| License: Creative Commons Attribution 4.0 (98MB) |
- URN to cite this document:
- urn:nbn:de:bvb:355-epub-370844
- DOI to cite this document:
- 10.5283/epub.37084
| Item type: | Thesis of the University of Regensburg (PhD) |
|---|---|
| Open Access Type: | Primary Publication |
| Date: | 4 March 2019 |
| Referee: | Prof. Dr. Klaus D. Grasser |
| Date of exam: | 20 February 2018 |
| Institutions: | Biology, Preclinical Medicine > Institut für Pflanzenwissenschaften > Lehrstuhl für Zellbiologie und Pflanzenphysiologie (Prof. Dr. Klaus Grasser) |
| Keywords: | Affinity-purification coupled to mass spectrometry; Chromatin; Epigenetic; Gene transcription; Histones; histone ubiquitination; mRNA export; mRNA processing; RNA polymerase II; SPT16; SSRP1; transcript elongation factor; transcript elongation complex |
| Dewey Decimal Classification: | 500 Science > 570 Life sciences |
| Status: | Published |
| Refereed: | Yes, this version has been refereed |
| Created at the University of Regensburg: | Yes |
| Item ID: | 37084 |
Abstract (English)
FACT, a heterodimer of SSRP1 and SPT16, is a conserved and essential histone chaperone. FACT binds to H2A-H2B dimers to reorganizes octameric nucleosomes and to make the genomic DNA accessible. For instance, FACT facilitates the progression of the transcription machinery through the chromatin template by destabilizing nucleosomes in the path of the elongating RNAPII. By the reverse action, ...
Abstract (English)
FACT, a heterodimer of SSRP1 and SPT16, is a conserved and essential histone chaperone.
FACT binds to H2A-H2B dimers to reorganizes octameric nucleosomes and to make the genomic
DNA accessible. For instance, FACT facilitates the progression of the transcription machinery
through the chromatin template by destabilizing nucleosomes in the path of the elongating
RNAPII. By the reverse action, FACT restores the chromatin structure in the wake of the
RNAPII and maintains the status quo.
By in vitro EMSA experiments, this study shows that the DNA- and nucleosome binding
properties of Arabidopsis SSRP1 were mediated by its C-terminal HMG-box domain. In vivo,
the loss of the HMG-box domain did not alter the subcellular localization of SSRP1 or the
nuclear protein dynamics/binding properties as shown by in-detail CLSM and FRAP analysis,
respectively. Additionally, immunoblot analysis showed that HMG-box-deficient SSRP1 was still
associated with SPT16 and the transcriptionally active RNAPII in vivo. Phenotyping of SSRP1
HMG-box deficiency mutants showed that the lack of the DNA-binding domain had no effect
on Arabidopsis growth and development. The in vitro data indicate that the binding of FACT
to H2A-H2B dimers in higher eukaryotes depends, in the first place, on the association of the
SSRP1 HMG-box domain with nucleosomal DNA. Nevertheless, the in vivo data suggest that
the loss of the SSRP1 HMG-box domain can be compensated in Arabidopsis by other unknown
factors as may be HMGB proteins that provide the DNA-binding function for FACT.
During transcript elongation, the histone chaperone FACT facilitates together with other
TEFs efficient mRNA synthesis by RNAPII. This study contributed to reveal the composition
of the Arabidopsis transcript elongation complex (TEC) by a proteomic approach using reciprocal
tagging in combination with affinity purification and mass spectrometry. The TEFs FACT,
PAF1-C, SPT4/5, SPT6, and TFIIS co-purified robustly with each other and the elongating
RNAPII, while P-TEFb was not among the interactors. Additionally, further chromatin modifying
factors including NAP1 and the Elongator were repeatedly co-purified with different TEFs.
