A transcriptional and regulatory map of mouse somite maturation

The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression and open chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals

Functional analysis of cis-regulatory elements during vertebrate development

Les éléments cis-régulateurs contrôlent l'expression spatio-temporelle des gènes au cours du développement. La segmentation du rhombencéphale en rhombomères (r) établit l'organisation du cerveau postérieur des vertébrés. La spécification et la formation des segments r3 et r5 sont gouvernées par l'expression du facteur de transcription Krox20/Egr2, sous le contrôle de plusieurs éléments cis-régulateurs. Les éléments B (dans r5) et C (dans r3 et r5) sont actifs au démarrage de l'expression de Krox20 alors que l'élément A (dans r3 et r5) est un élément autorégulateur. En générant la délétion de l'élément A dans le génome murin, nous avons montré qu'il est nécessaire au maintien de l'expression de Krox20 dans r3 et r5. De façon inattendue, la délétion de l'élément C n'impacte pas la phase de démarrage mais abolit le maintien de l'expression de Krox20 dans r3 de manière analogue à celle de l'élément A. J'ai mis en évidence par différentes approches génétiques que l'élément C coopère en cis avec l'élément A, lors de la phase d'autorégulation. Plusieurs approches de conformation et d'accessibilité de la chromatine au locus Krox20 indiquent que l'élément A interagit de façon constitutive avec le promoteur et que l'élément C est capable d'affecter l'accessibilité de l'élément A à distance. Parallèlement, j'ai montré que la délétion de l'élément C chez le poisson-zèbre n'entraîne pas de modification de l'expression du gène Krox20 contrairement à la souris malgré un patron d'expression similaire. Ces approches complémentaires ont permis de caractériser le rôle fonctionnel de plusieurs éléments cis-régulateurs contrôlant l'expression d'un gène clef du développement des Vertébrés.Cis-regulatory elements ensure the specifity and the maintenance of the spatio-temporal gene expression pattern throughout the development of an organism. Segmentation of the hindbrain into rhombomeres (r) establishes the posterior brain organisation in vertebrates. The specification and formation of r3 and r5 are governed by the expression of the transcription factor Krox20/Egr2, controlled by three cis-regulatory elements. Elements B (in r5) and C (in r3 and r5) are active during the initiation of Krox20 expression while element A (in r3 and r5) is an autoregulatory element. By studying the deletion of element A in the mouse genome, we have shown that Krox20 expression is not maintained in absence of this element. Unexpectedly, the deletion of element C does not impair Krox20 initiation but abolishes the maintenance of expression in r3, similarly to element A knock-out. I also showed by different genetic approaches that element C cooperates in cis with element A. Moreover, investigation of chromatin conformation and accessibility in the Krox20 locus indicate that element A and the promoter are engaged in a constitutive interaction and that element C is able to affect the accessibility of element A at a distance. In parallel, the deletion of element C in zebrafish did not reveal any modification in Krox20 expression, despite a similar expression pattern in mouse. These complementary approaches lead to the characterization of the functional role of several cis-regulatory elements driving the expression of a key developmental gene in vertebrates

Analyse fonctionnelle d'éléments cis-régulateurs lors du développement des Vertébrés

Cis-regulatory elements ensure the specifity and the maintenance of the spatio-temporal gene expression pattern throughout the development of an organism. Segmentation of the hindbrain into rhombomeres (r) establishes the posterior brain organisation in vertebrates. The specification and formation of r3 and r5 are governed by the expression of the transcription factor Krox20/Egr2, controlled by three cis-regulatory elements. Elements B (in r5) and C (in r3 and r5) are active during the initiation of Krox20 expression while element A (in r3 and r5) is an autoregulatory element. By studying the deletion of element A in the mouse genome, we have shown that Krox20 expression is not maintained in absence of this element. Unexpectedly, the deletion of element C does not impair Krox20 initiation but abolishes the maintenance of expression in r3, similarly to element A knock-out. I also showed by different genetic approaches that element C cooperates in cis with element A. Moreover, investigation of chromatin conformation and accessibility in the Krox20 locus indicate that element A and the promoter are engaged in a constitutive interaction and that element C is able to affect the accessibility of element A at a distance. In parallel, the deletion of element C in zebrafish did not reveal any modification in Krox20 expression, despite a similar expression pattern in mouse. These complementary approaches lead to the characterization of the functional role of several cis-regulatory elements driving the expression of a key developmental gene in vertebrates.Les éléments cis-régulateurs contrôlent l'expression spatio-temporelle des gènes au cours du développement. La segmentation du rhombencéphale en rhombomères (r) établit l'organisation du cerveau postérieur des vertébrés. La spécification et la formation des segments r3 et r5 sont gouvernées par l'expression du facteur de transcription Krox20/Egr2, sous le contrôle de plusieurs éléments cis-régulateurs. Les éléments B (dans r5) et C (dans r3 et r5) sont actifs au démarrage de l'expression de Krox20 alors que l'élément A (dans r3 et r5) est un élément autorégulateur. En générant la délétion de l'élément A dans le génome murin, nous avons montré qu'il est nécessaire au maintien de l'expression de Krox20 dans r3 et r5. De façon inattendue, la délétion de l'élément C n'impacte pas la phase de démarrage mais abolit le maintien de l'expression de Krox20 dans r3 de manière analogue à celle de l'élément A. J'ai mis en évidence par différentes approches génétiques que l'élément C coopère en cis avec l'élément A, lors de la phase d'autorégulation. Plusieurs approches de conformation et d'accessibilité de la chromatine au locus Krox20 indiquent que l'élément A interagit de façon constitutive avec le promoteur et que l'élément C est capable d'affecter l'accessibilité de l'élément A à distance. Parallèlement, j'ai montré que la délétion de l'élément C chez le poisson-zèbre n'entraîne pas de modification de l'expression du gène Krox20 contrairement à la souris malgré un patron d'expression similaire. Ces approches complémentaires ont permis de caractériser le rôle fonctionnel de plusieurs éléments cis-régulateurs contrôlant l'expression d'un gène clef du développement des Vertébrés

