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Interactome and transcriptome approaches to study HISTONE MONOUBIQUITINATION1 (HUB1) function in plant growth and development

Tommaso Matteo Boccardi UGent (2010)
abstract
The chromatin fiber is a protein-DNA association that allows to confine the DNA molecule within living cells. To give an idea, the Arabidopsis thaliana DNA is ca. 4 cm long (4,125 cm assuming the presence of 125´000´000 base pairs which are 3.3 Å long) and it has to fit in the nucleus which is just 1–10 μm long in diameter. The cell evolved the production of positively-charged proteins called histones that can be wrapped by the negatively-charged DNA filament resulting in a highly packed structure. At the same time, this tight association obstructs the contact of RNA Polymerase II with DNA, a multimeric complex that triggers gene transcription. When a gene has to be transcribed, chromatin must be in an open state. For this, histone post-translational modifications are epigenetic marks that regulate this process. For example, acetylation can change histone-DNA affinity by erasing the positive charge of chromatin. On the other hand, methyl group and ubiquitin are attached to the nucleosomes functioning as signals involved in RNA Polymerase II-driven gene transcription. Histone H2B monoubiquitination (H2BUb) has been extensively studied in Saccharomyces cerevisiae cells where it has a critical function in promoting gene transcription (reviewed in chapter 2). Like for polyubiquitination for degradation, H2BUb is catalyzed by three enzymes called E1 activating enzyme, E2 conjugating enzyme and the E3 ligase. In yeast, the two latter are called Rad6 and Bre1 respectively. This work aimed at investigating the role of the epigenetic mark H2BUb in the model species Arabidopsis thaliana. The following chapters will report a detailed investigation of the E3 ligase HISTONE MONOUBIQUITINATION1 (HUB1), the Arabidopsis Bre1 homologue. Our aims were: 1. to functionally characterize HUB1 with the use of the hub1-1 mutant 2. to identify target genes of H2BUb 3. to explore the HUB1 interactome 4. to functionally characterize newly discovered HUB1 interactors The hub1-1 mutant originates from an EMS mutagenesis screening for leaf mutants and, by mapbased cloning, we identified the gene in which the mutation occurred. A micro array was performed to characterize the molecular phenotype of hub1-1 and to learn about the pathway affected. In addition, a biochemical assay with recombinant proteins was done to confirm the role of HUB1 in histone H2B monoubiquitination (Chapter 3). For aim 2 (Chapter 4), our interest was to identify putative target genes of H2BUb and to decipher how specific is the occurrence of H2BUb at various gene sequences. The first task was done performing a micro array loop design with wild type, hub1-1 and OE-HUB1 lines. Regarding the specificity of H2BUb, ChIP experiments on a number of genes among the most differentially expressed in the micro array loop were performed. In addition, we checked the putative cross talk Aims 14 between H2BUb and H3K4me3 in Arabidopsis at H2BUb target sequences using the hub1-1 mutant in comparison to the wild type. Our next goal was then to identify new HUB1 interactors (chapter 5). To address this question, we took advantage of two in-house techniques such as Tandem Affinity Purification (TAP) and Yeast-2- Hybrid (H2Y). In the first case, Arabidopsis cell suspensions were transformed with a GS-TAP tagged version of HUB1. The proteins obtained in this experiment were then also fused with the same tag, an approach called “reverse TAP”. Subsequently, the Y2H assay was used to check pairwise interaction of all the proteins identified by means of TAP. To fulfill aim 4, we functionally characterize two of the consistent interactors of HUB1 using T-DNA insertion lines (Chapter 6). The phenotypic characterization was mainly focused on the flowering time and growth behavior of these mutant lines since these are two traits affected in the HUB1 mutant. In addition, a double mutant between HUB1 and its direct interactor SUESS was generated to support the Y2H data with putative genetic interaction between these two proteins.