The phenotypic analysis of Arabidopsis double mutants that are defective in different combinations
of TEFs revealed genetic interactions between the genes encoding subunits of FACT,
PAF1-C, and TFIIS, resulting in synergistic/epistatic effects on plant growth/development
Genome-wide transcriptome profiling of SSRP1- or SPT16-depleted plants in comparison to
wild-type revealed that almost the identical small set of genes was differentially expressed in both
mutants. Strikingly, genes encoding key anthocyanin biosynthesis enzymes were overrepresented
among the genes that were downregulated in both FACT mutants. A phenotypic analysis showed
that FACT-depleted plants have clear defects in the light-induced accumulation of anthocyanin
in their leaves. In response to high light (HL) stress, anthocyanin biosynthesis genes were upregulated
to a lesser extend in the FACT mutants compared to wild type. Additionally, the gene
expression of SSRP1 and SPT16 was upregulated upon HL stress. These data suggest that
FACT is novel factor required for the accumulation of anthocyanins in response to HL stress.
ENY2, an evolutionary conserved adaptor protein, links transcription by RNAPII with export
of the newly synthesized mRNA to the cytoplasm by being part of the transcriptional coactivator
SAGA and the NPC-associated mRNA export complex TREX-2. Histochemical GUS
staining revealed that the Arabidopsis ENY2 promoter is widely active during plant growth and
development. In the plant cells that are expressing ENY2, the protein is forming speckle-like
structures in the nucleoplasm and is highly mobile as shown by in-detail CLSM and FRAP
analysis, respectively.
Using reciprocal tagging of ENY2 and its putative interactors in combination with affinity
purification and mass spectrometry revealed that ENY2 associates with two components
/SGF11, UBP22) of the SAGA histone H2B deubiquitinase (DUB) module. Furthermore, no
subunits of other SAGA modules or the TREX-2 complex were co-purified with ENY2. Additionally,
several splicing complexes especially the U2, U5 and NTC/NTR were identified in the
affinity purification of ENY2. In accordance with these findings, ENY2 and the NTC component
MOS4 co-localized in splicing speckles, whereas no direct protein-protein (PPIs) interactions between
ENY2 and NTC/NTR components were found by Y2H and FRET. Three (ENY2, SGF11,
UBP22) of the four SAGA-DUB components that were identified in yeast, fruit-fly and humans
were highly conserved in Arabidopsis, the two adaptor proteins ENY2 and SGF11 as well as
enzymatically active UBP22. SGF73, the missing protein that links the DUB module to the
remaining SAGA complex in other organisms had no homolog in plants. Direct PPIs between
SGF11 and ENY2 as well as UBP22 could be detected by Y2H and FRET analysis.
The composition of the Arabidopsis SAGA complex was revealed by a proteomic approach
using reciprocal tagging of one representative of each bioinformatically predicted SAGA module
in combination with affinity purification and mass spectrometry. In total, 17 Arabidopsis SAGA
subunits were biochemically identified. The DUB module did almost not co-purify with the
other SAGA modules (HAT, SPT, TAF), which suggest that the plant DUB module can act in
a SAGA-independent manner. Additionally, several subunits of the spliceosome were repeatedly
co-purified with the DUB, HAT, and SPT modules of SAGA.
A reverse genetics approach revealed that knockdown of ENY2 by RNAi had no obvious effect
on plant growth and development, while the complete knockout of ENY2 by CRISPR/Cas9
induced a late flowering phenotype. The overexpression of ENY2 did not cause any obvious
phenotype. The knockdown of SGF11 by a T-DNA insertion resulted as well in a late flowering
phenotype and an upregulation of the floral repressor FLC. Moreover, the global H2Bub levels
were increased in ENY2- and SGF11-depleted plants.
Taken together, this study revealed the composition of the Arabidopsis SAGA complex.
ENY2 is part of a histone H2B de-ubiquitinating module that can exist most likely SAGAindependent
(Figure 9.1). DUB-defective plants show a late flowering phenotype. Additionally,
the DUB as well as other SAGA modules show a strong association to the pre-mRNA splicing
machinery. Surprisingly, Arabidopsis ENY2 is no part of the mRNA export complex TREX-2
as it was shown in other eukaryotes.
