Gene targeting | EPFL Graph Search

En biologie moléculaire, le gene targeting est une méthode permettant le remplacement d'un gène par une nouvelle séquence par le biais de la recombinaison homologue. La nouvelle séquence peut être un nouveau gène, une version modifiée du même gène ou tout autre séquence d'intérêt. Au sens large le gene targeting décrit tout remplacement ciblé d'une séquence dans un génome. Prix Nobel Le Prix Nobel de physiologie ou médecine 2007 fut décerné à Mario R. Capecchi, Martin J. Evans et Oliver Smithies pour leurs travaux sur les « principes pour l'introduction de modification génétique spécifique chez la souris par l'usage cellules souches embryonnaires ». Notes et références Articles connexes

Knock-out_(génétique)
Souris knock-out
Knock-in

Catégorie:Technique de biologie moléculaire

Transposable elements contribute to the spatiotemporal microRNA landscape in human brain development

Evaristo Jose Planet Letschert, Christopher James Playfoot, Shaoline Sheppard, Didier Trono

Transposable elements (TEs) contribute to the evolution of gene regulatory networks and are dynamically expressed throughout human brain development and disease. One gene regulatory mechanism influenced by TEs is the miRNA system of post-transcriptional control. miRNA sequences frequently overlap TE loci and this miRNA expression landscape is crucial for control of gene expression in adult brain and different cellular contexts. Despite this, a thorough investigation of the spatiotemporal expression of TE-embedded miRNAs in human brain development is lacking. Here, we identify a spatiotemporally dynamic TE-embedded miRNA expression landscape between childhood and adolescent stages of human brain development. These miRNAs sometimes arise from two apposed TEs of the same subfamily, such as for L2 or MIR elements, but in the majority of cases stem from solo TEs. They give rise to in silico predicted high-confidence pre-miRNA hairpin structures, likely represent functional miRNAs and have predicted genic targets associated with neurogenesis. TE-embedded miRNA expression is distinct in the cerebellum when compared to other brain regions, as has previously been described for gene and TE expression. Furthermore, we detect expression of previously non-annotated TE-embedded miRNAs throughout human brain development, suggestive of a previously undetected miRNA control network. Together, as with non-TE-embedded miRNAs, TE-embedded sequences give rise to spatiotemporally dynamic miRNA expression networks, the implications of which for human brain development constitute extensive avenues of future experimental research. To facilitate interactive exploration of these spatiotemporal miRNA expression dynamics, we provide the "Brain miRTExplorer" web application freely accessible for the community.

2022

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014

Genetically encoded multivalent sensors to detect bivalent epigenetic modifications in living stem cells

Aurore Marie-France Delachat

Eukaryotic DNA is organized in the form of chromatin whose basic unit is the nucleosome. The four core histones forming the nucleosome, H2A, H2B, H3 and H4 can be highly post-translationally modified, especially on their N-terminal tail protruding from the nucleosome particle. Histone post-translational modifications (PTMs) work combinatorially to establish chromatin states defined by specific gene expression status. Found in embryonic stem cells (ESCs) at promoters of key developmental genes, bivalent chromatin is the combination of the active chromatin mark, trimethylation of lysine 4 on histone H3 (H3K4me3) and the repressive mark, H3K27me3. Established and maintained by Polycomb (Pc) and Trithorax (Trx) proteins, bivalency is proposed to keep gene transcription repressed but poised for activation. How bivalent domains are organized within the nucleus and how it is installed by Pc and Trx is still unkown. In this work, we aim to answer these questions by designing probes that allow live cell imaging of bivalent domains and by studying the establishment and removal of H3K4 methylation on nucleosomes. The current lack of live cell imaging methods for PTM patterns prompt us to engineer genetically encoded sensors which bind to bivalent marks in a multivalent fashion. These sensors contain a fluorescent protein and two reader domains, joined by flexible linkers. Their selectivity for bivalent nucleosomes was tested in a pulldown assay with a library of differently modified reconstituted nucleosomes. For this, we obtained site-specifically modified histones via expressed protein ligation. The best probe was then applied in live ESCs to visualize bivalent domains. Subsequent imaging by confocal microscopy revealed the organization of bivalent chromatin into discrete and local clusters. Furthermore, this probe was employed to monitor loss of bivalency upon treatment with a small molecule epigenetic inhibitor. Then, we studied the histone-lysine N-methyltransferase Set1B and lysine-specific histone demethylase 1A (LSD1), enzymes which deposit and remove methyl groups of H3K4 respectively. We measured a mid-micromolar affinity for LSD1 to reconstituted nucleosomes using microscale thermophoresis. This interaction might have an impact on the recruitment of LSD1 to its target genes. We then measured the activity of Set1B complex on symmetrically modified H3K27me3 nucleosomes and on asymmetrically modified H3K4me3 nucleosomes. We showed that H3K27me3 does not influence the activity of Set1B complex whereas H3K4me3 activates Set1B-mediated deposition of methyl groups at K4 on the opposite H3 tail. This findings might have important implications concerning the establishment of bivalent chromatin. Together these results gave insights about multivalent binding of tandem reader domains, subnuclear bivalent chromatin organization and establishment of bivalent marks. In the future, we envisage to develop multivalent sensors for other PTM patterns of biological interest.

EPFL2017