Glutamine-Expanded Ataxin-7 Alters TFTC/STAGA Recruitment and Chromatin Structure Leading to Photoreceptor Dysfunction

Spinocerebellar ataxia type 7 (SCA7) is one of several inherited neurodegenerative disorders caused by a polyglutamine (polyQ) expansion, but it is the only one in which the retina is affected. Increasing evidence suggests that transcriptional alterations contribute to polyQ pathogenesis, although the mechanism is unclear. We previously demonstrated that theSCA7 gene product, ataxin-7 (ATXN7), is a subunit of the GCN5 histone acetyltransferase–containing coactivator complexes TFTC/STAGA. We show here that TFTC/STAGA complexes purified from SCA7 mice have normal TRRAP, GCN5, TAF12, and SPT3 levels and that their histone or nucleosomal acetylation activities are unaffected. However, rod photoreceptors from SCA7 mouse models showed severe chromatin decondensation. In agreement, polyQ-expanded ataxin-7 induced histone H3 hyperacetylation, resulting from an increased recruitment of TFTC/STAGA to specific promoters. Surprisingly, hyperacetylated genes were transcriptionally down-regulated, and expression analysis revealed that nearly all rod-specific genes were affected, leading to visual impairment in SCA7 mice. In conclusion, we describe here a set of events accounting for SCA7 pathogenesis in the retina, in which polyQ-expanded ATXN7 deregulated TFTC/STAGA recruitment to a subset of genes specifically expressed in rod photoreceptors, leading to chromatin alterations and consequent progressive loss of rod photoreceptor function

Essential role of alpha 6 integrins in cortical and retinal lamination

International audienceExtracellular matrix (ECM) is believed to play important roles in many aspects of nervous system development [1]. The laminins are ECM glycoproteins expressed in neural tissues and are potent stimulators of neurite outgrowth in vitro [1-3]. Genetic approaches using Drosophila and Caenorhabditis elegans have demonstrated a role for laminin and a laminin receptor in vivo in axon pathfinding and fasciculation, respectively [4,5]. In higher organisms, however, the role of laminins in the development of the nervous system is poorly understood. Integrins alpha 6 beta 1 and alpha 6 beta 4 are major laminin receptors. A role for the alpha 6 integrin in neurulation has been reported in amphibians [6]. We previously described mice lacking integrin alpha 6; these mice died at birth with severe skin blistering [7]. Detailed analyses of integrin alpha 6-/- mice reported here revealed abnormalities in the laminar organization of the developing cerebral cortex and retina. Ectopic neuroblastic outgrowths were found on the brain surface and in the vitreous body in the eye. Alterations of laminin deposition were found in mutant brains. Thus, this study provides evidence for an essential role of integrin-laminin interactions in the proper development of the nervous system. These observations are particularly significant given the recent report that human patients suffering from epidermolysis bullosa can carry mutations in ITGA6, the gene encoding the alpha 6 integrin chain [8,9]

Loss of a Specific Gene Compartmentalization in Rods from SCA7 Mice

<div><p>(A–F) BAC probes containing<i>Rho</i> (A–C) or<i>Acta2</i> (D–F) genes were hybridized to retinal cryosections from 2-mo-old WT (A), (B), (D), and (E) or R7E (C) and (F) animals. FISH signals appear in green whereas DAPI-stained photoreceptor nuclei were pseudo-colored in red for better visualization. Merged images were collected by confocal imaging analysis and showed co-localization of<i>Acta2</i> within the densely DAPI-stained heterochromatin region in WT rod nuclei (arrowheads in [D] and [E]). By contrast,<i>Rho</i> was excluded from this compact heterochromatin region. In R7E rod nuclei,<i>Rho</i> and<i>Acta2</i> showed a comparable random pattern of intranuclear distribution. Both could be found co-localizing within the central densely DAPI-stained region (arrowheads in [C] and [F]). Scale bars represent 2 μm.</p> <p>(G) Distribution of<i>Rho</i> and<i>Acta2</i> between heterochromatin and euchromatin territories in WT and R7E rod nuclei was estimated by counting the number of spots detected in the densely DAPI-stained region. Counting was performed on the projection of four consecutive<i>z</i> stacks (1.2 μm between each stack) taken through the retinal section such that only entire rod nuclei were included in the counting. More than 500 nuclei were analyzed, and each bar represents the mean value ± SEM of three independent experiments performed on two different animals.</p></div