Krox20 hindbrain regulation incorporates multiple modes of cooperation between cis-acting elements

International audienceDevelopmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between cis-acting elements are poorly documented, notably in vertebrates. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements

Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes

Schematic of the <i>cis</i>-regulation of <i>krox20</i> expression in r3 and r5, illustrating differences between zebrafish and mouse.

<p><i>Cis</i>-acting elements are indicated by light blue boxes along the locus, with their position with respect to the site of transcription initiation underneath. The different types of activities of the elements are represented by arrows originating from the element: enhancer activities involved in the initiation of <i>krox20</i> expression are indicated by green arrows pointing toward the promoter, enhancer activities corresponding to direct autoregulation are indicated by blue arrows pointing back to the element and the potentiator activity of element C is represented by red arrows pointing toward element A. Question marks indicate that the activity is suspected, but not confirmed.</p

Physical interactions within the <i>Krox20</i> locus.

<p><b>(A)</b> Alignment of data in the <i>Krox20</i> and adjacent loci from Hi-C in ES cells [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref011" target="_blank">11</a>], 4C-seq in E9.5 whole mouse embryos, using the <i>Krox20</i> and <i>Nrbf2</i> promoters as viewpoints (this work, 2 biological replicates) and CTCF ChIP-seq in E14.5 mouse brain (ENCODE, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref058" target="_blank">58</a>]). <b>(B)</b> Zoom in on the <i>Krox20</i> locus, showing 4C-seq data from the <i>Krox20</i> promoter, element A, element B and element C as viewpoints. CTCF ChIP-seq data in E14.5 mouse brain (ENCODE) are indicated below. Signals from simultaneously processed E9.5 whole embryo (dark blue) and E8.5 embryo head (light blue) samples are shown. On the right, normalized distributions of the 4C-seq signals in different genomic regions are indicated. TADs as defined in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.ref007" target="_blank">7</a>] or by our additional analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006903#pgen.1006903.s002" target="_blank">S2 Fig</a>) are indicated above, with dashed lines in the graphs demarcating TAD boundaries. Genes (black/red), <i>cis</i>-regulatory elements (orange) and genomic coordinates are indicated below each set of data. Arrowheads above each 4C track pinpoint viewpoints.</p

DNA accessibility and candidate enhancer sequences within and around the zebrafish <i>krox20</i> locus.

<p>UCSC genome browser view of the <i>krox20</i> locus showing gene positions (purple), repetitive sequences (black) and the sequences selected for enhancer activity tests (light blue), including those that showed activity (named A to F). Below are ATAC-seq data from experiments performed at the indicated stages, either on whole embryos (95% epiboly) or dissected hindbrain or posterior regions of the embryos (5s and 15s), as shown on the schematics on the right side. The seven mostly significant peaks located in non-coding sequences are highlighted in yellow. Underneath is a Vista browser view of sequence conservation between zebrafish and mouse (black) over the region.</p

<i>krox20</i> r5 expression involves cooperation between three enhancer elements.

<p>Embryos carrying combinations of deletions affecting both alleles of elements B, A and/or C, as indicated, were analysed for <i>krox20</i> expression by in situ hybridization at the indicated stages. Somatic deletions are indicated by the * symbol and positions of r3 and r5 are shown.</p