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Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts' maps could uncover functionally and clinically related genes.

Contains fulltext : 174530.pdf (Publisher’s version ) (Open Access) Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts' maps could uncover functionally and clinically related genes.

In female mammals, one of the two X chromosomes is transcriptionally silenced to equalize X-linked gene dosage relative to XY males, a process termed X chromosome inactivation. Mechanistically, this is thought to occur via directed recruitment of chromatin modifying factors by the master regulator, X-inactive specific transcript (Xist) RNA, which localizes in cis along the entire length of the chromosome. A well-studied example is the recruitment of polycomb repressive complex 2 (PRC2), for which there is evidence of a direct interaction involving the PRC2 proteins Enhancer of zeste 2 (Ezh2) and Supressor of zeste 12 (Suz12) and the A-repeat region located at the 5' end of Xist RNA. In this study, we have analyzed Xist-mediated recruitment of PRC2 using two approaches, microarray-based epigenomic mapping and superresolution 3D structured illumination microscopy. Making use of an ES cell line carrying an inducible Xist transgene located on mouse chromosome 17, we show that 24 h after synchronous induction of Xist expression, acquired PRC2 binding sites map predominantly to gene-rich regions, notably within gene bodies. Paradoxically, these new sites of PRC2 deposition do not correlate with Xist-mediated gene silencing. The 3D structured illumination microscopy was performed to assess the relative localization of PRC2 proteins and Xist RNA. Unexpectedly, we observed significant spatial separation and absence of colocalization both in the inducible Xist transgene ES cell line and in normal XX somatic cells. Our observations argue against direct interaction between Xist RNA and PRC2 proteins and, as such, prompt a reappraisal of the mechanism for PRC2 recruitment in X chromosome inactivation.

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Douze lignées de blé tendre en cours de sélection à l’INRA et 3 témoins ont été étudiés pour la stabilité du rendement dans le réseau multilocal INRA. Ce matériel a été évalué dans des dispositifs à 2 blocs randomisés, les parcelles élémentaires totalisant au moins 5,9 m2. Au total, 24 milieux diversifiés ont été étudiés durant les campagnes 1990-1991, 1991-1992 et 1992-1993. Le facteur milieu a été défini par la combinaison des niveaux des facteurs lieu, mode cultural et année. Dans chaque milieu, une description biologique a été réalisée pendant les phases de mise en place du nombre de grains par m2 et de remplissage du grain. L’analyse de la stabilité a pris en compte sous forme de covariables les caractéristiques du milieu définies au cours de la description précédente et a permis ainsi de constater que le comportement des génotypes se différencie uniquement pendant le remplissage. Par ailleurs, les 2 modèles d’analyse de l’interaction, régression conjointe et régression factorielle, ont permis de préciser que le comportement des lignées INRA est bien différent de celui des témoins. En effet, les génotypes sélectionnés à Rennes sont, à l’inverse des témoins, les plus résistants aux conditions défavorables de milieu. Deux autres lignées sélectionnées à Clermont-Ferrand et à Estrées-Mons sont à la fois productives et stables, présentant une bonne adaptabilité générale et un assez bon niveau de résistance aux conditions défavorables de milieu, ce qui laisse présager qu’il est possible d’associer rusticité et productivité en sélection., The yield stability of 12 lines of winter wheat bred by INRA (Institut National de Recherche Agronomique) and 3 controls has been studied in the INRA network. The material was evaluated in randomized 2-block designs, elementary plots measuring at least 5.9 m2. Twenty-four various environments have been studied over 3 seasons: 1990-1991, 1991-1992 and 1992-1993. They are combinations of levels of 3 factors: location, condition and year. Each environment has been biologically described during the period of the establishment of the number of grains per square meter and the grain-filling period. The analysis of stability included environmental characteristics as covariates. These were defined in a previous description and showed that the genotypes only behaved in a different way during the grain-filling period. In addition, the 2 models used for the analysis of interaction, joint regression and factorial regression showed that the behaviour of the INRA lines was quite different from that of controls. In contrast to controls, the genotypes selected in Rennes are the most resistant to unfavourable conditions of the environment. Two other entries, selected in Clermont-Ferrand and Estrées-Mons, are simultaneously productive, stable, with good general adaptability and a rather good resistance to unfavourable conditions of environment, which indicates that it is possible to associate hardiness and yield potential in a breeding programme.

