Appeal No. 0330: Bob Lane dba Bethel Oil & Gas v. J. Michael Biddison, Chief, Division of Oil and Gas

Chief\u27s Order 88-156

Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo

L’organisation du noyau au cours de la différenciation cellulaire dans le tissu mammaire

Dans de nombreux tissus, les noyaux des cellules présentent des caractéristiques spécifiques, notamment en ce qui concerne la nature et la répartition des compartiments nucléaires, la position des chromosomes et des gènes. Cette organisation spatiale du noyau laisse apparaître des domaines plus ou moins permissifs pour l’expression des gènes et constituerait un mécanisme épigénétique participant au maintien des profils d’expression tissu-spécifiques. La glande mammaire est un tissu complexe dans lequel les cellules épithéliales mammaires (CEMs), qui synthétisent et secrètent les composants du lait, interagissent avec différents autres types de cellules (myoépithéliales, adipocytes) et la matrice extracellulaire. Des cultures de CEMs reproduisent partiellement la différenciation cellulaire in vitro. Elles ont été utilisées pour suivre la mise en place et l’importance fonctionnelle de l’organisation du noyau. Elles ont permis de montrer comment les stimulations hormonales aboutissent au remodelage des domaines nucléaires et au repositionnement de gènes spécifiques de la glande mammaire, comme par exemple, ceux des protéines du lait. Moduler les conditions de croissance des cultures afin de replacer les cellules dans un micro-environnement dont les caractéristiques sont proches de celles du tissu mammaire, devrait ensuite permettre d’étudier le rôle de cet environnement cellulaire dans l’organisation nucléaire

Towards a stochastic model of the spatial organization and the activity of mammalian genomes

National audienceIn eukaryotes, DNA is confined in the cell nucleus. Therefore it is in the nucleus that the main functions involving DNA such as replication, repair and transcription take place. DNA is highly folded in the nucleus: the whole genome extends on about 2 meters while the nucleus is about 5-10 microns. The wrapping of DNA around protein complexes (nucleosomes), forming the chromatin fiber, induces a first level of DNA compaction. However folding at a higher level is necessary to reach observed levels of compaction. Furthermore it is believed that the nucleus is not spatially homogeneous with respect to transcription: the densities of transcription factors and chromatin vary spatially. Conversely the activity of the genome may play a major role in the setup of its spatial organization. In particular, active loci tend to cluster together and could be responsible for establishing chromatin loops. The interplay between the activity and the spatial organization of the genome has been investigated through diverse experimental methods. Modelling approaches have been developed to test hypotheses about rules gouverning the spatial organization. Models are generally stochastic and can be viewed as Gibbs models. Two types of geometry are considered: chains of fixed length segments (random walks) and sequences of points (worm-like chains). Different types of interactions are included: short-range repulsion (volume exclusion), attraction between consecutive points (compaction), long-range attraction between a subset of points (looping). Such models succeed in reproducing global features of the chromatin spatial organization. So far, models are low-dimensional and the chromatin fibers are supposed to be homogeneous. However recent hight-throughput technics are providing data at the scale of the whole genome showing differences between various chromatin regions. Hence current models should be extended involving heterogeneous fibers instead of homogeneous ones. This means that interaction parameters should be specific to each segment or point, leading to models with thousands of parameters! We will sketch such a model. The chromatin fibers will be represented as sequences of points (worm-like chains) where each point interacts specifically with other points and other nuclear components. In order to investigate the interplay between spatial organization and transcriptional activity, the transcription level of each point will be represented in the model as a mark. The different types of interactions to be considered will be modelled by potentials. This model does not raise new theoretical issues concerning Gibbs model. However it is much more complex than standard models. This is due to the number of types of interactions considered in the model, and to the fact that points are allowed to interact specifically with other elements. Several issues relative to simulation and fitting will be discussed

Modelling a 3D active genome as a Gibbs marked point process

International audienceWe will present a new model accounting for the spatial organization and the transcription of the genome in the mammalian cell nucleus. In this model, genome regions are represented as sequences of points with marks reflecting their transcriptional activity. The probabilistic distribution of the marked points is written as a Gibbs distribution with several potentials representing different types of interactions between nuclear elements. The considered interactions are: volume exclusion (limiting the local chromatin density), chromatin elasticity (limiting the distance between consecutive points lying on the same chromosome), binding of chromatin to the nuclear envelope, repression of transcription due to local crowding and clustering of highly transcribed genomic regions. In addition to encompass a wide range of interactions compared to previous 3D DNA models, our model takes into account the heterogeneity of the chromatin fibers concerning elasticity, transcription and interactions with the nuclear envelope. More precisely, some interaction parameters vary along the sequence of points. Hence the proposed model is of very high dimension with a number of parameters of the same order as the number of points. An algorithm for simulating the model together with some simulation results will be presented. Finally some issues relative to fitting from genome-wide contact data will be discussed