Translation of the abstract (German)
Das essentielle/konservierte Histon-Chaperon FACT besteht aus den Untereinheiten SSRP1 und SPT16. Um Nukleosomen zu erkennen, sie umzuformen und die genomische DNA zugänglich zu machen, bindet FACT an Histon H2A-H2B Dimere. Dadurch ermöglicht der FACT Komplex DNA-abhängige Prozesse wie zum Beispiel die Transkription von Genen. In der Elongationsphase der Transkription ermöglicht der FACT Komplex ...
Translation of the abstract (German)
Das essentielle/konservierte Histon-Chaperon FACT besteht aus den Untereinheiten SSRP1 und SPT16. Um Nukleosomen zu erkennen, sie umzuformen und die genomische DNA zugänglich zu machen, bindet FACT an Histon H2A-H2B Dimere. Dadurch ermöglicht der FACT Komplex DNA-abhängige Prozesse wie zum Beispiel die Transkription von Genen. In der Elongationsphase der Transkription ermöglicht der FACT Komplex zusammen mit weiteren Transkript-Elongationsfaktoren (TEF‘s) die effiziente Synthese von mRNA durch die RNA Polymerase II (RNAPII). In dieser Studie wurde die Zusammensetzung des Arabidopsis RNAPII Transkript-Elongationskomplexes mit Hilfe von Proteom-analytischen Methoden (Affinitätschromatografie und Massenspektrometrie) wie folgt aufgeklärt: Die RNAPII interagiert mit den TEF‘s FACT, PAF1-C, SPT4/5, SPT6 und TFIIS. Des Weiteren wurde die Rolle der HMG-box Domäne von Arabidopsis SSRP1 untersucht. In vitro Experimente zeigten, dass die HMG-box Domäne für die Bindung von SSRP1 an DNA/Nukleosomen wichtig ist. Auf der anderen Seite bewiesen in vivo Versuche, dass die HMG-box Domäne am C-Terminus von SSRP1 entbehrlich ist für die Funktion von SSRP1. In einem weiteren Projekt wurde der Zusammenhang zwischen dem Histon-Chaperon FACT und der Stress-induzierten Expression von Anthocyan Biosynthese-Genen untersucht. Dabei wurde gezeigt, dass der FACT Komplex eine wichtige Rolle in der Akkumulation von Anthocyan Pigmenten als Reaktion auf Starklicht-Stress spielt.
In Hefe und Metazoen ist das Protein ENY2 Bestandteil des Transkriptions-Coaktivators SAGA und des an der Kernpore lokalisierten TREX-2 Komplexes. Daher verbindet ENY2 die Transkription (RNAPII) im Zellkern mit dem Export der neu synthetisierten mRNA ins Zytoplasma. Diese Studie zeigt, dass Arabidopsis ENY2 im Nukleoplasma lokalisiert ist, aber nicht an der Kernhülle akkumuliert wie in Hefe/Metazoen. Mit Hilfe von Proteom-analytischen Methoden (Affinitätschromatografie und Massenspektrometrie) wurde gezeigt, dass ENY2 mit SAGA interagiert, aber nicht mit TREX-2. Außerdem konnte mit Proteom-analytischen Methoden die Zusammensetzung des Arabidopsis SAGA Komplexes aufgeklärt werden, welcher aus vier Modulen besteht (HAT, SPT, TAF und DUB). Das DUB Modul, bestehend aus ENY2, SGF11 und UBP22, ist in Pflanzen nur schwach/transient mit den übrigen SAGA Komplex assoziiert. SGF11 interagiert direkt mit ENY2 und UBP22. Arabidopsis Mutanten, in denen die DUB Komponente SGF11 oder ENY2 dezimiert ist, zeigen nur einen schwachen Phänotyp, weisen aber ein erhöhtes Level an mono-ubiquitiniertem Histon H2B auf. Dies deutet darauf hin, dass SAGA-DUB Histon H2B Deubiquitinase Aktivität in Arabidopsis besitzt. Zusätzlich zu Proteinen, die mit der Transkription in Zusammenhang stehen (z.B. TEFs, Mediator) wurden auch viele Spleiß-Faktoren mit dem SAGA Komplex in Verbindung gebracht.
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