Decreased RNA Polymerase II Occupancy at Promoters and Coding Regions of Genes Down-Regulated in SCA7 Mice

<p>(A–F) ChIP assays were performed using formaldehyde-fixed chromatin extracts of retina from 2-mo-old control (WT or R7N; dark grey) and R7E (light grey) mice. Specific regions within the different genes were analyzed, as depicted; within the<i>Rho</i> gene, each amplified regions were separated by 1.5 kb at least. Each bar represents the mean value ± SEM (<i>n</i> = 4–6). RNA polymerase II occupancy, and thus transcriptional activity, was assessed using an antibody directed against the heptapeptide repeat of its C-terminal domain (Pol II CTD). All genes down-regulated at the mRNA level in R7E retina<i>(Rho, Pde6b, Rbp3, Nrl,</i> and<i>Crx)</i> showed marked decrease in Pol II occupancy at their promoters.</p

Comprehensive Characterization of Specific Transcriptional Alterations in the Retina from SCA7 Mice

<div><p>mRNA levels were quantified by real-time PCR of reverse transcribed RNA from 2-mo-old WT and R7E retina. Expression levels are represented as a percentage of the mean of WT littermate mice after normalization to<i>Ppia</i> or<i>Arbp</i> levels. In each panel, each bar represents the mean value ± the standard error of the mean (SEM) (<i>n</i> = 3–6).</p> <p>(A) Down-regulation of genes encoding transcription factors,<i>Crx, Nrl,</i> and<i>Nr2e3,</i> involved in photoreceptor differentiation and maintenance.</p> <p>(B) Expression analysis of cone-specific genes,<i>Bcp, Gnat2,</i> and<i>Carr,</i> in WT and R7E animals.</p> <p>(C) Severe down-regulation of rod-specific genes,<i>Rho, Gnat1, Pde6b, Rhok, Rom,</i> and<i>Rbp3,</i> encoding components of the phototransduction machinery.</p></div

Severe Reorganization of Chromatin Territories in Rod Photoreceptors from SCA7 Mice

<div><p>(A) Ultrastructure of rod nuclei in R7E mice. Electron microscopy micrographs from 2-y-old R7N (panel a) and two distinct R7E (panels b and c) animals are shown. In R7N mice, rod nuclei are characterized by a large territory of centrally located heterochromatin. In R7E mice, rod nuclei were bigger and rounder, and displayed much more euchromatin, which appears lightly stained by electrons. Arrows indicate pale grey structures likely resulting from aggregation of mutant ATXN7 in rods from R7E mice. Scale bar represents 1 μm.</p> <p>(B) Ultrastructure of photoreceptor nuclei during disease progression in R7E mice. Electron microscopy micrographs from 2-y-old R7N (panel a), 2-mo-old R7E (panel b), and 2-y-old R7E (panel c) animals are shown. This time-course analysis revealed that alterations of rod nuclear architecture progressively worsened as retinopathy progressed in R7E mice. Cone photoreceptor nuclei (c), which are found in the outermost part of the ONL adjacent to inner segments (is), present a different architecture with several heterochromatin clumps surrounded by more euchromatin. As evidenced in panel c, rod nuclei in R7E mice look highly similar to cone nuclei. Scale bar represents 5 μm.</p> <p>(C) Ultrastructure of photoreceptor nuclei in SCA7 knock-in mice. Electron microsocopy micrographs from 3-mo-old WT (panels a and d) and two distinct age-matched SCA^7266Q/5Q (panels b, c, e, and f) animals are shown. Morphological appearance of the central densely stained heterochromatin territory is altered in mutant mice. At higher magnification (lower panels [e–f]), rod nuclei from heterozygous knock-in animals are slightly larger and contain more euchromatin than nuclei from control mice. Cone nuclei (c) and photoreceptor inner segments (is) are depicted. Scale bars represent 2 μm in upper panels (a–c) and 0.5 μm in lower panels (e–f).</p></div

