A bacterial surface display platform for the discovery of cytosine modification readers from cDNA libraries

Simple item page
dc.contributor.advisorSummerer, Daniel
dc.contributor.authorSchiller, Damian
dc.contributor.refereeMutschler, Hannes
dc.date.accepted2024-01-16
dc.date.accessioned2024-02-07T07:25:02Z
dc.date.available2024-02-07T07:25:02Z
dc.date.issued2023
dc.description.abstract5-methylcytosine (5mC) occurs in palindromic cytosine guanine dyads (CpGs) of mammalian genomes and is a key element of epigenetic transcription regulation. Central to these regulatory functions is the ability of cytosine modifications to modulate the interaction of chromatin proteins with DNA. In this context, reader proteins are known to selectively recognize the cytosine modification. Interactome profiling studies based on pulldowns of nuclear extracts in combination with mass spectrometry-based (MS) proteomics have been the main approach to discovering readers of 5mC. However, this approach may miss important (anti-)readers due to the competition of proteins for the probe and low expression levels. Moreover, direct readers cannot be distinguished from indirect binders of protein complexes. In the scope of this thesis, a bacterial surface display system for discovering novel reader candidates from human cDNA was established. In detail, our approach offers screenings of cDNA-encoded protein libraries independent of their endogenous expression levels and without competition with other proteins. It further allows for rapid iterative FACS selections with defined, fluorescently labeled on- and off-target DNA probes. Furthermore, the system benefits from the fast protein expression machinery and growth of bacterial cells. We created surface display libraries of full-length and fragmented coding sequences (CDS) from human cDNA and demonstrated the compatibility of the Intimin surface display platform with displaying human proteins and protein fragments. Moving on, we proved the functionality of our selection system by enriching known 5mC reader candidates from a display library of human thyroid cDNA. In addition, we were able to identify novel reader candidates. Therefore, this study offers a promising tool to complement existing efforts in discovering 5mC reader candidates. Furthermore, the tool bears the potential to be extended to the oxidized derivates of 5mC and their combinations in CpGs, which have not been investigated so far.en
dc.identifier.urihttp://hdl.handle.net/2003/42314
dc.identifier.urihttp://dx.doi.org/10.17877/DE290R-24151
dc.language.isoende
dc.subjectEpigeneticsen
dc.subjectCytosine 5-modificationsen
dc.subjectReader Proteinsen
dc.subjectScreeningen
dc.subjectBacterial surface displayen
dc.subjectcDNA librariesen
dc.subject.ddc570
dc.subject.ddc540
dc.subject.rswkEpigenetikde
dc.subject.rswkCytosinde
dc.subject.rswkModifikation <Biochemie>de
dc.subject.rswkScreeningde
dc.subject.rswkBakteriende
dc.subject.rswkZelloberflächede
dc.subject.rswkcDNSde
dc.subject.rswkDisplay libraryde
dc.subject.rswkMassenspektrometriede
dc.subject.rswkProteomanalysede
dc.titleA bacterial surface display platform for the discovery of cytosine modification readers from cDNA librariesen
dc.typeTextde
dc.type.publicationtypePhDThesisde
dcterms.accessRightsopen access
eldorado.secondarypublicationfalsede

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Dissertation_Schiller.pdf
Size:
14.91 MB
Format:
Adobe Portable Document Format
Description:
DNB
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
4.85 KB
Format:
Item-specific license agreed upon to submission
Description:
Download

Collections

Chemische Biologie