Please use this url to cite or link to this publication:
author
promoter
UGent and UGent
organization
year
type
dissertation (monograph)
subject
pages
236 pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Zwijnaarde : Technologiepark (FSVM building)
defense date
2010-04-09 16:00
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
3007329
handle
http://hdl.handle.net/1854/LU-3007329
date created
2012-10-05 11:13:39
date last changed
2012-10-08 10:15:12
@phdthesis{3007329,
  abstract     = {The chromatin fiber is a protein-DNA association that allows to confine the DNA molecule within living cells. To give an idea, the Arabidopsis thaliana DNA is ca. 4 cm long (4,125 cm assuming the presence of 125{\textasciiacute}000{\textasciiacute}000 base pairs which are 3.3 {\AA} long) and it has to fit in the nucleus which is just 1--10 \ensuremath{\mu}m long in diameter. The cell evolved the production of positively-charged proteins called histones that can be wrapped by the negatively-charged DNA filament resulting in a highly packed structure. At the same time, this tight association obstructs the contact of RNA Polymerase II with DNA, a multimeric complex that triggers gene transcription. When a gene has to be transcribed, chromatin must be in an open state. For this, histone post-translational modifications are epigenetic marks that regulate this process. For example, acetylation can change histone-DNA affinity by erasing the positive charge of chromatin. On the other hand, methyl group and ubiquitin are attached to the nucleosomes functioning as signals involved in RNA Polymerase II-driven gene transcription. Histone H2B monoubiquitination (H2BUb) has been extensively studied in Saccharomyces cerevisiae cells where it has a critical function in promoting gene transcription (reviewed in chapter 2). Like for polyubiquitination for degradation, H2BUb is catalyzed by three enzymes called E1 activating enzyme, E2 conjugating enzyme and the E3 ligase. In yeast, the two latter are called Rad6 and Bre1 respectively. This work aimed at investigating the role of the epigenetic mark H2BUb in the model species Arabidopsis thaliana. The following chapters will report a detailed investigation of the E3 ligase HISTONE MONOUBIQUITINATION1 (HUB1), the Arabidopsis Bre1 homologue. Our aims were: 1. to functionally characterize HUB1 with the use of the hub1-1 mutant 2. to identify target genes of H2BUb 3. to explore the HUB1 interactome 4. to functionally characterize newly discovered HUB1 interactors The hub1-1 mutant originates from an EMS mutagenesis screening for leaf mutants and, by mapbased cloning, we identified the gene in which the mutation occurred. A micro array was performed to characterize the molecular phenotype of hub1-1 and to learn about the pathway affected. In addition, a biochemical assay with recombinant proteins was done to confirm the role of HUB1 in histone H2B monoubiquitination (Chapter 3). For aim 2 (Chapter 4), our interest was to identify putative target genes of H2BUb and to decipher how specific is the occurrence of H2BUb at various gene sequences. The first task was done performing a micro array loop design with wild type, hub1-1 and OE-HUB1 lines. Regarding the specificity of H2BUb, ChIP experiments on a number of genes among the most differentially expressed in the micro array loop were performed. In addition, we checked the putative cross talk Aims 14 between H2BUb and H3K4me3 in Arabidopsis at H2BUb target sequences using the hub1-1 mutant in comparison to the wild type. Our next goal was then to identify new HUB1 interactors (chapter 5). To address this question, we took advantage of two in-house techniques such as Tandem Affinity Purification (TAP) and Yeast-2- Hybrid (H2Y). In the first case, Arabidopsis cell suspensions were transformed with a GS-TAP tagged version of HUB1. The proteins obtained in this experiment were then also fused with the same tag, an approach called {\textquotedblleft}reverse TAP{\textquotedblright}. Subsequently, the Y2H assay was used to check pairwise interaction of all the proteins identified by means of TAP. To fulfill aim 4, we functionally characterize two of the consistent interactors of HUB1 using T-DNA insertion lines (Chapter 6). The phenotypic characterization was mainly focused on the flowering time and growth behavior of these mutant lines since these are two traits affected in the HUB1 mutant. In addition, a double mutant between HUB1 and its direct interactor SUESS was generated to support the Y2H data with putative genetic interaction between these two proteins.},
  author       = {Boccardi, Tommaso Matteo},
  language     = {eng},
  pages        = {236},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {Interactome and transcriptome approaches to study HISTONE MONOUBIQUITINATION1 (HUB1) function in plant growth and development},
  year         = {2010},
}

Chicago
Boccardi, Tommaso Matteo. 2010. “Interactome and Transcriptome Approaches to Study HISTONE MONOUBIQUITINATION1 (HUB1) Function in Plant Growth and Development”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
Boccardi, T. M. (2010). Interactome and transcriptome approaches to study HISTONE MONOUBIQUITINATION1 (HUB1) function in plant growth and development. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
Boccardi TM. Interactome and transcriptome approaches to study HISTONE MONOUBIQUITINATION1 (HUB1) function in plant growth and development. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2010.
MLA
Boccardi, Tommaso Matteo. “Interactome and Transcriptome Approaches to Study HISTONE MONOUBIQUITINATION1 (HUB1) Function in Plant Growth and Development.” 2010 : n. pag. Print.