Biallelic non-productive enhancer-promoter interaction precedes imprinted expression of<i>Kcnk9</i>during mouse neural commitment

AbstractHow constitutive allelic methylation at imprinting control regions (ICRs) interacts with other levels of regulation to drive timely parental allele-specific expression along large imprinted domains remains partially understood. To gain insight into the regulation of thePeg13-Kcnk9domain, an imprinted domain with important brain functions, during neural commitment, we performed an integrative analysis of the epigenetic, transcriptomic and cis-spatial organisation in an allele-specific manner in a mouse stem cell-based model of corticogenesis that recapitulates the control of imprinted gene expression during neurodevelopment. We evidence that despite an allelic higher-order chromatin structure associated with the paternally CTCF-boundPeg13ICR, the enhancer-Kcnk9promoter contacts can occur on both alleles, although they are only productive on the maternal allele. This observation challenges the canonical model in which CTCF binding isolates the enhancer and its target gene on either side, and suggests a more nuanced role for allelic CTCF binding at some ICRs.</jats:p

Biallelic non-productive enhancer-promoter interactions precede imprinted expression of Kcnk9 during mouse neural commitment

Summary: It is only partially understood how constitutive allelic methylation at imprinting control regions (ICRs) interacts with other regulation levels to drive timely parental allele-specific expression along large imprinted domains. The Peg13-Kcnk9 domain is an imprinted domain with important brain functions. To gain insights into its regulation during neural commitment, we performed an integrative analysis of its allele-specific epigenetic, transcriptomic, and cis-spatial organization using a mouse stem cell-based corticogenesis model that recapitulates the control of imprinted gene expression during neurodevelopment. We found that, despite an allelic higher-order chromatin structure associated with the paternally CTCF-bound Peg13 ICR, enhancer-Kcnk9 promoter contacts occurred on both alleles, although they were productive only on the maternal allele. This observation challenges the canonical model in which CTCF binding isolates the enhancer and its target gene on either side and suggests a more nuanced role for allelic CTCF binding at some ICRs

Toward the identification of factors involved into concerted regulation of imprinted genes in brain

