Promjena vezanja pauzirajućih faktora i ekspresije gena ovisnih o starenju

master's thesis

• Title (english): Age-related changes in the expression and recruitment of transcription factors in murine liver
• Author: Dora Grbavac
• Mentor: Peter Tessarz (mentor)
• Mentor: Vlatka Zoldoš (sumentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Committee member: Ana Previšić (član povjerenstva)
• Committee member: Dunja Leljak-Levanić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-01-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Starenje dovodi do progresivnog narušavanja homeostaze organizma, te posljedično povećanog rizika
obolijevanja od različitih bolesti povezanih sa starenjem. Pravilna ekspresija gena ključna je za stanični
identitet, pa su stoga promjene regulacije ekspresije vjerojatno jedan od prvih događaja narušenih
starenjem. Posljednji korak kompleksnog procesa regulacije transkripcije gena - prijelaz RNA
polimeraze II (Pol II) iz koraka inicijacije u elongaciju - karakterizira pauziranje Pol II 20-60 pb
nizvodno od mjesta početka transkripcije. Tijekom starenja dolazi do globalnog smanjenja ovakvog
pauziranja proksimalno od promotora. U ovom diplomskom radu pratila sam promjene u ekspresiji gena
povezane sa starenjem, te u vezanju transkripcijskih faktora koji reguliraju pauziranje Pol II u jetri miša:
Spt4, NELF-A, PAF1, Spt16, Spt6. Analiza ekspresije transkripcijskih faktora pokazala je smanjenje
razine Spt16, podjedinice kompleksa FACT koji olakšava prolaz Pol II kroz nukleosome, povezano sa
starenjem. Kako bih pratila promjene u vezanju transkripcijskih faktora u promotorskoj regiji gena,
uspostavila sam protokol za kromatinsku imunoprecipitaciju popraćenu PCR metodom u stvarnom
vremenu (ChIP-qPCR), u mišjoj jetri. Eksperimenti ChIP-qPCR na odabranom lokusu Hsf1 pokazali su
smanjenje vezanja pauzirajućeg faktora Spt4 koji stabilizira pauzirani Pol II kompleks. Ovaj rezultat
upućuje na efikasnije otpuštanje pauzirane Pol II u produktivnu elongaciju i u skladu je sa zabilježenim
smanjenjem pauziranja Pol II proksimalno od promotora koje se dešava tijekom starenja. Nadalje, u
ovom radu sam priredila i biblioteku sekvenci za analizu metodom ChIP-seq koja bi mogla dati
pouzdanije podatke o smanjenju vezanja Spt4 ovisno o starenju.

Abstract (english)

Ageing leads to the progressive impairment of homeostasis and is secondary to multiple aberrant
processes. Proper gene regulation is a key to cellular identity, therefore an aberrant change in gene
expression is likely one of the initial processes associated with ageing. Regulation of transcription is a
complex process. The final regulatory step – the transition from initiation to elongation of RNA
Polymerase II (Pol II) – is characterized by Pol II pausing 20-60 bp downstream of the transcription
start site. This promoter-proximal pausing of Pol II is demonstrated to globally decrease during ageing.
Here, I monitor age-related changes in expression and recruitment of the transcription factors that
regulate Pol II pausing in murine liver: Spt4, NELF-A, PAF1, Spt16, Spt6. Western blot analysis of
transcription factor expression patterns revealed an age-related decrease in Spt16 abundance, a subunit
of the FACT complex that facilitates Pol II passage through nucleosomes. For monitoring the
recruitment of transcription factors to gene promoters, chromatin immunoprecipitation followed by
qPCR (ChIP-qPCR) was performed after establishing a ChIP protocol for murine liver tissue. ChIPqPCR
experiments at an exemplary locus Hsf1 demonstrated an age-related decrease in the recruitment
of Spt4, a pausing factor that stabilizes the paused Pol II complex. This observation suggests a more
efficient Pol II pause release with age and is in line with the observed age-related decrease in promoterproximal
Pol II pausing. Additionally, libraries for ChIP-seq data analysis, which is expected to confirm
the age-related decrease in Spt4 recruitment on a genome-wide level, have been established in this work.
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• Keywords: Pol II; pauziranje proksimalno od promotora; DSIF; NELF; FACT; Spt6; PAF; ChIP
• Keywords (english): Pol II; promoter-proximal pausing; DSIF; NELF; FACT; Spt6; PAF; ChIP-qPCR
• Language: croatian
• URN:NBN: urn:nbn:hr:217:644074
• 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: 2023-01-28
• Created on: 2021-02-12 14:03:45