The SAGA histone acetyltransferase module targets SMC5/6 to specific genes.

BACKGROUND: Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin. RESULTS: We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. CONCLUSION: Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading.

Long-term engraftment of systemically transplanted, gene-modified bone marrow-derived cells in the adult mouse retina.

AIMS: To provide evidence for a novel route of gene administration to normal and diseased retinas, we performed systemic transplantation of genetically engineered bone marrow-derived cells (BMDCs) to wild-type mice and to mutant mice with retinal degeneration. METHODS: Lethally irradiated recipient mice-C57BL/6 (wild-type), SCA7 (spinocerebellar ataxia type 7) and FVB/N (rd1 mutant)-were transplanted intravenously with 5x106 BMDCs, which were transduced with a retroviral vector to express the enhanced green fluorescent protein (GFP). Chimeras were killed at 1, 3, 8, 11, 12 and 15 months (wild-type) or at 8 and 12 months (mutants) after transplantation. Eyes were enucleated, and the retinas were analysed using immunohistochemistry. RESULTS: In wild-type retinas, BMDCs preferentially engrafted in the inner and outer plexiform layers, the ganglion cell layer and the optic nerve. No BMDCs were found in the photoreceptor layer. BMDCs were more common in the degenerating retinas of the mutant mice. The majority of BMDCs in the retina were identified as microglia based on morphology and immunophenotype. Approximately 8-16% of all CD11b(+) cells in the retina expressed GFP. None of the BMDCs expressed neuronal cell markers. GFP-expressing BMDCs were found to persist for more than 1 year after transplantation. CONCLUSIONS: We demonstrate that gene-modified BMDCs show long-term engraftment and stable expression of GFP from a retrovirus in both wild-type and mutant mouse retinas. Thus, BMDCs may be used as vehicles for gene delivery to the retina.

SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types.

Accumulation of expanded polyglutamine proteins and selective pattern of neuronal loss are hallmarks of at least eight neurodegenerative disorders, including spinocerebellar ataxia type 7 (SCA7). We previously described SCA7 mice displaying neurodegeneration with progressive ataxin-7 accumulation in two cell types affected in the human pathology. We describe here a new transgenic model with a more widespread expression of mutant ataxin-7, including neuronal cell types unaffected in SCA7. In these mice a similar handling of mutant ataxin-7, including a cytoplasm to nucleus translocation and accumulation of N-terminal fragments, was observed in all neuronal populations studied. An extensive screen for chaperones, proteasomal subunits and transcription factors sequestered in nuclear inclusions (NIs) disclosed no pattern unique to neurons undergoing degeneration in SCA7. In particular, we found that the mouse TAF(II)30 subunit of the TFIID initiation complex is markedly accumulated in NIs, even though this protein does not contain a polyglutamine stretch. A striking discrepancy between mRNA and ataxin-7 levels in transgenic mice expressing the wild-type protein but not in those expressing the mutant one, indicates a selective stabilization of mutant ataxin-7, both in this model and the P7E/N model described previously. These mice therefore provide in vivo evidence that the polyglutamine expansion mutation can stabilize its target protein.