Un challenge majeur concernant l’empreinte parentale est de comprendre comment l’expression des loci à empreinte peut être différentiellement régulée selon les stades de développement et les tissus. Cette question est importante dans le cerveau car l’expression précise de cette classe de gènes est impliquée dans de nombreux processus neurologiques. Récemment, l’équipe d’accueil a révélé que la chromatine bivalente aux régions de contrôle de l’empreinte (ICR), combinant les marques H3K4me3 (permissive) et H3K27me3 (répressive), contribue à l’expression tissu-spécifique des gènes soumis à empreinte. L’objectif de ma thèse est d’identifier les facteurs de transcription (FT) qui contrôlent la dynamique de H3K27me3 aux ICR dans les lignées neurales, en utilisant un modèle adapté de corticogenèse in vitro. Grâce à une première approche gène-candidat, j’ai identifié la protéine TET3 (ten-eleven translocation 3) comme étant impliquée dans le maintien du différentiel de méthylation ADN aux ICR Peg10, Impact et Zrsr1 dans les lignées neurales. De plus, TET3 régule indirectement d’autres loci d’empreinte, tels que Mest et Peg3, potentiellement en activant l’expression d’un gène codant pour une déméthylase de H3K27me3, Jmjd3 (jumonji domain containing 3). En parallèle, mon projet principal de thèse vise à identifier ces FT de manière exhaustive, grâce à la mise en place d’une approche non biaisée originale. Notre rationnel est que la régulation des gènes soumis à empreinte dépend de réseaux d’interaction médiés par les facteurs Polycomb et les FT qui régulent la dynamique de H3K27me3 aux ICR. Avec le modèle de corticogenèse, nous avons récemment généré une carte résolutive des signatures moléculaires et tridimensionnelles des loci à empreinte, en s’appuyant en particulier sur des expériences de 4C (circular chromosome conformation capture) alléliques dans lesquelles 10 ICR sont utilisées comme ancre. Grâce à une analyse préliminaire de ces données, nous avons pu observer que les ICR sont capables de s’associer physiquement avec certains promoteurs de gènes à empreinte en cis, et que ces contacts sont très fréquemment médiés par l’allèle paternel de l’ICR. Ces analyses questionnent sur le rôle potentiel d’enhancer des ICR. L’analyse complète des données haut-débit de RNA-seq, ChIP-seq, RRBS et 4C à deux étapes de la corticogenèse nous fournira une vue intégrative, allélique et dynamique des signatures linéaires, comme les marques histones et la transcription, mais aussi de l’organisation 3D des ICR. Ces ressources inédites seront traitées par modélisation mathématique afin d’identifier des candidats robustes impliqués dans l’expression cerveau-spécifique des gènes à empreinte. Une fois ce projet abouti, nous disposerons d'un outil pertinent qui fait défaut aujourd’hui, permettant de révéler les mécanismes contrôlant l'empreinte dans le cerveau dans un contexte sain et pathologique.One major challenge in genomic imprinting is to understand how the expression of imprinted loci is regulated at different developmental stages and tissues. This issue is of particular importance in brain where the fine-tuned regulation of imprinted expression is involved in various neurological processes. Recently, my host team revealed that the so-called bivalent chromatin structure at Imprinting Control Regions (ICR), combining the permissive H3K4me3 and repressive H3K27me3 marks, contributes to the appropriate tissue-specific expression of imprinted genes. My objective is to identify the transcription factors (TF) controlling H3K27me3 dynamic at ICR during neural lineage commitment by using a validated in vitro model of murine corticogenesis. By a candidate-based approach, I identified TET3 (ten-eleven translocation 3) as a regulator potentially involved in differential DNA methylation maintenance at Peg10, Impact and Zrsr1 ICR. Moreover, TET3 indirectly regulates other imprinted loci, such as Mest and Peg3, possibly by activating the H3K27me3-specific demethylase gene Jmjd3 (jumonji domain containing 3) in neural lineages. Meanwhile, the main part of my thesis aims to identify those TF exhaustively by conducting an original non-biased approach. Our rational is that the regulation of imprinted genes relies on Polycomb- and TF-mediated physical networks that regulate H3K27me3 dynamic at ICR. With the model of corticogenesis, we have recently generated a high-resolution map of allelic molecular signatures and genome architecture at imprinted domains, mainly by the mean of allelic 4C (circular chromosome conformation capture) experiments using 10 ICR as bait. In this manuscript, a preliminary analysis allowed us to observe that ICR are involved in cis-interactions with imprinted genes promoters, and that these contacts are frequently mediated by paternal unmethylated allele of ICR. Those observations raise the question about the potential enhancer function of ICR. Once high-throughput data from RNA-seq, ChIP-seq, RRBS and 4C at two steps of corticogenesis fully analyzed, it will provide an allelic and integrative view of linear genomics features dynamic, such as histone modifications and transcription, in combination with the dynamic of the 3D organization at ICR. This unprecedented resource will be treated by mathematical modeling to identify robust candidate actors of brain-specific imprinted expression. Outcome of this project will provide us relevant, and so far missing, tools to decipher the underlying mechanisms involved in brain-specific imprinting in normal and pathological contexts

TET3 controls the expression of the H3K27me3 demethylase Kdm6b during neural commitment

International audienceThe acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression

Biallelic non-productive enhancer-promoter interaction precedes imprinted expression of<i>Kcnk9</i>during mouse neural commitment

How constitutive allelic methylation at imprinting control regions (ICRs) interacts with other levels of regulation to drive timely parental allele-specific expression along large imprinted domains remains partially understood. To gain insight into the regulation of the Peg13-Kcnk9 domain, an imprinted domain with important brain functions, during neural commitment, we performed an integrative analysis of the epigenetic, transcriptomic and cis-spatial organisation in an allele-specific manner in a mouse stem cell-based model of corticogenesis that recapitulates the control of imprinted gene expression during neurodevelopment. We evidence that despite an allelic higher-order chromatin structure associated with the paternally CTCF-bound Peg13 ICR, the enhancer-Kcnk9 promoter contacts can occur on both alleles, although they are only productive on the maternal allele. This observation challenges the canonical model in which CTCF binding isolates the enhancer and its target gene on either side, and suggests a more nuanced role for allelic CTCF binding at some ICR