DS0405 - Génétique et génomique: relation génotype-phénotype, interactions génome-environnement, épigénétique

Transcriptional regulation by nucleolar sequestration – NucleoReg

Transcriptional regulation by nucleolar sequestration

The goal of this project is <br />to demonstrate the role of the nucleolus in genome organization and RNA polymerase II genes expression in plants. The <br />nucleolus is the largest nuclear compartment, implicated in ribosome biogenesis, but where RNA polymerase II is <br />excluded. However, genomic regions that do not contain ribosomal RNA genes associate with the nucleolus (Nucleolus- <br />Associated Domains or NADs). The working hypothesis is that genes are sequestered in the nucleolus to prevent <br />transcription.

This project aims to demonstrate whether nucleolus sequestration may be a way to regulate gene transcription.

Several layers of regulation are implicated for proper gene expression. Genome organization and gene positioning are <br />some of them and were clearly shown in plants to impact on light signalling and flowering time. The <br />nucleolus is the largest nuclear compartment, implicated in ribosome biogenesis, but where RNA polymerase II is <br />excluded. However, genomic regions that do not contain ribosomal RNA genes associate with the nucleolus (Nucleolus- <br />Associated Domains or NADs). The working hypothesis is that genes are sequestered in the nucleolus to prevent <br />transcription. <br />Our first NADs identification from Arabidopsis thaliana <br />leaves revealed that NADs are composed of heterochromatin and repetitive elements, but also euchromatic domains, some containing genes <br />unexpressed in leaves, associate with the nucleolus. Because nuclear organization is very dynamic, I propose to identify <br />genes present in the NADs in different tissues and at different developmental stages. Plants have to face environmental <br />challenges and their adaptation is crucial to survive. At the molecular level, stress response is accompanied by nuclear <br />and transcriptional reorganization. Analysing NADs composition upon stress will test the impact of gene regulation by <br />nucleolar sequestration during stress response, in the context of an entire organism. <br />To validate these genomic data, NAD nuclear positioning will be examined at the cell scale. These approaches will <br />allow us to follow the nucleolus association of NADs containing genes expressed after a stimulus. We will therefore <br />validate whether or not, upon transcriptional activation, the nucleolus association of a gene in a NAD is lost. To complete <br />this project, a genetic screen will be performed to discover factors implicated in NAD nucleolar sequestering. <br />Overall, this project aims to demonstrate the existence of a novel gene regulation pathway by nucleolus sequestering and <br />its role in plant development and adaptation.

We are identifying NADs in different cell-types and at different time-points during plant development. Plants expressing the nucleolar marker FIBRILLARIN2 under the control of ubiquitous or cell-specific promoters, already available, will be used to isolate nucleoli by FACS. The nucleolar DNA will then be sequenced by high-throughput sequencing and analysed to identify the NADs. In parallel, a transcriptomic analysis of these samples will be realized to corroborate the nucleolar association and the expression level of the genes present in NADs (by RNA-seq and quantitative PCR). In addition, to see whether nucleolar sequestration may at least partially impact on differential allelic expression in hybrid plants, I propose to identify NADs from Arabidopsis plants that have been subjected to intraspecific and interspecific crosses using the same approach.

In parallel, we will develop new approaches to visualize the nucleolar association of specific genes or clusters of genes directly in plant cells. We develop a non-intrusive DNA labelling system in plants, suitable for live single-copy gene detection in the context of an entire organism. Two approaches are currently developed: (i) a modified CRISPR-based technique and (ii) the ANCHOR system. (i) The type II CRISPR (clustered regularly interspaced short palindromic repeats) system uses an EGFP-tagged, endonuclease-deactivated Cas9 protein to recognize a genomic DNA target via a structurally optimized small guide (sg) RNA. (ii) In the ANCHOR system, a single copy small DNA fragment of 1 kb serves as a binding platform. In that case, oligomerisation of a binding factor (ParB, a bacterium-specific protein) creates fluorescent foci without altering the neighbouring genes chromatin context and transcriptional activity. This technology has been successfully used in yeast and human cells. A patent for which I signed an agreement and an MTA for its development in Arabidopsis thaliana restricts its use in eukaryotes.

We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.
In addition, our technical development of single copy-gnes detection in planta gave us promising preliminary data that should now be validated.

Our main results on NADs were recently published in Cell Reports (Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome. Pontvianne, Frédéric et al. Cell Reports , Volume 16 , Issue 6 , 1574 - 1587).

Our next goal is to determine our dynamic are NADs in different nuclear context and under stress conditions.

Pontvianne F#, Carpentier MC, Durut N, Pavlištová V, Jaške K, Schorová S, Parrinello H, Rohmer M, Pikaard CS, Fojtová M, Fajkus J and Sáez-Vásquez J. (2016) Identification of nucleolus-associated chromatin domains reveals the role of the nucleolus in the

Several layers of regulation are implicated for proper gene expression. Genome organization and gene positioning are some of them and were clearly shown in plants to impact on light signalling and flowering time. The goal of this project is to demonstrate the role of the nucleolus in genome organization and RNA polymerase II genes expression in plants. The nucleolus is the largest nuclear compartment, implicated in ribosome biogenesis, but where RNA polymerase II is excluded. However, genomic regions that do not contain ribosomal RNA genes associate with the nucleolus (Nucleolus-Associated Domains or NADs). The working hypothesis is that genes are sequestered in the nucleolus to prevent transcription.
Using novel protocols designed to isolate nucleoli from any plant tissue, I have identified NADs from Arabidopsis thaliana leaves. In addition to heterochromatin and repetitive elements, euchromatic domains, some containing genes unexpressed in leaves, associate with the nucleolus. Because nuclear organization is very dynamic, I propose to identify genes present in the NADs in different tissues and at different developmental stages. Plants have to face environmental challenges and their adaptation is crucial to survive. At the molecular level, stress response is accompanied by nuclear and transcriptional reorganization. Analysing NADs composition upon stress will test the impact of gene regulation by nucleolar sequestration during stress response, in the context of an entire organism.
To validate these genomic data, NAD nuclear positioning will be examined at the cell scale. Novel single-copy gene imaging approaches will be developed to allow gene-positioning analyses in planta and in real time. These approaches will allow us to follow the nucleolus association of NADs containing genes expressed after a stimulus. We will therefore validate whether or not, upon transcriptional activation, the nucleolus association of a gene in a NAD is lost. To complete this project, a genetic screen will be performed to discover factors implicated in NAD nucleolar sequestering.
Overall, this project aims to demonstrate the existence of a novel gene regulation pathway by nucleolus sequestering and its role in plant development and adaptation.

Project coordinator

Monsieur Frédéric PONTVIANNE (Laboratoire Génome et Développement des Plantes)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LDGP Laboratoire Génome et Développement des Plantes

Help of the ANR 229,107 euros
Beginning and duration of the scientific project: December 2015 - 42 Months

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