DOMAIN-SPECIFIC INTERACTIONS OF BPM1 WITH DMS3 AND RDM1 IN RNADIRECTED DNA METHYLATION

doctoral thesis

• Title (english): INTERAKCIJE DOMENA PROTEINA BPM1 S PROTEINIMA DMS3 I RDM1 U RNAPOSREDOVANOJ METILACIJI DNA
• Author: Mateja Jagić
• Mentor: Dunja Leljak-Levanić (mentor)
• Committee member: Biljana Balen (predsjednik povjerenstva)
• Committee member: Petra Peharec Štefanić (član povjerenstva)
• Committee member: Zdravko Lorković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-04-21, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology
• Universal decimal classification (UDC): 577 - Biochemistry. Molecular biology. Biophysics

Abstract

BPM1 protein belongs to the MATH-BTB protein family, characterized as adaptors of CUL3-
dependent E3 ubiquitin ligase complex responsible for recognizing and directing substrates for
proteasomal degradation. An initial mass spectrometry experiment identified a key component of
the RdDM, the DMS3 protein, as a potential BPM1 partner. Since RdDM is the major epigenetic
mechanism responsible for de novo DNA methylation in plants, the goal of this work was to further
investigate the role of BPM1 in RdDM. By mass spectrometry, along DMS3, another component
of RdDM, the RDM1, was determined in complex with BPM1. Their direct interactions with BPM1
were confirmed by Y2H, pull-down, microthermophoresis and FRET-FLIM. Colocalization of
BPM1, DMS3, and RDM1 was observed in the nucleus, indicating the site of their common
function. The reduced activity of the RdDM-controlled FWA gene promoter in the BPM1-
overexpressing line together with protein-protein interaction data strongly suggest a role for BPM1
in controlling gene expression as part of RdDM.

Abstract (croatian)

Protein BPM1 pripada porodici proteina MATH-BTB koji su opisani kao adapteri u CUL3-
ovisnom kompleksu ubikvitinskih ligaza E3 odgovornih za prepoznavanje i usmjeravanje supstrata
na proteasomalnu razgradnju. U preliminarnom eksperimentu spektrometrije masa kao potencijalni
partner proteina BPM1 utvrđena je važna komponenta mehanizma RdDM, protein DMS3. Budući
da je RdDM glavni epigenetski mehanizam odgovoran za metilaciju DNA de novo u biljkama, cilj
ovog rada bio je detaljnije razjasniti ulogu BPM1 u sklopu RdDM-a. Spektrometrijom masa u
kompleksu s proteinom BPM1 uz DMS3, utvrđena je i druga komponenta RdDM-a, protein RDM1.
Njihove izravne interakcije s BPM1 potvrđene su metodama sustava dvaju kvašćevih hibrida,
proteinske kopurifikacije, mikrotermoforeze te metodom FRET-FLIM. Kolokalizacija proteina
BPM1, DMS3 i RDM1 utvrđena je u jezgri što ukazuje na mjesto njihove zajedničke funkcije.
Smanjena aktivnost promotora gena FWA kontroliranog RdDM-om u liniji s prekomjernom
ekspresijom gena BPM1 zajedno s rezultatima proteinskih interakcija snažno upućuju na ulogu
proteina BPM1 u kontroli genske ekspresije u sklopu mehanizma RdDM.

• Keywords: MATH-BTB; BPM1; RdDM; DDR complex; protein interactions; Arabidopsis thaliana
• Keywords (croatian): MATH-BTB; BPM1; RdDM; kompleks DDR; interakcije proteina; Arabidopsis thaliana
• Language: english
• URN:NBN: urn:nbn:hr:217:359683
• Promotion: 2023
• Study programme: Title: Biology; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija (doktor/doktorica znanosti, područje prirodnih znanosti, polje biologija)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-05-04 13:18:51