Multi-omics studies of transcription regulatory mechanisms during human cell fate transition
von Kseniia Lysakovskaia
Datum der mündl. Prüfung:2023-02-20
Erschienen:2024-02-16
Betreuer:Prof. Dr. Patrick Cramer
Gutachter:Prof. Dr. Patrick Cramer
Gutachter:Prof. Dr. Matthias Dobbelstein
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-10357
Dateien
Name:Dissertation_Kseniia_Lysakovskaia.pdf
Size:3.34Mb
Format:PDF
Description:Doctoral thesis
Diese Datei ist bis 19.02.2025 gesperrt.
Zusammenfassung
Englisch
RNA polymerase II (Pol II) transcription is an essential process in living organisms that requires accurate regulation and coordination in order to produce protein-coding and many types of non-coding transcripts. One of the highly regulated transcriptional steps is promoter-proximal Pol II pausing that occurs shortly after initiation of transcription. Paused Pol II can either be released into productive elongation of transcription or terminated prematurely. Promoter-proximal Pol II pausing was demonstrated to tune gene expression in the cell fate transition processes ensuring controlled and rapid transcriptional response. However, the exact mechanisms underlying these processes remain to be further elucidated in a quantitative manner. Here, we investigate promoter-proximal Pol II regulation throughout the cell type switching by employing a reprogrammable human cell transdifferentiation as a model system. In order to assess the kinetics of Pol II transcription, we apply a multi-omics approach combining a measurement of new RNA synthesis by TT-seq and profiling of transcriptionally engaged Pol II by mNET-seq. We additionally introduce the estimation of promoter-proximal Pol II stability at terminal time points of transdifferentiation using a high-resolution ChIP-nexus technique with the time series of initiation inhibition treatment. We show the global changes in the transcription kinetic parameters including productive initiation frequency (pIF) and apparent pause duration (aPD) for the defined gene groups across the time course of transdifferentiation. Strikingly, the pausing-related factors occupancies analyzed by ChIP-seq exhibit the highest correlation with the pIF and partial correlation with the aPD parameters. Our stability measurements from the ChIP-nexus data indicate that Pol II is highly dynamic and relatively short-lived in the pausing region. Importantly, we suggest distinct mechanisms of promoter-proximal Pol II regulation for the upregulated (iMac) and downregulated (pre-B) gene groups based on our Pol II kinetics and stability estimations during the transdifferentiation. The productive transcription of pre-B genes appears to be downregulated through an increase of the prematurely terminating Pol II fraction rather than stable Pol II pausing. Also, the upregulation of the iMac genes is mediated by an increase of the actual transcription initiation rate along with modulating the productive RNA synthesis via prematurely terminating Pol II fraction in the promoter-proximal region. Taken together, our results illuminate the importance of the Pol II premature termination in transcriptional regulation of the cell stage-specific gene groups throughout the cell fate transition process. We note that our findings can be transferred to the biologically relevant systems for the future medical applications and drug development.
Keywords: Gene regulation; Transcription; Transcription initiation; Promoter-proximal Pol II pausing; Premature termination; Cell type transition; Transdifferentiation; Precursor B-cell leukemia; Macrophage