Régulation de la mémoire épigénétique (la dynamique de la méthylation de l'ADN et son couplage aux modifications de la chromatine)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Modelling a 3D active genome as a Gibbs marked point process

International audienceWe will present a new model accounting for the spatial organization and the transcription of the genome in the mammalian cell nucleus. In this model, genome regions are represented as sequences of points with marks reflecting their transcriptional activity. The probabilistic distribution of the marked points is written as a Gibbs distribution with several potentials representing different types of interactions between nuclear elements. The considered interactions are: volume exclusion (limiting the local chromatin density), chromatin elasticity (limiting the distance between consecutive points lying on the same chromosome), binding of chromatin to the nuclear envelope, repression of transcription due to local crowding and clustering of highly transcribed genomic regions. In addition to encompass a wide range of interactions compared to previous 3D DNA models, our model takes into account the heterogeneity of the chromatin fibers concerning elasticity, transcription and interactions with the nuclear envelope. More precisely, some interaction parameters vary along the sequence of points. Hence the proposed model is of very high dimension with a number of parameters of the same order as the number of points. An algorithm for simulating the model together with some simulation results will be presented. Finally some issues relative to fitting from genome-wide contact data will be discussed

Towards a stochastic model of the spatial organization and the activity of mammalian genomes

National audienceIn eukaryotes, DNA is confined in the cell nucleus. Therefore it is in the nucleus that the main functions involving DNA such as replication, repair and transcription take place. DNA is highly folded in the nucleus: the whole genome extends on about 2 meters while the nucleus is about 5-10 microns. The wrapping of DNA around protein complexes (nucleosomes), forming the chromatin fiber, induces a first level of DNA compaction. However folding at a higher level is necessary to reach observed levels of compaction. Furthermore it is believed that the nucleus is not spatially homogeneous with respect to transcription: the densities of transcription factors and chromatin vary spatially. Conversely the activity of the genome may play a major role in the setup of its spatial organization. In particular, active loci tend to cluster together and could be responsible for establishing chromatin loops. The interplay between the activity and the spatial organization of the genome has been investigated through diverse experimental methods. Modelling approaches have been developed to test hypotheses about rules gouverning the spatial organization. Models are generally stochastic and can be viewed as Gibbs models. Two types of geometry are considered: chains of fixed length segments (random walks) and sequences of points (worm-like chains). Different types of interactions are included: short-range repulsion (volume exclusion), attraction between consecutive points (compaction), long-range attraction between a subset of points (looping). Such models succeed in reproducing global features of the chromatin spatial organization. So far, models are low-dimensional and the chromatin fibers are supposed to be homogeneous. However recent hight-throughput technics are providing data at the scale of the whole genome showing differences between various chromatin regions. Hence current models should be extended involving heterogeneous fibers instead of homogeneous ones. This means that interaction parameters should be specific to each segment or point, leading to models with thousands of parameters! We will sketch such a model. The chromatin fibers will be represented as sequences of points (worm-like chains) where each point interacts specifically with other points and other nuclear components. In order to investigate the interplay between spatial organization and transcriptional activity, the transcription level of each point will be represented in the model as a mark. The different types of interactions to be considered will be modelled by potentials. This model does not raise new theoretical issues concerning Gibbs model. However it is much more complex than standard models. This is due to the number of types of interactions considered in the model, and to the fact that points are allowed to interact specifically with other elements. Several issues relative to simulation and fitting will be discussed

MethylQuant: a sensitive method for quantifying methylation of specific cytosines within the genome

Here we present MethylQuant, a novel method that allows accurate quantification of the methylation level of a specific cytosine within a complex genome. This method relies on the well-established treatment of genomic DNA with sodium bisulfite, which converts cytosine into uracil without modifying 5-methyl cytosine. The region of interest is then PCR-amplified and quantification of the methylation status of a specific cytosine is performed by methylation-specific real-time PCR with SYBR Green I using one of the primers whose 3′ end discriminates between the methylation states of this cytosine. The presence of a locked nucleic acid at the 3′ end of the discriminative primer provides the specificity necessary for accurate and sensitive quantification, even when one of the methylation states is present at a level as low as 1% of the overall population. We demonstrate that accurate quantification of the methylation status of specific cytosines can be achieved in biological samples. The method is high-throughput, cost-effective, relatively simple and does not require any specific equipment other than a real-time PCR instrument

Les cellules souches germinales et leur épigénétique chez l'embryon de poulet

International audienc