT3L, te
„multi-guide“ modul koji može primiti do 11 gRNA. Od nekoliko kandidat gena koji sudjeluju
u epitelno-mezenhimskoj tranziciji (EMT), odabrala sam ZEB1 i SNAI1 za daljnje epigenetičke
manipulacije u staničnoj liniji HepG2. Njihovi CpG otoci su istovremeno ciljani s DNMT3AdCas9
u kombinaciji s određenom dCas9-ED fuzijom kako bi se istražilo među-djelovanje
DNA metilacije i modifikacija histona. Kombinacije su imale sinergistički učinak na ekspresiju
gena i utjecale su na EMT fenotip. Simultano uvedene modifikacije pokazale su sinergistički
efekt na razini metilacije DNA na određenom lokusu, koji nije bio osjetljiv na povećanje
globalne acetilacije histona. Kombinacije dCas9 fuzija nisu djelovale na ekspresiju ciljanih
gena u A549 staničnoj liniji, što ukazuje na specifičnost epigenetičkih regulatornih putova.

• Keywords: DNA methylation; histone modifications; epigenetic crosstalk; CRISPR/dCas9 epigenetic engineering; CpG island; EMT
• Keywords (croatian): metilacija DNA; histonske oznake; epigenetičke interakcije; CRISPR/dCas9 epigenetičko inženjerstvo; CpG otok; EMT
• Language: english
• URN:NBN: urn:nbn:hr:217:444849
• Promotion: 2024
• Project: Number: KK.01.1.1.04.0085; Title: Genomsko inženjerstvo i genska regulacija u staničnim linijama i modelnim organizacijama tehnologija CRISPR / Cas9 CasMouse; Title: Genomic engineering and gene regulation in the cell lines model organisms CRISPR / Cas9 CasMouse technology; Acronym: CRISPR/Cas9-CasMouse; Leader: Vlatka Zoldoš; Jurisdiction: Croatia; Funder: EK; Funding stream: EFRR
• Study programme: Title: Doctoral study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti u području prirodnih znanosti (doktor/doktorica znanosti u području prirodnih znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2026-03-21
• Created on: 2024-04-02 10:13:19