Zusammenfassung (Englisch)
The landscape of transcript elongation factors associated with transcribing Pol II contains multiple factors with AIDs which potentially interact with the basic interface of the unwinding nucleosome: ELF1, SPT5, CTR9, SSRP1, SPT16 and SPT6 (Ehara et al., 2019). Here, four of those factors have been targeted by AP-MS approaches (ELF1, SPT6l, SSRP1 and SPT16) in the Arabidopsis cell culture system. ...
Zusammenfassung (Englisch)
The landscape of transcript elongation factors associated with transcribing Pol II contains multiple factors with AIDs which potentially interact with the basic interface of the unwinding nucleosome: ELF1, SPT5, CTR9, SSRP1, SPT16 and SPT6 (Ehara et al., 2019). Here, four of those factors have been targeted by AP-MS approaches (ELF1, SPT6l, SSRP1 and SPT16) in the Arabidopsis cell culture system. Indeed, each dataset contained histones, Pol II and other transcript elongation factors. In line, direct interaction of ELF1 with both DNA and histones was shown here and/or by students supervised in this thesis (Markusch, 2020; Thorbecke, 2019). Transient binding of histone and subsequent release qualify ELF1 as a histone chaperone. Concomitantly, double mutant analysis of ELF1 with other histone chaperones showed synergistic effects (Markusch, 2020), potentially indicating a concerted action in in tethering the nucleosome in the process of transcription. Additionally, it was shown that histone chaperone function of the FACT complex is regulated by post-translational modifications. Prior to this work, post-translational modifications of FACT were detected by a shotgun proteomics approach (Holzinger, 2015), leading to the detection of phosphorylations in the acidic region of SPT16 and acetylations in the DNA binding region of SSRP1. For SPT16 phospho-insensitive/unphosphorylated variants led to a decrease, phospho-mimetic variants however led to an increase in histone binding affinity, both in vivo and in vitro. Creation of phospho-insensitive and phospho-mimetic SPT16 transgenes complementing spt16-1 showed that altering the charge has an impact on the rescue-ability of the respective transgene. Particularly the phospho-insensitive variant was affected. H3 ChIP-Seq analysis revealed altered H3 occupancy for the phospho-insensitive variant, highlighting the need of SPT16 phosphorylation. Particularly the region upstream of TSS containing the fragile -1 nucleosome was affected, indicating a role for clearing the nucleosome depleted region. Changing the acetylation pattern in SSRP1, led to a loosening of chaperone/DNA contacts for acetylation-mimetic variants, which translated into a reduction of histone pulldown efficiency compared to the fully charged variant. Acetylation-insensitive and acetylation-mimetic SSRP1 transgenes complementing ssrp1-1 led to only mild phenotypic alterations. Finally, DOG1, the quantitative trait locus for seed dormancy was shown to be particularly affected by FACT function in the ssrp1-2 mutant.
Übersetzung der Zusammenfassung (Deutsch)
-Untersuchung der Transkriptionselongationsfaktoren ELF1, SSRP1, SPT16 und SPT6 mit AP-MS
-Die Rolle von ELF1 in Arabidopsis
-Die Rolle von PTMs auf die Histone Chaperon FACT (Acetylierung von SSRP1 und Phosphorylierung von SPT16)
-SSRP1 und Keimruhe
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