Increased TFTC Recruitment and Histone Acetylation at a Specific Subset of Deregulated Genes in R7E Retina

<div><p>ChIP assays were performed using formaldehyde-fixed chromatin extracts of retina from 2-mo-old control (WT or R7N; dark grey) and R7E (light grey) mice. Primers were selected to amplify promoter or enhancer regions of the specified genes, as depicted.</p> <p>(A and B) ChIPs using an antibody against a TFTC-specific subunit (Spt3) revealed an increased recruitment of Spt3 in R7E retina, at promoters from genes normally highly and specifically expressed in differentiated rods, namely<i>Rho, Pde6b,</i> and<i>Rbp3.</i> No such differences could be observed at promoters from two genes regulating rod terminal differentiation,<i>Crx</i> and<i>Nrl,</i> and at the intronic enhancer region of a house-keeping gene,<i>Ncl.</i></p> <p>(C and D) ChIPs using an antibody against acetylated lysines 9 and 14 of histone H3 (Ac H3) revealed an increased acetylation of histone H3 in R7E retina, specifically at the same promoters, which showed increased Spt3 binding<i>(Rho, Pde6b,</i> and<i>Rbp3)</i>. Promoters from<i>Crx, Nrl,</i> and<i>Ncl</i> did not show any differences in histone H3 acetylation.</p> <p>The amount of immunoprecipitated DNA was quantified by real-time PCR and normalized to the amount of DNA present in a fraction of the input chromatin extract. Values are expressed as fold enrichment over background IP signals obtained in corresponding no antibody ChIP experiments (see<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040067#sg009" target="_blank">Figure S9</a>). Each bar represents the mean value ± SEM (<i>n</i> = 4).</p></div

TFTC Subunit Composition and HAT Activity Are Not Altered by PolyQ-Expanded ATXN7 in R7E Retina

<div><p>(A) Western blot analysis of TFTC-type complexes immunopurified from 4-wk-old R7N or R7E retinal homogenates using an anti-ATXN7 antibody. ATXN7 IPs revealed comparable levels of Trrap, Gcn5, and Taf12 in complexes purified from R7N or R7E retina.</p> <p>(B and C) Whole retinal extracts from 4-wk-old R7N and R7E animals were immunoprecipitated with an anti-SPT3 (B) and anti-TRRAP antibody (C). Using the same antibodies, complexes were also immunoprecipitated from HeLa cell nuclear extract as positive controls. The retinal extracts (Input) and the immunopurified complexes were analyzed by immunoblotting with an anti-ATXN7 antibody. Complexes contained similar amounts of normal or mutant ATXN7.</p> <p>(D) Western blot analysis using anti-TRRAP and anti-GCN5 antibodies on complexes immunoprecipitated with an anti-TRRAP antibody from 10-wk-old R7N and R7E retinal homogenates. Using the same antibody, complexes were also immunoprecipitated from HeLa cell nuclear extract as positive controls. Comparable levels of Gcn5 and Trrap were detected in purified complexes from R7N and R7E retina.</p> <p>(E–G) HAT activities of immunopurified complexes bound to either the anti-ATXN7 pAb (E), anti-SPT3 mAb (F), or anti-TRRAP mAb beads (G) from R7N and R7E retina were measured by an in vitro HAT assay performed on free core histones. Histones were separated by SDS-PAGE and stained by Coomassie Brilliant Blue (CBB) and acetylated histones were visualized by fluorography (Fluorogr). The position of each histone is indicated. The pattern of histone acetylation was identical to that of a highly purified TFTC fraction used as a positive control. No histone acetylation could be detected when the antibody was omitted. The age of the mice are indicated in weeks in brackets. These results are representative of three independent experiments. Quantification of all HAT assays performed on R7N and R7E retina is provided in<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040067#sg006" target="_blank">Figure S6</a>A.</p></div