National audience; The yield stability of 12 lines of winter wheat bred by INRA (Institut National de Recherche Agronomique) and 3 controls has been studied in the INRA network. The material was evaluated in randomized 2-block designs, elementary plots measuring at least 5.9 m2. Twenty-four various environments have been studied over 3 seasons: 1990-1991, 1991-1992 and 1992-1993. They are combinations of levels of 3 factors: location, condition and year. Each environment has been biologically described during the period of the establishment of the number of grains per square meter and the grain-filling period. The analysis of stability included environmental characteristics as covariates. These were defined in a previous description and showed that the genotypes only behaved in a different way during the grain-filling period. In addition, the 2 models used for the analysis of interaction, joint regression and factorial regression showed that the behaviour of the INRA lines was quite different from that of controls. In contrast to controls, the genotypes selected in Rennes are the most resistant to unfavourable conditions of the environment. Two other entries, selected in Clermont-Ferrand and Estrées-Mons, are simultaneously productive, stable, with good general adaptability and a rather good resistance to unfavourable conditions of environment, which indicates that it is possible to associate hardiness and yield potential in a breeding programme.; Douze lignées de blé tendre en cours de sélection à l’INRA et 3 témoins ont été étudiés pour la stabilité du rendement dans le réseau multilocal INRA. Ce matériel a été évalué dans des dispositifs à 2 blocs randomisés, les parcelles élémentaires totalisant au moins 5,9 m2. Au total, 24 milieux diversifiés ont été étudiés durant les campagnes 1990-1991, 1991-1992 et 1992-1993. Le facteur milieu a été défini par la combinaison des niveaux des facteurs lieu, mode cultural et année. Dans chaque milieu, une description biologique a été réalisée pendant les phases de mise en place du nombre de grains par m2 et de remplissage du grain. L’analyse de la stabilité a pris en compte sous forme de covariables les caractéristiques du milieu définies au cours de la description précédente et a permis ainsi de constater que le comportement des génotypes se différencie uniquement pendant le remplissage. Par ailleurs, les 2 modèles d’analyse de l’interaction, régression conjointe et régression factorielle, ont permis de préciser que le comportement des lignées INRA est bien différent de celui des témoins. En effet, les génotypes sélectionnés à Rennes sont, à l’inverse des témoins, les plus résistants aux conditions défavorables de milieu. Deux autres lignées sélectionnées à Clermont-Ferrand et à Estrées-Mons sont à la fois productives et stables, présentant une bonne adaptabilité générale et un assez bon niveau de résistance aux conditions défavorables de milieu, ce qui laisse présager qu’il est possible d’associer rusticité et productivité en sélection.

Copy number variants (CNVs) are major contributors to genomic imbalance disorders. Phenotyping of 137 unrelated deletion and reciprocal duplication carriers of the distal 16p11.2 220 kb BP2-BP3 interval showed that these rearrangements are associated with autism spectrum disorders and mirror phenotypes of obesity/underweight and macrocephaly/microcephaly. Such phenotypes were previously associated with rearrangements of the non-overlapping proximal 16p11.2 600 kb BP4-BP5 interval. These two CNV-prone regions at 16p11.2 are reciprocally engaged in complex chromatin looping, as successfully confirmed by 4C-seq, fluorescence in situ hybridization and Hi-C, as well as coordinated expression and regulation of encompassed genes. We observed that genes differentially expressed in 16p11.2 BP4-BP5 CNV carriers are concomitantly modified in their chromatin interactions, suggesting that disruption of chromatin interplays could participate in the observed phenotypes. We also identified cis- and trans-acting chromatin contacts to other genomic regions previously associated with analogous phenotypes. For example, we uncovered that individuals with reciprocal rearrangements of the trans-contacted 2p15 locus similarly display mirror phenotypes on head circumference and weight. Our results indicate that chromosomal contacts’ maps could uncover functionally and clinically related genes.

