14 research outputs found
In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications
BACKGROUND: The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity. METHODOLOGY: We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30 nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin. CONCLUSIONS: Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains
Structural and functional studies of chromatin in rods photoreceptor nuclei from SCA7 and wild type mice
Les noyaux de bâtonnets de rétines de souris saines présentent une organisation caractéristique dans laquelle la chromatine condensée occupe 70% de leur volume. Chez des souris SCA7, modèles de la maladie humaine Ataxie-SpinoCérébelleuse de type 7, les noyaux de bâtonnets présentent une décondensation de la chromatine associée à des défauts transcriptionnels. Les gènes fortement transcrits dans les bâtonnets de souris saines sont sous-exprimés chez les souris SCA7. La protéine ataxine 7 mutée, responsable de cette maladie, appartient au complexe SAGA qui facilite la transcription notamment en acétylant les histones. La structure de la chromatine est connue pour réguler la transcription et il est généralement admis que la chromatine décondensée est transcriptionnellement active. Le phénotype observé dans les noyaux SCA7 est donc paradoxal. Pour comprendre ce phénotype, des techniques de cryo-préparation des rétines pour la microscopie électronique sont optimisées. L'organisation 3D de la chromatine est déterminée par tomographie. Les ARN polymérases II et les histones modifiées sont cartographiées par immunomarquage à l'or colloïdal. A l'aide de ces méthodes, l'organisation structurale de la chromatine est corrélée in vivo aux modifications des histones et à l'activité transcriptionnelle. La comparaison des noyaux d'animaux SCA7 à ceux d'animaux sains, permet de caractériser des remaniements chromatiniens. L'étude des protéines associées à la chromatine montre une diminution drastique de l'histone H1 dans les noyaux SCA7. Nos travaux suggèrent que l'histone H1, connue pour favoriser la compaction de la chromatine, joue un rôle clé dans le phénotype des souris SCA7.The nuclei of rod photoreceptors in the retina of mice display a characteristic organization with condensed chromatin occupying 70% of their volume. In SCA7 mice, which are models for human type 7 SpinoCerebellar Ataxia, rod nuclei display decondensed chromatin which is associated with defects in transcription. Highly expressed genes in rods of healthy mice are poorly expressed in SCA7 mice. The ataxin 7protein which is mutated in SCA7 disease belongs to the SAGA complex which enhances transcription in particular through histone acetyltransferase activity. Chromatin structure is known to regulate transcription andit is generally thought that decondensed chromatin is transcriptionally active. The phenotype of SCA7 nucleiistherefore paradoxical. To understand this phenotype, methods for the cryo-preparation of retinas and their visualization by electron microscopy were optimized. The 3D organization of chromatin is determined by electron tomography. RNA polymerase II molecules and modified histones are mapped by immuno-gold labeling. Using these techniques, the structural organization of chromatin is correlated in vivo with histone modifications and transcriptional activity. The comparison of nuclei from SCA7 animals to those of healthy animals allows the characterization of chromatin reorganization. The analysis of proteins associated with chromatin demonstrates a drastic decrease in histone H1 in the nuclei of SCA7 mice. Our studies suggest that histone H1, known to be involved in chromatin compaction, plays a key role in the phenotype of SCA7 mice
Structural and functional studies of chromatin in rods photoreceptor nuclei from SCA7 and wild type mice
Les noyaux de bâtonnets de rétines de souris saines présentent une organisation caractéristique dans laquelle la chromatine condensée occupe 70% de leur volume. Chez des souris SCA7, modèles de la maladie humaine Ataxie-SpinoCérébelleuse de type 7, les noThe nuclei of rod photoreceptors in the retina of mice display a characteristic organization with condensed chromatin occupying 70% of their volume. In SCA7 mice, which are models for human type 7 SpinoCerebellar Ataxia, rod nuclei display decondensed ch
Structural and functional studies of chromatin in rods photoreceptor nuclei from SCA7 and wild type mice
Les noyaux de bâtonnets de rétines de souris saines présentent une organisation caractéristique dans laquelle la chromatine condensée occupe 70% de leur volume. Chez des souris SCA7, modèles de la maladie humaine Ataxie-SpinoCérébelleuse de type 7, les noyaux de bâtonnets présentent une décondensation de la chromatine associée à des défauts transcriptionnels. Les gènes fortement transcrits dans les bâtonnets de souris saines sont sous-exprimés chez les souris SCA7. La protéine ataxine 7 mutée, responsable de cette maladie, appartient au complexe SAGA qui facilite la transcription notamment en acétylant les histones. La structure de la chromatine est connue pour réguler la transcription et il est généralement admis que la chromatine décondensée est transcriptionnellement active. Le phénotype observé dans les noyaux SCA7 est donc paradoxal. Pour comprendre