Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause de-repression of DNA-methylated genes and TEs, but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes.

Ablation of Mer function in mer(kd) mice results in a retinal phenotype almost identical with that of RCS rats. The similarity in phenotypes between the two rodent models suggests that an RPE phagocytic defect is a feature of all types of retinal degeneration caused by loss of function of Mer tyrosine kinase, perhaps including mutations in human MERTK.

The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.T he Royal College of Surgeons (RCS) rat strain displays an unusual retinal phenotype in which shed photoreceptor (PR) outer segment (OS) debris accumulates in the subretinal space (1). PR disk shedding normally commences at postnatal day (P)12 in the rat (2), and an OS debris layer is readily apparent in the eyes of RCS animals at P20. Genetic chimera studies suggest that the defect is a cell-autonomous function of the RPE, not the PRs (3). Indeed, in vivo (4) and RPE cell culture studies (5) revealed a defect in the ability of RCS RPE to phagocytize shed OS membranes. Despite the RPE-specific nature of the defect, death strikes the PRs first, through the process of apoptosis (6). The time course of PR degeneration is rapid, beginning around P20, with few PR nuclei remaining in the outer nuclear layer (ONL) by P60. Thinning and atrophy of the RPE begins after onset of PR degeneration. The cause of PR cell death is not well understood; the debris zone acting as a diffusion barrier to metabolites (7), disruption of the interphotoreceptor matrix (8), and PR hypoxia due to diminished oxygen diffusion (9) have been proposed as explanations.In contrast to the extensively studied retinal phenotype of the RCS rat, the nature of the genetic defect has remained obscure until recently. Early genetic linkage studies showed that the phenotype is complet...

The RCS rat is a widely studied model of recessively inherited retinal degeneration. The genetic defect, known as rdy (retinal dystrophy), results in failure of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segment membranes. We previously used positional cloning and in vivo genetic complementation to demonstrate that Mertk is the gene for rdy. We have now used a rat primary RPE cell culture system to demonstrate that the RPE is the site of action of Mertk and to obtain functional evidence for a key role of Mertk in RPE phagocytosis. Phagocytosis is a process by which large particles are internalized by cells to form phagosomes. The process can be divided into three phases: binding, ingestion, and digestion. Retinal pigment epithelial (RPE) 1 cells, which form a polarized epithelium between the photoreceptor cells and the choroid in the outer retina, phagocytize more biomass than any other mammalian cell type (1). The RPE phagocytizes photoreceptor outer segment (OS) membranes (2) that are shed as part of the normal ongoing process of photoreceptor OS renewal (3). Failure of OS membrane uptake leads to photoreceptor cell death (4), as illustrated by the RCS rat, a widely used model for recessively inherited retinal degeneration. The RCS mutation rdy (retinal dystrophy) causes, either directly or indirectly, a defect in RPE phagocytosis (4). This defect leads to an accumulation of shed OS membranes in the subretinal space (4) and a rapid and progressive degeneration of photoreceptor cells (5).The molecular mechanisms of RPE phagocytosis are unclear. Studies of the internalization of exogenous OS by cultured primary RPE cells suggested a receptor-mediated process (6 -8). Inhibition of the RPE cell culture phagocytic assay by antireceptor antibodies or competitive ligands suggested several specific proteins that might play a role in the process, including the mannose receptor (9, 10), CD36 (11), and ␣ v ␤ 5 integrin (12-14). Inhibition of ␣ v ␤ 5 integrin function disrupts the OS binding phase of RPE phagocytosis, whereas the mannose receptor and CD36 have been implicated in both OS binding and ingestion. Cultured RCS RPE cells bind exogenous OS at wildtype levels. However, only a small percentage of bound OS are ingested by RCS RPE cells (15), indicating that the protein encoded by the rdy locus is critical, directly or indirectly, for OS uptake.The gene corresponding to rdy remained unknown until recently. The mannose receptor protein and messenger RNA are present in the RPE of both wild-type and RCS rats from postnatal day (P) 5 to adult (16). CD36 null mice have been reported to have normal electroretinography and retinal histology (17). These data suggest that neither the mannose receptor nor CD36 is the gene mutated in the RCS rat. We used positional cloning to identify a mutation in the receptor tyrosine kinase gene Mertk in the RCS rat. A deletion of RCS genomic DNA results in expression of an aberrant Mertk transcript with a translation termination signal after codon 20 (18),...