RNA polymerase III (Pol III) synthesizes short noncoding RNAs, many of which are essential for translation. Accordingly, Pol III activity is tightly regulated with cell growth and proliferation by factors such as MYC, RB1, TRP53, and MAF1. MAF1 is a repressor of Pol III transcription whose activity is controlled by phosphorylation; in particular, it is inactivated through phosphorylation by the TORC1 kinase complex, a sensor of nutrient availability. Pol III regulation is thus sensitive to environmental cues, yet a diurnal profile of Pol III transcription activity is so far lacking. Here, we first use gene expression arrays to measure mRNA accumulation during the diurnal cycle in the livers of (1) wild-type mice, (2) arrhythmic Arntl knockout mice, (3) mice fed at regular intervals during both night and day, and (4) mice lacking the Maf1 gene, and so provide a comprehensive view of the changes in cyclic mRNA accumulation occurring in these different systems. We then show that Pol III occupancy of its target genes rises before the onset of the night, stays high during the night, when mice normally ingest food and when translation is known to be increased, and decreases in daytime. Whereas higher Pol III occupancy during the night reflects a MAF1-dependent response to feeding, the rise of Pol III occupancy before the onset of the night reflects a circadian clock-dependent response. Thus, Pol III transcription during the diurnal cycle is regulated both in response to nutrients and by the circadian clock, which allows anticipatory Pol III transcription.

Mouse liver regeneration after partial hepatectomy involves cells in the remaining tissue synchronously entering the cell division cycle. We have used this system and H3K4me3, Pol II and Pol III profiling to characterize adaptations in Pol III transcription. Our results broadly define a class of genes close to H3K4me3 and Pol II peaks, whose Pol III occupancy is high and stable, and another class, distant from Pol II peaks, whose Pol III occupancy strongly increases after partial hepatectomy. Pol III regulation in the liver thus entails both highly expressed housekeeping genes and genes whose expression can adapt to increased demand.

The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genome-wide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that only ∼40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.

Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) experiments are widely used to determine, within entire genomes, the occupancy sites of any protein of interest, including, for example, transcription factors, RNA polymerases, or histones with or without various modifications. In addition to allowing the determination of occupancy sites within one cell type and under one condition, this method allows, in principle, the establishment and comparison of occupancy maps in various cell types, tissues, and conditions. Such comparisons require, however, that samples be normalized. Widely used normalization methods that include a quantile normalization step perform well when factor occupancy varies at a subset of sites, but may miss uniform genome-wide increases or decreases in site occupancy. We describe a spike adjustment procedure (SAP) that, unlike commonly used normalization methods intervening at the analysis stage, entails an experimental step prior to immunoprecipitation. A constant, low amount from a single batch of chromatin of a foreign genome is added to the experimental chromatin. This “spike” chromatin then serves as an internal control to which the experimental signals can be adjusted. We show that the method improves similarity between replicates and reveals biological differences including global and largely uniform changes.

Genome-wide rhythms in RNA polymerase II loading and dynamic chromatin remodeling underlie periodic gene expression during diurnal cycles in the mouse liver., Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver., Author Summary In mammalian organs such as the liver, many metabolic and physiological processes occur preferentially at specific times during the 24-hour daily cycle. The timing of these rhythmic functions depends on a complex interplay between the endogenous circadian clock and environmental timing cues relayed through the master circadian clock in the suprachiasmatic nucleus, or via feeding rhythms. These rhythms can be implemented on several regulatory levels, and here we aimed at a better understanding of the transcriptional and epigenetic changes that regulate diurnal rhythms. We performed genome-wide analysis of the locations of RNA polymerase II (Pol II) and the epigenetic histone modifications H3K4me3 and H3K36me3 at specific times of day, relating these data to mRNA expression levels. Our analyses show that Pol II transcriptional rhythms are biphasic in mouse liver, having predominant peak activities in the morning and evening. Moreover, dynamic changes in histone marks lag transcription rhythms genome-wide, indicating that the epigenetic landscape can be remodeled during the 24-hour cycle. Finally, a quantitative analysis of temporal Pol II and mRNA accumulation profiles indicates that posttranscriptional regulation significantly contributes to the amplitude and phase of mRNA accumulation profiles. While many studies have analyzed how transcription and chromatin states are modified during irreversible cell differentiation processes, our work highlights how these states can evolve reversibly in a system exhibiting periodicity in time.