Structural and biochemical studies on chromatin-protein complexes in transcription initiation
Kumulative Dissertation
Datum der mündl. Prüfung:2023-12-08
Erschienen:2024-04-02
Betreuer:Prof. Dr. Patrick Cramer
Gutachter:Prof. Dr. Kai Tittmann
Gutachter:Prof. Dr. Argyris Papantonis
Gutachter:Dr. Marieke Oudelaar
Gutachter:Prof. Dr. Hauke Hillen
Gutachter:Dr. Juliane Liepe
Dateien
Name:PhD_Thesis_AbrilGarrido.pdf
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Format:PDF
Zusammenfassung
Englisch
Gene expression is an essential process in the cell for the development and homeostasis of any organism. The eukaryotic genome is tightly packed with nucleosomes, the basic unit of chromatin, which protects DNA integrity. Chromatin compaction regulates the DNA accessibility of genes, thus having an effect on transcription and gene regulation. Gene promoters are flanked by the -1 and +1 nucleosome upstream and downstream of the transcription start site (TSS), respectively, while the promoter region is usually nucleosome-depleted to facilitate the assembly of the transcription initiation machinery. In eukaryotes, the DNA-dependent RNA polymerase II (Pol II) transcribes protein-coding genes, which can initiate transcription at promoters only in complex with the general transcription factors (GTFs) TBP, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. Their recruitment nucleates the assembly of the RNA polymerase II pre-initiation complex (PIC), known to be one of the major transcription regulation checkpoints, so that RNA synthesis can begin. Previously, it was shown that the nucleosome is a key repressor of transcription initiation, yet the regulatory mechanism is unclear. To study the regulatory role of the +1 nucleosome on transcription initiation, I performed functional studies and investigated the inherent mechanism by electron cryo-microscopy (cryo-EM). First, we correlated RNA synthesis (TT-seq) with the position of the +1 nucleosome (MNase-seq) in HEK293 cells. We describe that promoter proximity of the +1 nucleosome correlates with a reduction of transcription in vivo in a gradual manner. I corroborated these results in vitro and determined two cryo-EM structures of a mammalian PIC engaged with a promoter-proximal +1 nucleosome. The PIC assembles productively when the edge of the +1 nucleosome is located ~20 bp downstream of the TSS. However, when the nucleosome shifts its position ~10 bp upstream, the PIC forms in an inactive conformation. The transcription factor IIH (TFIIH) is found in its closed state where only one lobe of its ATPase subunit XPB contacts DNA, incompatible with DNA opening. Therefore, I provide here a regulation mechanism by which a promoter-proximal +1 nucleosome can impair productive PIC assembly and thereby reduce transcription. Together with recent structural studies, it emerges that the effect of the +1 nucleosome on transcription depends on its relative position to the gene promoter and PIC.
Keywords: Transcription initiation; RNA polymerase II pre-initiation complex; Promoter-proximal +1 nucleosome; Gene regulation; Transcription reduction