ce phénotype, des techniques de cryo-préparation des rétines pour la microscopie électronique sont optimisées. L'organisation 3D de la chromatine est déterminée par tomographie. Les ARN polymérases II et les histones modifiées sont cartographiées par immunomarquage à l'or colloïdal. A l'aide de ces méthodes, l'organisation structurale de la chromatine est corrélée in vivo aux modifications des histones et à l'activité transcriptionnelle. La comparaison des noyaux d'animaux SCA7 à ceux d'animaux sains, permet de caractériser des remaniements chromatiniens. L'étude des protéines associées à la chromatine montre une diminution drastique de l'histone H1 dans les noyaux SCA7. Nos travaux suggèrent que l'histone H1, connue pour favoriser la compaction de la chromatine, joue un rôle clé dans le phénotype des souris SCA7.The nuclei of rod photoreceptors in the retina of mice display a characteristic organization with condensed chromatin occupying 70% of their volume. In SCA7 mice, which are models for human type 7 SpinoCerebellar Ataxia, rod nuclei display decondensed chromatin which is associated with defects in transcription. Highly expressed genes in rods of healthy mice are poorly expressed in SCA7 mice. The ataxin 7protein which is mutated in SCA7 disease belongs to the SAGA complex which enhances transcription in particular through histone acetyltransferase activity. Chromatin structure is known to regulate transcription andit is generally thought that decondensed chromatin is transcriptionally active. The phenotype of SCA7 nucleiistherefore paradoxical. To understand this phenotype, methods for the cryo-preparation of retinas and their visualization by electron microscopy were optimized. The 3D organization of chromatin is determined by electron tomography. RNA polymerase II molecules and modified histones are mapped by immuno-gold labeling. Using these techniques, the structural organization of chromatin is correlated in vivo with histone modifications and transcriptional activity. The comparison of nuclei from SCA7 animals to those of healthy animals allows the characterization of chromatin reorganization. The analysis of proteins associated with chromatin demonstrates a drastic decrease in histone H1 in the nuclei of SCA7 mice. Our studies suggest that histone H1, known to be involved in chromatin compaction, plays a key role in the phenotype of SCA7 mice
Automatic segmentation of high pressure frozen and freeze-substituted mouse retina nuclei from FIB-SEM tomograms
International audienceFocused Ion Beam milling combined with Scanning Electron Microscopy is a powerful tool to determine the 3-D organization of whole cells and tissue at an isotropic resolution of 3-5 nm. This opens the possibility to quantify several cellular parameters and to provide detailed phenotypic information in normal or disease states. Here we describe Biocomputing methods to extract in an automated way characteristic features of mouse rod photoreceptor nuclei such as the shape and the volume of the nucleus; the proportion of heterochromatin; the number, density and distribution of nuclear pore complexes (NPC). Values obtained on five nuclei show that the number of NPC (348 ± 8) is the most conserved feature. Nuclei in higher eukaryotes show large variations in size and rod nuclei are amongst the smallest reported (32 ± 3 lm 3). Despite large species-and cell-type-specific variations in size, the density of NPC (about 15/lm 2) is highly conserved
Immunolabelling of RNA Polymerase II.
<p>(A) Rod nuclei immunogold labelled with antibodies directed against the total pool of RNA Pol II molecules. (B) Rod nuclei immunogold labeled with an antibody directed against the transcriptionally active RNA Pol II in which the CTD heptapeptide is phosphorylated on Ser5. (C) Histogram showing the distances between the EC/HC interface and the immunogold particles in the case of the total pool of RNA Pol II (blue) and the actively transcribing RNA Pol II (red). The grey histogram represents a simulated random distribution of the particles in the same EC domains. The bar represents 200 nm.</p
Radial distribution of the labelling densities within rod nuclei.
<p>(A) Table showing the labelling densities found for each antibody in the different chromatin compartments. (B) Graph representing the labelling density of the silencing histone marks in concentric nuclear compartments. From left to right the graph represents the nuclear envelope-associated heterochromatin (NE-HC), the euchromatin compartment (EC) and five concentric rings of the central heterochromatin domain that are separated by 150nm as indicated on panel B. The darkest bar positions the EC/HC interface. The labelling densities were normalized by the average labelling density of the central HC domain. (C) Same representation as in (B) for the histone marks specific for active transcription. (D) Schematic representation of a rod nucleus showing the euchromatin (white) and the heterochromatin (grey) territories. The central heterochromatin domain is separated into 5 concentric rings separated by 150 nm and analyzed independently to determine the radial variation in labelling density. The coloured dots represent schematically the distribution of the histone marks and respect the colour code used in panel (A).</p