Epigenetics; Genome regulation; Heterochromatin; Non-coding RNA; genome-engineering
Flemr Matyas, Bühler Marc (2015), Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells, in Cell Reports
, 12(4), 709-716.
|Persistent Identifier (PID)
Gene Expression Omnibus
- 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
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.
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.