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Functional significance and evolutionary conservation of HP1 - RNA interactions

English title Functional significance and evolutionary conservation of HP1 - RNA interactions
Applicant Bühler Marc
Number 155940
Funding scheme Project funding (Div. I-III)
Research institution Friedrich Miescher Institute for Biomedical Research
Institution of higher education Institute Friedrich Miescher - FMI
Main discipline Molecular Biology
Start/End 01.01.2015 - 31.12.2017
Approved amount 703'000.00
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All Disciplines (2)

Discipline
Molecular Biology
Biochemistry

Keywords (5)

Epigenetics; Genome regulation; Heterochromatin; Non-coding RNA; genome-engineering

Lay Summary (German)

Lead
Ribonukleinsäuren (RNA) entstehen in allen lebenden Zellen wenn Gene abgelesen werden und dienen als Matrizen für die Synthese von Proteinen. Es gibt aber auch RNAs die nicht für Proteine kodieren. Die biologische Funktion ist für die meisten dieser RNAs unbekannt. Dieses Projekt leistet einen Beitrag zu deren besserem Verständnis.
Lay summary

Funktionelle Bedeutung und evolutionäre Konservierung von HP1-RNA Interaktionen

Inhalt und Ziele des Forschungsprojekts

Die Molekularbiologie hat in den letzten Jahren revolutionäre Erkenntnisse über die Komplexität des Erbgutes gewonnen. Nur 2% des menschlichen Genoms kodiert für Proteine und es wird geschätzt, dass ein großer Teils des Genoms für RNAs codiert die nicht als Matrizen für die Proteinsynthese dienen. In den meisten Fällen ist es aber völlig unklar welche funktionellen Eigenschaften diese RNAs aufweisen. Das Projekt befasst sich mit einer erst vor kurzem in Spalthefen Entdeckten Eigenschaft von RNAs.

In einem ersten Teil des Projekts wird die Regulation von einem wichtigen Erbsubstanz-bindenden Protein (HP1) durch RNA genauer untersucht. Es werden genetische und molekularbiologische Experimente in Spalthefe (Schizosaccharomyces pombe), sowie biochemische und biophysikalische Messungen im Reagenzglas durchgeführt. In einem zweiten Teil wird untersucht, ob sich die gewonnen Erkenntnisse aus Spalthefen auf Säugetiere, dessen Zellen dieselben HP1 Proteine herstellen, übertragen lassen. Dazu werden revolutionäre genetische Methoden verwendet die das Verändern des Erbguts in Zellkulturen erlauben.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Das Projekt befasst sich mit Grundlagenforschung und verzichtet gezielt auf das verwenden von genetisch veränderten Tieren. Anstelle werden Experimente in Hefen- und Mauszellen durchgeführt, um ganz grundlegende Fragen über die Rolle von nicht protein-kodierenden RNAs zu adressieren. Dies ist ein wichtiger erster Schritt um zu verstehen inwiefern nicht protein-kodierende RNAs zu Krankheiten führen könnten.

Key words

non-coding RNA, epigenetics, genome-engineering, heterochromatin, genome regulation

Direct link to Lay Summary Last update: 02.10.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells
Flemr Matyas, Bühler Marc (2015), Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells, in Cell Reports, 12(4), 709-716.

Datasets

Genomics datasets

Author Mohn, Fabio
Publication date 23.05.2018
Persistent Identifier (PID) GSE97945
Repository Gene Expression Omnibus
Abstract
- Expression profiling by high throughput sequencing - Genome binding/occupancy profiling by high throughput sequencing- Overall design: ChIP-seq, RNA-seq and ATAC-seq experiments were performed in mouse ES cells of isogenic background, each experiment was performed in biological replicates.- 82 samples were deposited and described in detail on GEO

The HP1 protein interactome of mouse embryonic stem cells

Author Iesmantavicius, Vytautas
Publication date 22.05.2018
Persistent Identifier (PID) PXD006226
Repository ProteomeXchange, PRIDE repository
Abstract
The family of Heterochromatin Protein 1 (HP1) consists of highly conserved proteins, which have important functions in the nucleus of eukaryotic cells. In mammals there are three HP1 paralogs: HP1(alpha), Hp1(beta), and Hp1(gamma)They are encoded by the Cbx5, Cbx1, and Cbx3 genes, respectively. Hp1 and Hp1 stably interact with Chd4 and Adnp to form the ChAHP complex. In this project, Chd4, Adnp, and the three Cbx genes were endogenously tagged with a FLAG-Avi tag in mouse embryonic stem cells. The tagged proteins were subjected to tandem-affinity purification and analysis by mass spectrometry.

Collaboration

Group / person Country
Types of collaboration
Nicolas Thomä, Friedrich Miescher Institute for Biomedical Research Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Sebastian Hiller, Biozentrum, University of Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Beat Fierz, EPFL, Lausanne Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Jörg Betschinger, Friedrich Miescher Institute for Biomedical Research Switzerland (Europe)
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Max Planck Freiburg 5th Epigenetic Meeting Talk given at a conference Insights into ChAHP complex function 05.12.2018 Freiburg i.B., Germany Bühler Marc;
Genetics and Autism, learning from cancer and other diseases: the ADNP syndrome as a case study Talk given at a conference Discovery and function of the ChAHP complex 10.05.2018 Tel Aviv, Israel Bühler Marc;


Abstract

Heterochromatin conformation is essential for proper structure and function of centromeres and telomeres. In addition, heterochromatin contributes to the silencing of unwanted and potentially harmful genomic regions, such as retroelements. During development, heterochromatin formation at selected protein-coding genes helps to establish cell type-specific gene expression programs upon cell differentiation. Hence, perturbation of heterochromatin structure and function can result in developmental defects and disease.From fission yeast to metazoans, heterochromatin is characterized by a set of specific histone marks: trimethylation of lysine 9 in H3 (H3K9me3) and the absence of lysine acetylation - as well as the presence of HP1 proteins, which are conserved from fission yeast to mammals. HP1 was originally thought to exert its repressive function by promoting formation and spreading of a H3K9me-enriched rigid chromatin structure inaccessible to the transcription machinery. However, multiple studies, including our own, have shown that HP1 association with heterochromatin is highly dynamic and that transcription within heterochromatin is possible, arguing against the idea of a static and transcriptionally inactive heterochromatin domain. Importantly, yeast and mouse HP1 proteins have been demonstrated to interact with RNA. However, the precise mechanism and functional relevance of HP1-RNA interactions have remained poorly understood. The main goal of this project is to investigate HP1-RNA interactions with biochemical and biophysical tools in vitro to identify and functionally characterize specific HP1 mutants that fail to bind RNA in vivo, in both yeast and mammalian cells:•Affinity chromatography approaches combined with mass-spectrometry and next generation sequencing will be used to determine the RNA and protein “interactome” of HP1 proteins.•Chemical chromatin synthesis and reconstitution will be combined with biophysical approaches to characterize the kinetics of the interactions of HP1 proteins with chromatin and the regulation thereof by RNA.•Yeast and mouse embryonic stem cells expressing specific HP1 mutants will be generated to perform loss-of-function-studies. The research that we have planned addresses fundamental questions in a new field of research, which is crucial for both a complete understanding of genome regulatory networks in the cell and our ability to develop novel therapeutic approaches to treat diseases that have been linked to perturbed chromatin regulation. I anticipate that our analyses will significantly advance our understanding of RNA-mediated heterochromatin regulation and accelerate discovery in many other disciplines of chromatin and RNA research.
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