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 toPpia orArbp levels. In each panel, each bar represents the mean value ± the standard error of the mean (SEM) (n = 3–6).
(A) Down-regulation of genes encoding transcription factors,Crx, Nrl, andNr2e3, involved in photoreceptor differentiation and maintenance.
(B) Expression analysis of cone-specific genes,Bcp, Gnat2, andCarr, in WT and R7E animals.
(C) Severe down-regulation of rod-specific genes,Rho, Gnat1, Pde6b, Rhok, Rom, andRbp3, encoding components of the phototransduction machinery.

(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 theRho gene, each amplified regions were separated by 1.5 kb at least. Each bar represents the mean value ± SEM (n = 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(Rho, Pde6b, Rbp3, Nrl, andCrx) showed marked decrease in Pol II occupancy at their promoters.

(A–F) BAC probes containingRho (A–C) orActa2 (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 ofActa2 within the densely DAPI-stained heterochromatin region in WT rod nuclei (arrowheads in [D] and [E]). By contrast,Rho was excluded from this compact heterochromatin region. In R7E rod nuclei,Rho andActa2 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.
(G) Distribution ofRho andActa2 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 consecutivez 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.

(A) Histological examination of retina from 2-mo-old R7N (panels 1 and 3) and R7E (panels 2 and 4) mice. Toluidin-blue staining of semi-thin sections revealed a discontinuous pattern of segment loss which paralleled an irregular appearance of rod chromatin decondensation in R7E animals (upper panels). High magnification micrographs (lower panels) showed that rods with preserved nuclear morphology are connected with inner segments of normal appearance. Scale bars represent 20 μm in upper panels and 10 μm in lower panels.
(B–D) Rhodopsin immunolocalization in 2-mo-old R7N and R7E retina.
(B) Immunohistochemistry of paraffin-embedded sections was performed using an anti-rhodopsin antibody (4D2). In R7E retina, residual staining was observed in the innermost part of the ONL and in folds in which normal condensed rod nuclei were typically located. Scale bar represents 10 μm.
(C and D) Immunofluorescence of retinal cryosections were performed using an anti-rhodopsin antibody (4D2) and DAPI to visualize rod nuclei. Images were collected by confocal imaging analysis, and merged images (rhodopsin in red, DAPI in grey scale) are shown. (C) Very few cells retained rhodopsin staining, which circumscribes the nucleus in the ONL. (D) Higher magnification images demonstrated that residual staining was exclusively found in rods showing a preserved nuclear morphology (arrows), whereas no rhodopsin could be detected in rods showing an altered chromatin organization (arrowheads). Scale bar represents 5 μm.
INL, inner nuclear layer; IS, inner segments; RPE, retinal pigment epithelium.

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.
(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, namelyRho, Pde6b, andRbp3. No such differences could be observed at promoters from two genes regulating rod terminal differentiation,Crx andNrl, and at the intronic enhancer region of a house-keeping gene,Ncl.
(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(Rho, Pde6b, andRbp3). Promoters fromCrx, Nrl, andNcl did not show any differences in histone H3 acetylation.
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 (seeFigure S9). Each bar represents the mean value ± SEM (n = 4).

(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.
(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.
(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).

(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.
(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.
(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.
(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 inFigure S6A.