L’organisation fonctionnelle et spatiale de la chromatine pendant le développement des lymphocytes T

Chromosome folding takes place at different hierarchical levels, with various topologies correlated with control of gene expression. Despite the large number of recent studies describing chromatin topologies and their correlations with gene activity, many questions remain, in particular how these topologies are formed and maintained. To understand better the link between epigenetic marks, chromatin topology and transcriptional control, we use CHi-C technique based on the chromosome conformation capture (3C) method. By using two capture strategies targeting two different chromatin structures (chromatin loops and topological domains), we have been able to decipher the chromatin structure associated with thymocyte differentiation and to highlight mechanisms for the transcriptional control of certain genes. Future experiments of the lab will examine mechanisms other than transcription which may influence chromatin architecture, such as differential binding of CTCF, and how these may interplay with transcriptional control and chromatin architecture.Malgré les vastes études démontrant le rôle de la conformation génomique dans le contrôle transcriptionnel, de nombreuses questions restent en suspens, et en particulier, comment ces structures chromatiniennes sont formées et maintenues. Pour mieux comprendre les liens entre l’état de la chromatine au niveau des éléments régulateurs, la topologie de la chromatine et la régulation de la transcription, nous utilisons la technique CHi-C basée sur la technologie de capture de la conformation chromosomique (3C). En utilisant deux stratégies de capture ciblant deux différentes structure chromatiniennes (les boucles chromatiniennes et les domaines topologiques), nous avons pu décrypter la structure chromatinienne associée à la différenciation des thymocytes et mettre en évidence des mécanismes de contrôle transcriptionnel de certains gènes. Les expériences futures de l’équipe vont consister à examiner les facteurs (hors transcription) qui peuvent influencer l'architecture de la chromatine, comme la liaison différentielle des CTCF, et comment ces facteurs peuvent être coordonnés par le contrôle de transcription

4See: A Flexible Browser to Explore 4C Data

It is established that transcription of many metazoan genes is regulated by distal regulatory sequences beyond the promoter. Enhancers have been identified at up to megabase distances from their regulated genes, and/or proximal to or within the introns of unregulated genes. The unambiguous identification of the target genes of newly identified regulatory elements can thus be challenging. Well-studied enhancers have been found to come into direct physical proximity with regulated genes, presumably by the formation of chromatin loops. Chromosome conformation capture (3C) derivatives that assess the frequency of proximity between different genetic elements is thus a popular method for exploring gene regulation by distal regulatory elements. For studies of chromatin loops and promoter-enhancer communication, 4C (circular chromosome conformation capture) is one of the methods of choice, optimizing cost (required sequencing depth), throughput, and resolution. For ease of visual inspection of 4C data we present 4See, a versatile and user-friendly browser. 4See allows 4C profiles from the same bait to be flexibly plotted together, allowing biological replicates to either be compared, or pooled for comparisons between different cell types or experimental conditions. 4C profiles can be integrated with gene tracks, linear epigenomic profiles, and annotated regions of interest, such as called significant interactions, allowing rapid data exploration with limited computational resources or bioinformatics expertise

VING: a software for visualization of deep sequencing signals.

International audienceNext generation sequencing (NGS) data treatment often requires mapping sequenced reads onto a reference genome for further analysis. Mapped data are commonly visualized using genome browsers. However, such software are not suited for a publication-ready and versatile representation of NGS data coverage, especially when multiple experiments are simultaneously treated. We developed 'VING', a stand-alone R script that takes as input NGS mapping files and genome annotations to produce accurate snapshots of the NGS coverage signal for any specified genomic region. VING offers multiple viewing options, including strand-specific views and a special heatmap mode for representing multiple experiments in a single figure. VING produces high-quality figures for NGS data representation in a genome region of interest. It is available at http://vm-gb.curie.fr/ving/ . We also developed a Galaxy wrapper, available in the Galaxy tool shed with installation and usage instructions

Context-dependent transcriptional remodeling of TADs during differentiation

Metazoan chromosomes are organized into discrete domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between TAD organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow TAD dynamics during development, we used Capture Hi-C to interrogate the TADs around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and sub-domains were formed around some activated genes without changes in CTCF binding. The ectopic induction of one gene was sufficient to recapitulate microdomain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin

Transcription induces context-dependent remodeling of chromatin architecture during differentiation.

Metazoan chromosomes are organized into discrete spatial domains (TADs), believed to contribute to the regulation of transcriptional programs. Despite extensive correlation between domain organization and gene activity, a direct mechanistic link is unclear, with perturbation studies often showing little effect. To follow chromatin architecture changes during development, we used Capture Hi-C to interrogate the domains around key differentially expressed genes during mouse thymocyte maturation, uncovering specific remodeling events. Notably, one TAD boundary was broadened to accommodate RNA polymerase elongation past the border, and subdomains were formed around some activated genes without changes in CTCF binding. The ectopic induction of some genes was sufficient to recapitulate domain formation in embryonic stem cells, providing strong evidence that transcription can directly remodel chromatin structure. These results suggest that transcriptional processes drive complex chromosome folding patterns that can be important in certain genomic contexts

A multiple super-enhancer region establishes inter-TAD interactions and controls Hoxa function in cranial neural crest

Abstract Enhancer-promoter interactions preferentially occur within boundary-insulated topologically associating domains (TADs), limiting inter-TAD interactions. Enhancer clusters in linear proximity, termed super-enhancers (SEs), ensure high target gene expression levels. Little is known about SE topological regulatory impact during craniofacial development. Here, we identify 2232 genome-wide putative SEs in mouse cranial neural crest cells (CNCCs), 147 of which target genes establishing CNCC positional identity during face formation. In second pharyngeal arch (PA2) CNCCs, a multiple SE-containing region, partitioned into Hoxa Inter-TAD Regulatory Element 1 and 2 (HIRE1 and HIRE2), establishes long-range inter-TAD interactions selectively with Hoxa2, that is required for external and middle ear structures. HIRE2 deletion in a Hoxa2 haploinsufficient background results in microtia. HIRE1 deletion phenocopies the full homeotic Hoxa2 knockout phenotype and induces PA3 and PA4 CNCC abnormalities correlating with Hoxa2 and Hoxa3 transcriptional downregulation. Thus, SEs can overcome TAD insulation and regulate anterior Hoxa gene collinear expression in a CNCC subpopulation-specific manner during craniofacial development